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Article
Simultaneous Exfoliation and Functionalization of 2H-MoS2 by
Thiolated Surfactants: Applications in Enhanced Antibacterial Activity
Subbaraj Karunakaran, Subhendu Pandit, Bikramjit Basu, and Mrinmoy De
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08994 • Publication Date (Web): 07 Sep 2018
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Page 1 of 12 Journal of the American Chemical Society

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Simultaneous Exfoliation and Functionalization of 2H-MoS2 by
8 Thiolated Surfactants: Applications in Enhanced Antibacterial
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10 Activity
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12 Subbaraj Karunakaran, Subhendu Pandit, Bikramjit Basu and Mrinmoy De.
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14 Department of Organic Chemistry, Laboratory for Biomaterials, Materials Research Centre, Centre for Biosystems Sci-
15 ence and Engineering, Indian Institute of Science, Bangalore 560012, India.
16 KEYWORDS. Functionalization, 2H-MoS2, Thiolated surfactant, Nano-antibiotics
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18 ABSTRACT: Two-dimensional transition metal dichalcogenides (TMDs), such as MoS2, generally exists in two different pol-
19 ymorphic structures, metallic (1T phase) and semiconducting (2H phase). In context of their wide spectrum of applications
20 ranging from electronic to biomedicine, the aspects of ligand conjugation and solution processability are highly significant.
21 In addition, the assessment of their antibacterial property and biocompatibility is equally important to explore their bio-
22 medical applications. Here we report a new method for the exfoliation and direct functionalization of 2H-MoS2 using surfac-
tant molecules with thiol functionality. We found that the exfoliated MoS2 using thiolated ligands are functionalized with
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desired functionality and the processing scheme can be extended to other TMDs. Functionalized 2H-MoS2 exhibits highly
24
enhanced antibacterial efficiency compared to similarly functionalized metallic 1T-MoS2 against pathogenic bacteria. The
25 newly synthesized functionalized 2H-MoS2 exhibit better hemocompatibility which makes it suitable for in vivo applications.
26 This convenient functionalization method opens the door for many other applications of functionalized semiconducting 2H
27 MoS2 and other TMDs.
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31 Introduction TMDs are prepared by direct sonication of bulk materials
32 Over the past few years, single layer nanosheets of transi- in suitable solvents such as water, N-methyl pyrrolidine,
33 tion metal dichalcogenides (TMDs) have attracted a great isopropanol, etc.20-23 Some surfactants or mixed solvent
assisted exfoliations are also utilized. For an example, eth-
34 attention in many applications, ranging from electronics,
sensing, catalysis, biomedical, energy storage etc.1-7 Lay- anol in water can exfoliate and stabilize single to few lay-
35
ered TMDs are mainly present in two polymorphs (2H and ers of TMDs through their close surface energy.24-28 Mac-
36 romolecule assisted exfoliation in the presence of chitosan,
37 1T), depending on the coordination geometry of metal and
chalcogen atoms. In the 2H structure, one metal atom is Protein (BSA) and lignin are also reported.29-31
38
coordinated with six chalcogens in trigonal prismatic (D3h) However, in all cases exfoliated 2H TMDs are not cova-
39 geometry with d-orbital splitting [dz2 (a1), dx2-y2, dxy (e) lently functionalized, which may cause instability over the
40 and dxz, yz (e’)] leading to semiconducting and photolumi- time and limits their applications. In our earlier study, we
41 nescence property. In the 1T structure, the metal coordina- had reported the covalent functionalization of 1T-MoS2 by
42 tion is in the octahedral (D3d) geometry with d-orbital using thiol ligand which was adopted for many applica-
43 splitting [dz2, dx2-y2 (eg) and dxy,xz,yz (t2g)], which results in tions.32 The covalent functionalization has been reported
44 the metallic character.8-11 Depending on the inherent prop- to enhance the stability and applicability.33-35 A few at-
45 erties of those polymorphs, these kinds of materials are tempts of functionalizing 2H-MoS2 using small thiolated
46 used for a wide spectrum of applications. For example, 1T- molecules was reported involving a two-step method: lay-
47 MoS2 with metallic conductivity has been used in hydrogen ered 2H-MoS2 preparation (solvent assisted or physical
48 evolution reaction (HER)12 and cell destruction using NIR vapor deposition) followed by conjugation with a suitable
49 photothermal effect.13 On the other hand, 2H-MoS2 with ligand. However these methods are limited by large-scale
50 finite band gap and semiconducting property is utilized in production and practical utility.33,36-40 So far, small thiolat-
wide-ranging applications, including electronic devices14 ed ligands, such as cysteine41 and lipoic acid42 have been
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and visible light-induced water disinfection.15 used for direct exfoliation and functionalization, but those
52 resulted in multilayer TMDs. The mode of functionalization
53 For many of the above applications, the control over the
preparation of 2H phase of MoS2 with the desired func- for cysteine is found to be physisorption, which compro-
54 mises the extended stability. However, the utilization of
tionalization poses a great challenge due to their inert na-
55 thiolated surfactant for dual purpose i.e. one step process
ture, compared to 1T polymorph.16,17 It is well known that
56 1T TMDs are prepared in bulk quantities in solution by Li- for exfoliation and direct functionalization of 2H-MoS2, is
57 intercalation method, using organolithium compounds limited. Herein, we hypothesized that surfactant thiol lig-
58 such as n-butyl lithium or other alkali metal intercalating and with amphiphilic property could provide efficient ex-
59 (sodium napthalenide).18,19 Similarly, liquid phase 2H foliation and functionalization of 2H-MoS2 simultaneously
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(Figure 1a). In literature, thiol surfactants have been ex- cessed 1T-MoS2 and that was adapted for various applica-
1 tensively used as a stabilizer for noble nanomaterials like tions, such as making highly efficient antibiotics.53 That
2 gold, platinum etc.43,44 Similar ligands have been also used method was further extended by several other groups for
3 to fabricate other nanomaterials such as CdSe.44 But the many more applications.54-56 Hence, we have first consid-
4 use of thiol surfactants in preparation of nanomaterials ered three thiol surfactants with different charge-based
5 along with stabilization through functionalization has rare- head group according to the earlier reported method, due
6 ly been reported.45 The thiol group present at the hydro- to their high biocompatibility and ability to impart stability
7 phobic end of the ligand is likely to be anchored to the sur- to functionalized materials in biological media.32,53 Also, to
8 face of MoS2 as we observed earlier and the desired head validate the importance of thiol group, we have synthe-
group at hydrophilic end will introduce the suitable func- sized similar surfactants without thiol group (Supporting
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tionality in exfoliated materials (figure 1b). Information, Figure S1-S4). In ligand design, we have used
10 C-11 alkyl chain and tetra ethylene glycol as a spacer to
11 In this report, we have used this novel strategy to exfoli-
ate and functionalize 2H-MoS2 using different thiol surfac- obtain a desirable combination of hydrophobicity and hy-
12 drophilicity. For negatively charged head group we have
tants, providing long term (> 8 months) stability for the
13 considered carboxylate functionality. In addition, trime-
first time. Another novel aspect of the present work is to
14 establish the enhanced bactericidal and biocompatibility of thylammonium and hydroxyl groups are used as positively
15 the functionalized 2H-MoS2 solution. Following several charged and neutral head group respectively (Figure 2a).
16 research reports, nanomaterial-based antibiotics are Once all ligands were synthesized and characterized by
17 emerging to combat multidrug-resistant bacteria.46-52 Spe- NMR, they were used for exfoliation of bulk MoS2 by the
18 cifically, Liu et al. have shown that vertically aligned 2H- sonochemical method.
19 MoS2 used in water disinfection and exploited that semi-
20 conducting phase of the materials as better ROS
21 generator15, which inspired us to test the antibacterial ac-
22 tivity. We have tested the ability of these functionalized
23 materials towards antibacterial activity against Gram-
24 positive and Gram-negative multidrug resistant bacteria.
Interestingly, the positively charged 2H-MoS2 shows better
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antibacterial activity with lower dosage even when com-
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pared to the similar positively charged 1T-MoS2, which is
27 known as a highly efficient antibacterial agent from our
28 previous study.53
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38 Figure 2. Monitoring ligand dependent rate of exfoliation
of MoS2 by UV-Vis Spectroscopy. a) The structure of am-
39 phiphilic thiol and non-thiol ligands with three differently
40 charged head groups utilized for exfoliation of the bulk MoS2.
41 b) Temporal evolution of absorbance at 674 nm of exfoliated
42 MoS2 by thiol and non-thiol ligand with a different time inter-
43 val of sonication.
44 In a typical sonochemical exfoliation experiment, 5 mg of
45 bulk MoS2 was added to a 15 ml of aqueous solution con-
46 taining 1.5 equivalents of thiol and non-thiol ligands w.r.t
47 the molecular weight of MoS2. This mixture was pulse
48 Figure 1. Scheme for the exfoliation of 2H-MoS2 with am- sonicated by probe sonicator over a period of 80 minutes
49 phiphilic ligand. a) General ligand structure with amphiphilic and the progress of exfoliation was monitored by UV-Vis
50 nature composed of hydrophobic moieties near thiol (red spectroscopy (Figure 2b), which correlates well with the
51 circle) and hydrophilic tetra ethylene glycol bearing function- amount of exfoliated MoS2 in solution. The optimization of
52 al group (blue oval). b) Schematic representation of the ligand ligand concentration for exfoliation was estimated by UV-
53 induced exfoliation and possible modes of MoS2 surface func- Vis spectroscopy (Supporting Information, Figure S5). The
54 tionalization. spectroscopy result indicates the absorbance evolution at
674 nm, increases as the concentration of ligand increases
55 Results and Discussion:
from 0.5 to 1.5 equivalence and remains saturated at 2
56 Ligand Induced exfoliation of TMDs. We have shown equivalences. As shown in Figure 2b, different ligands ex-
57 that thiolated ligand can functionalize the solution pro-
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hibit different influence on the extent of exfoliation of bulk non-thiolated ligands, where flocculation was observed
1 MoS2, depending on their solubility and interaction after the dialysis (See Figure 3a). This is likely due to the
2 through different head group. The neutral ligands (ligand fact that thiol ligands functionalize and stabilize the
3 1 and ligand 4) exfoliate better than the negatively exfoliated MoS2, preventing the aggregation. Since it is well
4 charged ligand (ligand 2 and ligand 5), followed by posi- known that during exfoliation of MoS2 sulfur deficiencies
5 tive ligands (ligand 3 and ligand 6). But in all cases the will be formed at edges, these sites can be easily occupied
6 rate of exfoliation is higher in the presence of ligands com- by thiol ligands resulting functionalization. Alternatively,
7 pared to the control (no ligands), indicating the crucial role the formation of sulfur-sulfur bond on the basal plane may
8 of ligands in disrupting the van der Waal force between also happen, as reported in earlier studies.42 The presence
two layers of MoS2. The best exfoliation rate of ligand 1 and the attachment of ligand are confirmed by 1H-NMR
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and ligand 4 could be attributed to the hydrophobicity of (Supporting Information, Figure S7-S9), where the TEG
10 the ligand compared to the charged ligands. The favorable linkage of ligands is exposed outside, exhibiting the strong
11 interaction of neutral ligands with MoS2 is distinctively 1H signal but the hydrophobic carbons are buried inside,
12 observed during mixing the ligands and bulk MoS2 (Sup- giving the very weak 1H signal. However, the non-thiolated
13 porting Information, Figure S6). After sonication, further ligands help in exfoliation but lacks conjugation owing to
14 purification was done by centrifugation at 2000 rpm for 10 the absence of thiol. This observation is further confirmed
15 mins. Finally, the samples were subjected to dialysis to by the zeta potential measurement (Figure 3b). The thiol
16 remove the excess of ligands and proceeded for characteri- ligands induced exfoliated MoS2 (after purification) shows
17 zation of all samples. corresponding charges according to the head group, but
18 non-thiol ligands induced exfoliated MoS2 shows negative
19 charges similar to non-functionalized MoS2, as reported
20 earlier.32 This clearly indicates that the thiol ligands func-
21 tionalize and stabilize the exfoliated MoS2. Thermogravi-
metric analysis of functionalized 2H-MoS2 (Supporting In-
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formation, Figure S10) further supports the presence of
23 attached ligands and indicates the presence of ~70% of the
24 ligand by weight on the surface of 2H-MoS2. X-ray diffrac-
25 tion (XRD) study used to understand the mode of function-
26 alization between edge vs basal plane. The XRD pattern
27 (Figure 3c) indicates that functionalization is mainly hap-
28 pened at the edges, since there is no change in (002) peak
29 at 14.40 in all the functionalized MoS2 when compared to
30 JCPDS #37-1492 (14.340 for 2H-MoS2) database.
31 Further characterization was then performed to ensure
32 that the exfoliated materials are single layers and 2H
33 phase. AFM images of the exfoliated MoS2 (by ligand 3 as
34 representative image) (Figure 4a) revealed that the exfoli-
35 ated materials are single layers with height 1.5 nm along
36 with the ligand, as shown in the height profile diagram
37 (Inset Figure 4a). Further, UV-Visible spectra show the
presence of 2H-MoS2 with A and B excitons peaks at 674
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nm and 613 nm which are attributed to the direct excitonic
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transitions at the K point of Brillouin zone. Also, indirect
40 transition arises at Γ & Ʌ point in Brillouin zone from C and
41 Figure 3. Stability of functionalized 2H-MoS2 and under- D exciton at 457 and 398, which are present in contrast to
42 standing the mode of functionalization. a) Colloidal stabil- the 1T-MoS2 (Figure 4b).57-59 The Raman spectra (Figure
43 ity of functionalized (Ligand 3) and non-functionalized MoS2 4c) show a corresponding mode of vibration of 2H-MoS2
44 (Ligand 6) over a period of more than eight months. b) Zeta (out-of-plane A1g-384 & in-plane E2g-409), with the ab-
45 potential of ligand induced exfoliated MoS2 by thiol ligands sence of 1T associated peaks (at ≈ J1-157, J2-226 and J3-330
and non-thiol ligands. Different charges with thiol ligands
46 cm-1) indicates predominantly 2H-MoS2 was obtained by
indicate the surface functionalization. c) XRD pattern of exfo-
47 liated MoS2 by using thiol ligands which compared with JCPDS
all the ligands.60 The presence of 2D-flake nature of MoS2
48 with average size 100-200 nm confirmed by low resolution
standard of MoS2 indicates mode of functionalization is on the
49 edges.
Transmission microscopy (TEM), as shown in Figure 4d.
50 The high-resolution TEM images (Figure 4e) indicate the
The exfoliation of MoS2 was carried with both thiolated layered nature and zoomed inset images clearly showed
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and non-thiolated ligands with same head group, but in- the hexagonally symmetric structure of 2H-MoS2.30 Also,
52 terestingly we found that between thiolated and non- large area HRTEM (Supporting information, Figure S11)
53 thiolated ligands, there is no significant difference in the could not reveal any evidence of structural distortion in
54 rate of exfoliation, as shown in Figure 2b. But we have not- the basal plane by this ligand induced exfoliation, when
55 ed that the exfoliation of MoS2 in the presence of thiol lig- compared to lithium intercalated exfoliation.61,62 The small
56 ands results in high colloidal stability for an extended angle X-ray diffraction pattern (Figure 4f) indicates that
57 period of time (more than eight months) compared to the single crystalline nature is not affected during sonication.
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21 Figure 4. Characterization of 2H Phase of MoS2 and morphological characteristic of ligand 3 functionalized MoS2. a) AFM
22 image with inset height profile diagram indicates the formation of a single layer with functionalization. b) Comparative UV-Visible
23 spectra of 1T-MoS2 and 2H-MoS2 indicate the presence of direct excitonic transitions in 2H phase. Inset picture presents the visual
24 appearance of two different materials at same concentration (1 mg/ml). c) Raman spectra of exfoliated MoS2 by different thiol lig-
ands indicates the absence of 1T phase. d-e) TEM, HRTEM and small angle X-ray diffraction pattern of functionalized MoS2. Inset
25 zoomed image indicates the hexagonal phase of 2H MoS2.
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27 Further, the exfoliated MoS2 with different thiol ligand ent concentration of functionalized 2H-MoS2. The detailed
28 shows photoluminescence peaks at 670 nm (Supporting procedure, growth curves are mentioned in supporting
29 Information, Figure S12), which confirms the 2H phase of information (Figure S17). Among the all functionalized 2H-
MoS2. The exfoliated MoS2 by various ligands, morpholo- MoS2, only ligand 3 functionalized positively charged 2H-
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gies are also studied (Supporting Information, Figure S13- MoS2 shows antibacterial activity against both bacterial
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S14). Also, to test the versatility of our method we utilized strains. But unconjugated 2H-MoS2, neutral (ligand 1 con-
32 ligand 4 to exfoliate other TMDs (MoSe2, WS2, and WSe2). jugated) and negative (ligand 2 conjugated) 2H-MoS2 do
33 It is found that this method is versatile for other TMDs, not shown any inhibitory action in our concentration range
34 which is confirmed by AFM and TEM analysis (Supporting (Table 1). This result is consistent with our previous re-
35 Information, Figure S15-S16). The concentration of exfoli- port of 1T functionalized MoS2. However, positively
36 ated MoS2 with different ligands was estimated by ICP-MS. charged 2H-MoS2 shows antibacterial activity against both
37 Thus, by using thiol surfactant, we efficiently exfoliated MRSA and P. aeruginosa with lower inhibitory concentra-
38 2H-MoS2 within a short period of time (~ 1 hour) with di- tion. Notably, positively charged 1T-MoS2 did not show
39 rect functionalization. effective antibacterial activity against P. aeruginosa, but
40 Antibacterial Activity of functionalized 2H-MoS2. It is positively charged 2H-MoS2 exhibits relatively higher anti-
41 evident that both 1T63 and 2H phase15 exhibit potential bacterial activity against both the strains of bacteria.
42 antibacterial activity and can be enhanced by many folds In our earlier study, we have also shown that by increas-
43 through surface functionalization (~1000 times for 1T- ing the hydrophobicity of positively charged MoS2, we can
44 MoS2).53 To explore the potential of the material-core apart improve the antibacterial activity by increased depolariza-
45 from surface functionalization we have tested the antibac- tion of the bacterial membrane.53 Hence to further enhance
46 terial activity of the 2H functionalized materials. We have the antibacterial activity of 2H-MoS2, we have prepared
considered two different bacterial strains, Gram-positive functionalized MoS2 by cationic thiol ligand with increasing
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methicillin-resistant Staphylococcus aureus (MRSA) and hydrophobicity, ligand 7-MoS2 and ligand 8-MoS2 (Figure
48 Gram-negative Pseudomonas aeruginosa (P. aeruginosa) as 6a). Similar sonochemical method was used to prepare the
49 two representatives of ESKAPE pathogens (Enterococcus functionalized 2H-MoS2 using those ligands and confirmed
50 faecium, Staphylococcus aureus, Klebsiella pneumoniae, by zeta potential measurement (Supporting Information,
51 Acinetobacter baumannii, Pseudomonas aeruginosa, and Figure S18). As hypothesized, the ligand 7 and ligand 8
52 Enterobacter species). These pathogens are identified as conjugated 2H-MoS2 shows enhanced antibacterial activity
53 most threatening pathogens by the Infectious Disease So- than ligand 3 conjugated 2H-MoS2. Also, it was found that
54 ciety of America (IDSA) due to the rapid development of the MIC of MRSA and P. aeruginosa of 2H MoS2 conjugated
55 antibiotic resistance.64 The minimum inhibitory concentra- with ligand 3, ligand 7 and ligand 8 was 6.3 times, 16.5
56 tion (MIC) of all 2H functionalized MoS2 against bacteria
57 was determined using broth dilution method with differ-
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Table1. MIC and MBC of functionalized 2H-MoS2 and comparison with functionalized 1T-MoS2 against MRSA and P.
1 aeruginosa.
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3 2H-MoS2 MRSA (MIC) P. aeruginosa (MIC) MRSA (MBC) P. aeruginosa (MBC)
4 2H 1T* 2H 1T* 2H 1T* 2H 1T*
5 Control > 4.75 ppm > 15 ppm > 4.75 ppm > 15 ppm NA NA NA NA
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7 Ligand 1 NI NI NI NI NA NA NA NA
8 Ligand 2 NI NI NI NI NA NA NA NA
9 Ligand 3 297 ppb 1.88 ppm 594 ppb > 15 ppm 297 ppb 3.75 ppm 4.75 ppm NA
10 Ligand 7 9.45 ppb 156 ppb 9.45 ppb 156 ppb 18.75 ppb 156 ppb 18.75 ppb 156 ppb
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12 Ligand 8 9.45 ppb 78 ppb 9.45 ppb 78 ppb 9.45 ppb 78 ppb 9.45 ppb 78 ppb
13 * The value for 1T MoS2 is adapted from our previous report.53 In the table concentration is expressed based on Mo only. No In-
14 hibition-NI and Not Applicable-NA.
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39 Figure 5. Colony forming ability of MRSA and P. aeruginosa after treating with different positive MoS2 for MBC estimation.
40 The bacterial strains streaked on agar plate after 20hr incubation with positive MoS2. The functionalized MoS2 reduces the CFU of
41 bacterial strain, in vitro.
42 times, 8.25 times, lower than corresponding 1T-MoS2, re- 2H MoS2 against P. aeruginosa (10x concentration) and
43 spectively. The minimum bactericidal concentration (MBC) ligand 7 functionalized 2H MoS2 against MRSA (2X con-
44 is the complementary measurement to MIC. The MBC is the centration). It is known that the antibacterial agents are
45 concentration at which the viability of bacteria is reduced considered bactericidal, only if the MBC is less than four
46 to zero, which is generally estimated by quantifying colony times of the MIC. To this end, only ligand 3 functionalized
47 forming units in the agar plate. This infers that even at 2H MoS2 does not show bactericidal activity. Importantly,
48 MIC, the bacterial growth is inhibited but the microbial ligand 8 functionalized 2H MoS2 shows similar bacterio-
49 death may not happen. In that situation, plating the treated static and bactericidal effect against both gram-positive
50 solution onto agar might still result in bacterial growth by and gram-negative bacteria. Further, the growth curve was
51 colony formation. Hence, the MBC test will indicate the used to extract the growth kinetics and doubling time
52 bactericidal effect of our newly developed antibiotics. We (specific time interval between two subsequent binary
have found that functionalized 2H-MoS2 is equally effective fission), as reported by Rasool et al.65 Table S1-S2 clearly
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and notably better than 1T-MoS2, as shown in Table 1 and show that there is no growth at the MIC in both the bacte-
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figure 5. As we can see from figure 5, the colony forming rial strain. This indicates the complete inhibition of bacte-
55 unit (CFU) is zero at MBC concentration. We have also ob- rial function. The growth kinetics and doubling time are
56 served in most cases the MIC and MBC values are same found only at very low concentrations as in ligand 8-MoS2
57 except the antibacterial activity of ligand 3 functionalized (4.72 ppb), ligand 6-MoS2 (4.72 ppb), and ligand 3-MoS2
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Figure 6. Mechanistic study for understanding the antibacterial property of functionalized MoS2. a) Structural representa-
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tion positively charged MoS2 with varied hydrophobicity of ligand 3-MoS2, ligand 7-MoS2 and ligand 8-MoS2. b) Abiotic oxidative
27 stress estimation by Ellman’s assay with 0.4 mM glutathione, 10x MIC of functionalized MoS2 and 1 mM of 5,5′-dithio- bis (2- nitro-
28 benzoic acid). The positive control is 10 mM H2O2 and without MoS2 is considered as a negative control. All values are statistically
29 significant (P<0.0001) with respect to negative control. c) Estimation of the type of ROS species by Ellman’s assay in the presence
30 of different ROS scavengers, 0.4 mM of isopropanol, oxalate, sodium chromate, TEMPO and 10 µm of catalase. All the values with
31 respect to without quencher are statistically significant (P<0.0001). d) Intracellular ROS estimation with fluorescent probe DCFDA
32 (2mM) with MRSA with excitation wavelength 485nm and emission wavelength 527nm. Ligand 7 and Ligand 8-MoS2 statistically
33 significant (***, P<0.0001), ligand 3-MoS2, 2H-MoS2 are not significant (ns) with respect to control. e) Quantification of membrane
depolarization of MRSA. Pelleted MRSA stained with 50 µM DISC3(5) fluorescent probe than added 10x MIC of 2H-MoS2 and func-
34 tionalized MoS2. The fluorescence was monitored with an excitation wavelength of 622 nm and an emission wavelength of 670 nm.
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36 (148 ppb for MRSA and 296 ppb for P. aeruginosa). Even at ($0.4/g based on Sigma-Aldrich) compare to commonly
37 the above-mentioned low concentrations, functionalized used antibiotics such as Vancomycin ($150/g based on
38 MoS2 effectively reduces the growth rate and increases the Sigma-Aldrich). Overall, this study clearly indicates that
doubling time of both the strains, when compared to the the positively charged 2H-MoS2 is better antibacterial
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control. For ligand 8-MoS2 at 4.72 ppb treated MRSA, agent compare to 1T-MoS2. The enhanced activity most
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growth rate is 0.288 h-1 and doubling time is 2.4 h. But for likely, is due to the synergistic effect of 2H phase of the
41 control without MoS2 growth rate is 1.11 h-1 and doubling MoS2 and the functionalized ligands.
42 time is 0.6 h. Further, we have taken an effort to under- Mechanistic study of the high antibacterial activity. The
43 stand the effect of the free ligand on microbial toxicity. We high antibacterial activity of positively charged 2H-MoS2
44 have found that the free ligands (both with and without can be attributed by the two factors. The first one is that
45 thiol) with a positively charged head group can also show the core of the MoS2 has different phase i.e. semiconduct-
46 antibacterial activity. But the MIC value is very high com- ing 2H phase, which may cause enhanced oxidative stress.
47 pared to the functionalized MoS2, which are estimated by Another factor is the extent of ligand functionalization on
48 bacterial growth curve analysis (Supporting Information, the 2H-MoS2, which is responsible for the membrane dam-
49 Figure S19, and Table S3). This is an important finding age and hence results in enhanced activity. Liu et al. re-
50 since these ligands are widely used in gold and other na- ported that vertically aligned 2H-MoS2 generates a various
51 nomaterial functionalization and used as a potent antimi- type of ROS15, which are playing the crucial role in the
crobial agent against multidrug-resistant bacteria.46 More
52 antibacterial activity. Hence, initially abiotic oxidative
significantly, the functionalized 2H-MoS2 is found to be a stress, mediated by functionalized 2H-MoS2 was estimated
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more potent antibacterial agent with low dosage when by Ellman’s assay. From Figure 6b, it is clear that 2H-MoS2
54 compared to 1T-MoS2, all nanomaterial based antibacterial
55 results in more loss of glutathione (GSH) after 15 mins
agent and even small molecule based antibiotics (Support- (~78 %), when compared to 1T-MoS2 (~56 %).53 The
56 ing Information, Figure S20). Also in view of cost effective- smaller extent of ROS (may be peroxide) is plausibly gen-
57 ness, the starting material MoS2 is very inexpensive
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erated in case of 1T-MoS2 due to the electron transfer from cells, as the surface possess net negative charge due to the
1 the biological sample to the oxygen via the metallic plat- presence of negatively charged component, such as
2 form.66 The suitable conduction band of 2H-MoS2 (which is teichoic acid. Aggregation within the confined membrane
3 lower than the redox potential of selective ROS formation usually results in fluorescence self-quenching. Any materi-
4 reaction) helps to generate many ROS species.15 Hence, all al uptake, results in membrane depolarization causes re-
5 positively charged 2H-MoS2 are responsible for higher oxi- lease of dye, which can be easily estimated directly from
6 dative stress than that of similarly functionalized 1T- fluorescence enhancement study. Figure 6e indicates that
7 MoS2.53 Particularly after 15 mins, ligand 8 conjugated 2H- the positively charged 2H-MoS2 with higher
MoS2 shows ~40% loss of GSH, while corresponding 1T- hydrophobicity (ligand 8) shows greater membrane depo-
8
MoS2 showed very negligible loss of GSH. Over the period larization than intermediate (ligand 7) and least hydro-
9
of 3 hr, the loss of GSH increase in both the 2H and 1T- phobic (ligand 3) functionalized 2H-MoS2. Since high hy-
10 MoS2 in a similar trend, but notably for all 2H-MoS2 results drophobic MoS2 strongly adheres to lipid components of
11 in more GSH loss compared to 1T-MoS2, in vitro. Hence, microbial cell membranes, it is resulting in disruption of
12 this study clearly reveals that all positively charged 2H- the membrane. In contrast, only 2H-MoS2 did not show any
13 MoS2 generates more ROS than 1T-MoS2.53 effect on membrane depolarization. In comparison to our
14 Further to estimate the type of ROS species which are previous report, the extent of membrane depolarization is
15 contributing to oxidative stress, we have used the different very similar to the functionalized 1T-MoS2. Thus, we can
16 scavengers such as catalase (peroxide), isopropanol (hy- conclude from the above observation that the higher anti-
17 droxyl radical), oxalate (hole), sodium chromate (electron) bacterial activity of positively charged 2H-MoS2 than 1T-
18 and TEMPO (superoxide anion radical) in the Ellman’s as- MoS2 is mainly due to the elevated generation of various
19 say, as shown in Figure 6c. All the scavengers are effective ROS species. However, the extent of membrane depolariza-
20 in reducing GSH loss, particularly the catalase shows a high tion is similar, which is mainly attributed to externally
functionalized ligands, which are same in both types of
21 reduction of GSH loss. This clearly indicates that peroxide
is one of the dominant species compared to all other inves- MoS2 materials. Thus, apart from external
22
tigated ROS species. It is consistently found that all fabri- functionalization, the core of the material plays an im-
23 portant role in the bactericidal property.
24 cated 2H-MoS2 in the presence of different scavengers
causes a reduction in the loss of GSH. This indicates that Further to support the mechanism visually, the change of
25
ROS species are one of the important mode causing bacte- membrane morphologies is imaged by atomic force mi-
26
ricidal property of positively charged 2H-MoS2. We have croscopy (AFM) and scanning electron microscopy (SEM),
27 also estimated the type of ROS generated by 1T-MoS2 as shown in figure 7. Both AFM and SEM for untreated
28 (Supporting Information, Figure S21) for comparison. In- MRSA indicates clear spherical shape of bacteria with dis-
29 terestingly we have found that only catalase shows the tinct membrane integrity (Figure 7a, & 7e), whereas MRSA
30 reduction in loss of GSH. Hence, peroxide is the only one treated with ligand 3 functionalized MoS2 (Figure 7b & 7f)
31 type ROS species, which is generated in 1T-MoS2 due to the causes wrinkling with minimal damage due to masking of
32 electron transfer from biological components.66 Further, bacterial membrane surface (result in oxidative stress). In
33 the fluorescent probe 2′,7′–dichlorofluorescin diacetate contrast, ligand 7 functionalized 2H-MoS2 (Figure 7c, &
34 (DCFDA) is used to estimate the intracellular ROS genera- 7g) and ligand 8 functionalized 2H-MoS2 (Figure 7d, & 7h)
35 tion by functionalized 2H-MoS2. The dye (DCFDA) has cell causes severe damage to bacterial membrane and causes
36 penetrating ability and can generate fluorescence in the cell lysis. Transmission electron microscopy (TEM) images
37 presence of ROS species such as hydroxyl, peroxyl etc. in also supports the effect of functionalized 2H-MoS2 on bac-
the cell. The dye diffusion to the cell results in deacylation terial membrane integrity, which is composed of tri-layer,
38
of DCFDA followed by oxidation with ROS yields 2′,7′– plasma membrane followed by cell wall and the outer layer
39 dichlorofluorescein (DCF). DCF is a highly fluorescent capsule.68 It is evident from the figure 8a, that the untreat-
40 compound, which is used to estimate ROS by using fluores- ed P. aeruginosa as a rod-shaped morphology with capsule
41 cence spectrometer with excitation and emission wave- layer. Whereas ligand 3-MoS2 treated strains shows only
42 length of 485 nm and 527 nm, respectively. The intracellu- binding at the outer membrane. This induces a loss in cap-
43 lar ROS generation is found to be high (Figure 6d) in lig- sule layer, without any loss in membrane integrity due to
44 and 8-MoS2, ligand 7-MoS2 than ligand 3-MoS2. Whereas intact cell wall and plasma membranes (Figure 8b). But
45 only 2H-MoS2 doesn’t have any significant enhancement, ligand 8-MoS2 results in complete damage of membrane
46 when compared to the control (only dye). This may be at- (Figure 8c) and release of vital components from the cyto-
47 tributed to the evidence that the positively charged func- plasm (such as protein, ribosome, ATP, DNA etc.) leads to
48 tionalized 2H-MoS2 can effectively interact with bacterial the bacteriolysis. Similar effect noted in MRSA treated with
49 surface and leads to high intracellular ROS species. But 2H- positively charged 2H-MoS2 (Supporting information, S22).
50 MoS2 with negative charge is non-interactive and hence no Elemental mapping was carried out to locate the presence
intercellular ROS was generated. of MoS2 in treated sample. Figures 8e and 8f clearly show
51
To probe the effect of 2H-MoS2 with varied hydrophobi- the evidence for the presence of Mo and S on the P. aeru-
52 ginosa externally as well as internally. The merged ele-
53 city on the bacterial membrane, we have assessed the
membrane depolarization assay using 3,3´- mental mapping image and energy dispersive spectrosco-
54 py (EDS) analysis further support the above observation
dipropylthiadicarbocyanine iodide (DISC3(5)) fluorescent
55 (Figure S23-S24). Hence, one can conclude that functional-
probe.67 DISC3(5) is a lipophilic cationic dye, which accu-
56 mulates in the confined interior membrane of bacterial ized 2H-MoS2 binds to the bacteria selectively, while
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20 Figure 7. Morphological changes of MRSA after treatment with functionalized MoS2 studied by AFM and SEM images. a) &
21 e) Control only MRSA. b) & f) Ligand 3-MoS2 treated MRSA. c) & g) Ligand 7-MoS2 treated MRSA. d) & h) Ligand 8-MoS2 treated
MRSA. AFM and SEM images respectively. The analysis provides signature of membrane alteration upon treatment with functional-
22
ized MoS2.
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47 Figure 8. TEM based analysis with elemental mapping of P. aeruginosa, provides evidence for the interaction of function-
48 alized MoS2 with bacteria. a) Control only P. aeruginosa. b) Ligand 3-MoS2 treated P. aeruginosa. c) Ligand 8-MoS2 treated P.
49 aeruginosa. d) Ligand 3-MoS2 treated P. aeruginosa. e) elemental mapping of Molybdenum in P. aeruginosa treated with Ligand 3-
50 MoS2. f) elemental mapping of sulfur in P. aeruginosa treated with Ligand 3-MoS2.
51 inducing bacterial toxicity. The overall study concludes mainly due to modulation/upregulation in oxidative stress
52 that ligand 7 and ligand 8 functionalized MoS2 drive with different ROS species and membrane depolarization.
53 through the dual effect of membrane depolarization and Hemocompatibility of positively charged 2H-MoS2: To
54 oxidative stress. While ligand 3-MoS2 functionalized inhib- test the bio-compatibility of positively charged 2H-MoS2,
55 its bacterial function only by ROS. Hence, the mechanism of we performed hemolytic assay using rabbit erythrocytes.
56 the bactericidal property of 2H positively charged MoS2 is The in vitro hemolysis assay evaluates hemoglobin release
57 (spectrophotometrically measured at 570nm) in the plas-
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ma as an indicator of red blood lysis. In this protocol, iso- sible in vivo aptness and related applications which are
1 lated rabbit erythrocytes are incubated with functionalized currently in progress.
2 positively charged 2H-MoS2 for 1 hr. PBS was used as nega- Conclusion: In summary, we have demonstrated that ex-
3 tive control and 0.1% of triton-X as a positive control. After foliation and desired functionalization of 2H MoS2 could be
4 one-hour of incubation, intact erythrocytes are pelleted achieved efficiently within a short period of time using
5 and the supernatants containing hemoglobin released thiol surfactant in the sonochemical method. The effect of
6 from lysed erythrocytes estimated photometrically by a surfactant was illustrated using non-thiolated ligands,
7 plate reader at 570nm. Experimentally, 8x concentration of which only assisted in exfoliate but without functionaliza-
8 three functionalized 2H-MoS2 exhibits minimal lysis to tion. We have also demonstrated that this method can be
erythrocytes by the ligand 3-MoS2 (2%), ligand 7-MoS2 extended to other TMDs for further applications in many
9
(4%) and ligand 8-MoS2 (5%), as shown in Figure 9a. The fundamental studies. As an example, we have quantified
10 functionalized MoS2 with different concentration on RBC
11 the antibacterial activity of functionalized 2H-MoS2 and
shows absence of released hemoglobin (Figure 9b). This compared it with the 1T phase of the same material. We
12 study indicates excellent hemocompatibility of functional- have found that the positively charged 2H-MoS2 exhibits
13 ized 2H-MoS2. Upon of 24 hour incubation, only 8x MIC of higher antibacterial activity compared to the similarly
14 all three positively charged 2H-MoS2 are cytotoxic to rabbit functionalized 1T-MoS2. Additionally, increasing the hy-
15 erythrocytes (Supporting Information, Figure S25). drophobicity in the positive head group can enhance the
16 antibacterial activity by enhancing cell membrane depolar-
17 ization. The mechanistic study indicates that the ROS in-
18 duced oxidative stress is more in 2H-MoS2, when com-
19 pared to 1T-MoS2 due to the semiconducting nature of the
20 2H-phase. However, the extent of membrane depolariza-
21 tion remains same in both type of MoS2 since the external
22 functionalization remains same as in 1T-MoS2. Thus, we
23 have concluded that change in the core material also can
24 enhance the antibacterial activity along with surface modi-
fication in nano-antibiotics. This fundamental study on
25
alteration of core material, as well as surface functionality
26
with 2D scaffold, can offer an opportunity for developing
27 new materials towards many promising applications in
28 protein recognition, delivery, imaging, and therapy. Cur-
29 rent studies are underway to explore the possible use of
30 these materials for in vivo applications such as cutaneous
31 wound healing.
32 Experimental Section.
33
Inductively coupled plasma mass spectrometry
34 (ICPMS) for Mo concentration determination.
35
ICP-MS was analyzed using quadrupole Inductively Cou-
36
pled Plasma Mass Spectrometer. The unknown samples for
37 analysis were prepared by adding 100 µl of concentrated
38 HNO3 in a 15 ml vial containing 50 µl of functionalized and
39 Figure 9. Hemocompatibility of functionalized 2H-MoS2. a) incubated overnight at 1000C for digestion. The digested
40 Hemolysis assay of positively charged 2H-MoS2 and b) Func- samples were made up to 10mL by adding DI water. Am-
41 tionalized MoS2 incubated with RBC centrifuged and collected
monium heptamolybdate (NH4)6Mo7O24·4H2O was used to
supernatant to detect cell-free hemoglobin after 1hr incuba-
42 prepare known concentration of Mo as an external stand-
tion. For negative control, only PBS was used without MoS2
43 and 0.1% of TritonX-100 was used as a positive control.
ard. The external standards accuracy used for ICP-MS was
44 confirmed by the linear regression fit to ICP-MS values of
45 Also, we have tested the hemolytic activity of free ligands external standards (R2>99.99). Molybdenum concentra-
46 at a different time interval. The result indicates within tion of the stock solution was determined by multiplying
47 1hour time frame, significant amount of hemolysis noted the dilution factor consistent with the procedure described
with different free ligands (Supporting Information, Fig- above.
48
ureS26-S27). Hence, this study indicates that the grafting
49 of ligands on MoS2 considerably reduces the toxicity to-
Estimation of oxidative stress and types of reactive
50 active species (ROS) generated by functionalized MoS2.
wards rabbit erythrocytes, compared to free ligands. This
51 can be well attributed to the known property of nano- Oxidative stress induced by functionalized MoS2 was stud-
52 materials-based drugs to reduce non-specific systemic ied by Ellman’s assay. In procedure, 10xMIC of MRSA has
53 toxicity. This highlights the importance of functionalized been used, that is Positive ligand 3, 7 and 8 conjugated
54 nanomaterials towards biological applications. Also the MoS2 and 3ppm of 2H-MoS2 was taken in vial. Then added
0.4mM GSH which was dissolved in 50mM bicarbonate
55 enhanced antibacterial activity and in vitro biocompatibil-
buffer with pH 8.6. In the negative control, no MoS2 was
56 ity of the reported materials prompt us to explore the pos-
added and in the positive control, 10mM H2O2 was added.
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The tubes were wrapped with aluminum foils to prevent a certain time compared to zero min has been taken and
1 any photochemical oxidation and solutions were incubated plotted.
2 at 37˚C. From the stock at different time interval (15min, TEM sample preparation.
3 30min, 45min, 1hr, 2hr, and 3hr) 100μL solution added to
The bacteria harvested at mid log phase with OD ~ 0.5 and
4 100μL of 2mM 5,5′-dithio- bis (2- nitrobenzoic acid)
washed with PBS thrice and suspended in PBS buffer. Then
5 (DTNB, SRL Chem) in 50mM TRIS-HCl (pH 8.3, SRL Chem)
added functionalized MoS2 with concentration equivalent
6 buffer taken in a 96 well plate. Then the absorbance of the
to MIC and incubated in 370C shaking incubator. Then drop
7 resulting solution was measured immediately after 5
casted 3.5 ul of sample on blow discharged copper grid and
minutes at 412nm using a UV-visible spectrometer by
8 blotted immediately. Further the staining of the sample
plate reader.
9 was done by using uranyl acetate for 5sec.
10 The percentage loss of glutathione was calculated as:

  412
  ℎ        Characterization: UV-Vis-NIR Spectrophotometer (Shi-
11
1 −  × 100%

  412
 
  
  0
madzu UV-Vis-NIR Spectrophotometer), Zeta Potential
12 (Malvern Zetasizer Nano UK), XRD (Bruker X-ray diffrac-
13 For ROS estimation, the experimental procedure is exactly tometer D8 Advance), Fluorescence measurement (Ther-
14 similar to that of Ellman’s assay, but the only difference is mo Scientific Varioskan Flash Multimode Reader), Bacteri-
15 that the assay is carried in the presence of different al optical density (Eppendorf Bio Spectrometer UV-Vis
16 quenchers added to the vial containing glutathione and spectrometer), AFM (JPK instruments), Raman and photo-
17 10xMIC MoS2 such as hole quencher sodium oxalate (0.4 luminescence (LabRAM HR), TEM (200 kV, FETEM), SEM
18 mM), electron quencher sodium chromate (0.4 mM), su- (FEI Sirion XL30 FEG SEM).
19 peroxide anion radical quencher TEMPO (0.4 mM), perox-
20 ide quencher catalase (1µM) and hydroxyl radical quench- ASSOCIATED CONTENT
er isopropanol (0.4 mM). The reduction in the percentage Supporting Information. Synthetic procedure of non-thiol
21
loss of GSH in the presence of respective quencher which ligand, Ligand optimization, NMR of ligand conjugated MoS2,
22
implies the type of ROS species responsible for the AFM & SEM of TMDs, Photoluminescence, MIC, Zeta potential,
23 oxidative stress of 2H-MoS2. The concentration of all the Growth kinetics, doubling time, ROS quenching study, TEM of
24 quenchers mentioned above is the final concentrations. MRSA, Elemental mapping and EDS spectra, Hemolytic assay.
25 This material is available free of charge via the Internet at
Intracellular ROS Study with DCFDA probe.
26 http://pubs.acs.org.
27 The mid log phase culture (A600nm~0.5) of bacteria was
harvested and suspended in PBS buffer. Then 20µl of AUTHOR INFORMATION
28
100mM of the DCFDA dye in DMSO added to 1ml of bacte-
29 Corresponding Author
rial suspension. And added 100 ul of bacteria to 96 well
30 plate and added 1ppm of functionalized MoS2 and non- * E-mail: md@iisc.ac.in.
31 functionalized MoS2.The sample without MoS2 considered
32 Notes
as a control. The experiment performed are triplicate. The The authors declare no competing financial interest.
33 fluorescence was monitored with an excitation wavelength
34 of 485 nm and an emission wavelength of 527 nm, for a ACKNOWLEDGMENT
35 period of 3 hour with lag time of 15 minutes. Increase in
MD thanks DST-SERB (SB/FT/CS-139/2014) for financial
36 fluorescence indicates ROS generation in the presence of
support. BB thanks the Department of Science and Technology
37 materials. (DST, Government of India) and Council for Scientific and In-
38 Estimation of membrane depolarization affinity of dustrial Research (CSIR, Government of India) for their major
39 functionalized MoS2. financial support. We are grateful to the Department of Bio-
40 For quantification of membrane depolarization of bacteria technology (DBT, Government of India) for financial support
41 by this functionalized MoS2. The MRSA was harvested at to the “Centers of Excellence and Innovation in Biotechnology”
42 mid-log phase culture (A600nm~0.3) and centrifuged at the scheme through the center of excellence project: “Transla-
tional Center on Biomaterials for Orthopedic and Dental Ap-
43 5000 rpm for 5 min. The pellet was washed with 5 mM
plications”. We acknowledge Dr. Somnath Dutta for helping
44 glucose and 5 mM HEPES buffer (pH 7.2) mixed in 1:1 ra-
in bacterial TEM analysis. S.K. thanks DST-INSPIRE for doc-
45 tio and the washed pellet was re-suspended in 5 mM
toral fellowships.
46 HEPES buffer, 5 mM glucose, and 100 mM KCl solution
47 mixed in a 1:1:1 ratio. Then 100 µl of bacterial suspension ABBREVIATIONS
taken in 96 well plate and added 2 µl of 5 mM of the DISC3
48 ICPMS, inductively coupled plasma mass spectrometry; ROS,
dye (3,3′-dipropylthiadicarbocyanine iodide, TCI Chemi-
49 reactive active species; TEMPO,(2,2,6,6-Tetramethylpiperidin-
cals) and the plate was incubated for 30 min. Then added
50 10xMIC of Positive ligand 3, 7, and 8 conjugated MoS2 and
1-yl)oxyl; DISC3(5), 3,3´-dipropylthiadicarbocyanine iodide;
51 3 ppm 2H-MoS2 were added to the 96 well pate containing
MRSA, methicillin-resistant Staphylococcus aureus; MIC, min-
52 imum inhibitory concentration; MBC, minimum bactericidal
bacterial suspension and DISC3 dye. After addition of the concentration; TEM, transmission electron microscope; SEM,
53 MoS2, the fluorescence was monitored with an excitation scanning electron microscope; AFM; atomic force microscopy;
54 wavelength of 622 nm and an emission wavelength of 670 DCFDA, 2′,7′–dichlorofluorescin diacetate.
55 nm, for next 2 hour with a lag time of 10 minutes. As the
56 increase in fluorescence indicates membrane depolariza- REFERENCES
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