Functionalized Two-Dimensional MoS2 with Tunable Charges for

Dec 17, 2018 - Recent advances in the synthesis and functionalization of ... Glancing Back at a Successful 2018 and Looking Ahead to the New Year: Our...
4 downloads 0 Views 3MB Size
Download PDF
This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Article Cite This: ACS Omega 2018, 3, 17532−17539

http://pubs.acs.org/journal/acsodf

Functionalized Two-Dimensional MoS2 with Tunable Charges for Selective Enzyme Inhibition Subbaraj Karunakaran, Subhendu Pandit,† and Mrinmoy De* Department of Organic Chemistry, Indian Institute of Science, Bangalore 560012, India

ACS Omega 2018.3:17532-17539. Downloaded from pubs.acs.org by 46.161.56.164 on 12/29/18. For personal use only.

S Supporting Information *

ABSTRACT: Recent advances in the synthesis and functionalization of two-dimensional (2D) nanomaterials have allowed us to explore their interaction with biological systems. 2D nanomaterials, owing to their unique layered structure and a high surface-to-volume ratio, are very promising systems for various biological applications. Transition metal dichalcogenides (TMDs) are a unique class of 2D nanomaterials known for their easy surface functionalization using thiol ligands. Interactions of functionalized MoS2, a TMD, with different proteins are worth exploring as that might give us an overall insight about the interaction of these materials with biomacromolecules. Here, we have chemically exfoliated MoS2 and functionalized it with surface groups having different charges (negative, neutral, positive, and zwitterionic). Interaction of these functionalized MoS2 with two model proteins, chymotrypsin (ChT) and β-galactosidase (β-gal), were explored. Positively charged MoS2 inhibited the enzymatic activity of βgal, whereas the activity of ChT was inhibited by negatively charged MoS2. Polyethylene glycol-functionalized neutral MoS2 did not affect any of the proteins, but the zwitterionic ligand-functionalized MoS2 inhibits ChT and causes hyperactivity in β-gal. The nature of the inhibition was found to be noncompetitive. We also studied the changes in the secondary structure of proteins upon inhibition to assess their biocompatibility. In brief, we have explored interactions between differently functionalized MoS2 nanomaterials and two model proteins, ChT and β-gal, as a proof of concept study toward the future development of 2D material-based enzyme inhibitors and for their other biological applications.

■

INTRODUCTION Nowadays, two-dimensional (2D) materials are gaining considerable attention in molecular interaction because of their inherent properties, which show enhancement in a wide range of applications compared to their other dimensional counterpart nanomaterials.1−5 Protein surface recognition using an artificial receptor always plays a crucial role in many biological applications for controlling intracellular and extracellular processes such as transcription regulation, enzymatic inhibition, delivery, sensing, and so forth.6−8 The advantage and success of using nanomaterials as artificial receptors mainly lie on the tunable size, structure, and functionality according to the target molecules. As a 2Dlayered material, pristine and modified graphene oxides (GOs) were explicitly used as biomolecular receptors.9,10 In this regard, several kinds of biomolecules are targeted, such as enzymes, peptides, DNA and RNA, cells, and so forth.11−17 Similar to GO, transition metal dichalcogenides (TMDs), especially MoS2, can be obtained in large quantities of singlelayer sheets by chemical exfoliation methods, often using lithium intercalation.18 Postmodification of the surface functionality can also be done in a very convenient way using thiol chemistry.19−25 Compared to GO, the ability to functionalize MoS2 sheets through facile thiol chemistry is © 2018 American Chemical Society

more suitable for easy surface modification according to the target molecules and for making it a potential candidate for biomolecular targets.26−28 Surface functionalization holds a key role in biomolecular recognition and other applications of nanomaterials, and the same implies for 2D nanomaterials.29,30 For targeting other biomolecules such as negatively charged active center like β-galactosidase (β-gal), surface modification is needed. Hence, the surface-modified GO can specifically inhibit β-gal rather than ChT.31 However, the surface modification of GO is a challenging job and creates a hurdle for altering GO as a biomolecular interaction material. In contrast, TMDs can be surface modified very conveniently, and here we want to explore the suitability of the tunable chargefunctionalized 2D MoS2 as an artificial receptor and control the enzymatic activity. To investigate, we have considered four different functionalized 2D MoS2 solutions based on the surface charges (Figure 1a). Other than positive- and negativefunctionalized materials, we have considered two different neutral ligand-functionalized MoS2; polyethylene glycol and a zwitterionic ligand. For the target enzyme, we have considered Received: September 30, 2018 Accepted: November 28, 2018 Published: December 17, 2018 17532

DOI: 10.1021/acsomega.8b02598 ACS Omega 2018, 3, 17532−17539

ACS Omega

Article

water addition, which generates hydrogen gas and mixed-phase 2D MoS2 layers. During this violent reaction, the resultant MoS2 layers are hydrophilic in nature with many lattice defects originated from Mo and thiol vacancies. This leads to two important properties. First, the chemically exfoliated MoS2 (ceMoS2) layers are negatively charged, and the surface is reactive toward electron pair-donating ligands such as thiol. We characterized the exfoliated MoS2 by using atomic force microscopy (AFM) (Figure 2a), which indicates the layer

Figure 1. (a) Structure of different ligands utilized for functionalization of chemically exfoliated 2D MoS2 with tunable charges. (b) Structure of ChT and β-gal highlighted with active site(s) (red circle), negatively charged residues (blue circle), and positively charged residues (green circle).

Figure 2. (a) AFM image of ce-MoS2; the inset height profile diagram indicates the single layer of exfoliated MoS2. (b) SEM image of ceMoS2 confirms the layered structure with a flexible foldable backbone. (c) Zeta potential of exfoliated and various functionalized MoS2 in phosphate buffer solution.

two model proteins, which can be inhibited through chargebased complexation. We have considered β-gal for positively charged nanomaterials and ChT for negatively charged nanomaterials (Figure 1b) in the same way as they were used for GO-based 2D materials. Apart from their wellestablished structure and activity, both proteins have also played a crucial role in many biomedical applications. For instance, inhibition of β-gal, which is used to estimate the activity of β-galactocerebrosidase, is clinically used to diagnose Krabbe disease.32 Other than that, developing inhibitors is a common strategy to combat β-lactamase, which induces bacterial resistance.33 β-Lactamase is a class of enzyme produced by drug-resistant bacteria to destroy beta-lactam antibiotics.9 The deficiency of proteolytic enzyme (e.g., ChT) inhibitors causes a wide range of diseases such as emphysema, diabetes, Alzheimer’s disease, and so forth.34−36 On the basis of these aspects, our developed, functionalized MoS2 as an enzyme inhibitor could be useful for developing highly efficient artificial enzyme inhibitors.

morphology and a thickness of ∼1.2 nm, which specifies the single-layer formation as shown in the height profile diagram (Figure 2a, inset). Further, the size distribution analysis (Supporting Information, Figure S3) indicates that MoS2 with a lateral diameter of 300−500 nm is predominant. Scanning electron microscopy (SEM) (Figure 2b) also indicates a layered morphology of exfoliated MoS2 with a very high surface-to-volume ratio. There are already few studies that are reported based on surface functionalization of ce-MoS2 using thiol chemistry, after the first experimental report by us.19 Here, we have utilized a similar strategy of functionalization and with various thiol ligands (synthesis procedure, Supporting Information, Figures S1 and S2) for modifying the surface charge of exfoliated 2D MoS2 (Figure 1a). According to this method, the exfoliated MoS2 generally results in a sulfur vacancy. So, the thiol ligands are used to repair the vacant position of exfoliated MoS2 and to enhance the stability and specificity of MoS2. The functionalized MoS2 are characterized by AFM (Supporting Information, Figure S4), which indicates a height increment (∼2 nm) on the MoS2 surface after ligand conjugation. Further, the zeta potential measurement shows a distinct charge difference from the ce-MoS2 (Figure 2c). From here onward, the positive, negative, neutral, and zwitterionic ligand conjugated MoS2 will be referred to as positive MoS2, negative MoS2, neutral MoS2, and zwitterionic MoS2, respectively. From Figure 2c, we can observe that the negative MoS2 is more negatively charged compared to exfoliated MoS2, and the stability was enhanced extensively over the period and

■

RESULTS AND DISCUSSION Exfoliation and Functionalization of MoS2. To develop the 2D MoS2-based materials for biomolecular recognition and biological application, we considered both nonfunctionalized and functionalized exfoliated 2D MoS2 layers. We chemically exfoliated (ce) bulk MoS2 to single-layer MoS2 (ce-MoS2) with a scalable quantity by using n-butyllithium, according to the reported method with some modifications. In this method, Mo4+ is reduced to Mo3+ by lithium intercalation followed by 17533

DOI: 10.1021/acsomega.8b02598 ACS Omega 2018, 3, 17532−17539

ACS Omega

Article

Figure 3. (a,b) Enzymatic activity of ChT as a function of inhibitor concentrations in 5 mM sodium phosphate buffer (pH 7.4) using SPNA as a substrate. The activities were normalized to that of only ChT. (c,d) Enzymatic activity of β-gal as a function of the same inhibitor concentrations in 5 mM sodium phosphate buffer (pH 7.4) using ONPG as a substrate. The activities were normalized to that of only β-gal.

protein. Further, the protein recognition using the zwitterionic ligand can hold a crucial role because zwitterionic ligands are well-known for their high stability and nontoxicity and exhibit antibacterial, antifouling, and antibiofilm activities.40−44 However, the protein interaction with zwitterionic materials is very rarely reported.45,46 So, we have prepared zwitterionic MoS2 and studied the effect on the enzymatic activity. We found that only ChT is inhibited selectively, whereas β-gal exhibits hyperactivity (∼180%) (Figure 4) in the presence of

in different buffer solutions. This clearly indicates the effective functionalization. A similar outcome was also observed in the case of positive MoS2, which exhibits the overall positive charge of +19 mV. As expected, the neutral MoS2 shows ∼0 mV, but the zwitterionic MoS2 exhibits a slender negative charge of −26 mV, most likely due to the presence of the outer sulfate group. Also, thermogravimetric analysis was used to estimate the ligand grafting on MoS2, which is found to be 55% for neutral ligand-conjugated MoS2 (Supporting Information, Figure S5). Functionalized MoS2 as a Protein Surface Recognition Material. To test the efficacy and suitability of the functionalized material for biomolecular recognition, we have chosen two model proteins, ChT and β-gal. The structure and activity of ChT (pI = 8.752) are well-studied, and it is wellknown that it can be inhibited by negatively charged macromolecules.37 Alternatively, β-gal with pI = 4.61 can be complexed with positively charged nanomaterials (Figure 1b), and the activity will be diminished.38 To estimate the extent of complexation by functionalized MoS2, enzymatic activity assays were carried out. The substrate used was N-succinyl-L-alaninep-nitroanilide (SPNA) for ChT assay and o-nitrophenyl-β-Dgalactopyranoside (ONPG) for β-gal. SPNA and ONPG are chromogenic substrates, and the rate of hydrolysis of these substrates were used to estimate the enzymatic activity. The experiments were carried out by incubating proteins (ChT 2.5 μM and β-gal 0.25 nM) with functionalized MoS2 and ce-MoS2 with varying concentrations from 0 to 25 μg/mL (based on Mo, determined by inductively coupled plasma mass spectrometry) of the inhibitor with the corresponding substrate. Out of all functionalized MoS2, as hypothesized, the negative MoS2 inhibited the ChT activity up to 90% by 3 μg/mL (6.4 μg/mL of the functionalized materials) of Mo concentration and positive MoS2 does not have any effect on the protein activity (Figure 3a). Negative MoS2 is ∼2 times more effective compared to GO, which is known as the best artificial inhibitor for ChT. Interestingly, even though ce-MoS2 is negatively charged (−39.1 mV), it does not have a great impact on the ChT activity at 3 μg/mL concentration. It can only alter the activity of ChT at higher concentration >25 μg/ mL.39 Similarly, the positive MoS2 inhibits the activity of β-gal around 90% with 4 μg/mL of Mo concentration, and negative MoS2 does not affect the enzymatic activity (Figure 3b) even at a very high concentration. In both cases, the neutral MoS2 does not have any significant effect on the activity of either of the

Figure 4. Enzymatic activity of ChT and β-gal in the presence of zwitterionic MoS2 as a function of the Mo concentration in 5 mM sodium phosphate buffer (pH 7.4) using SPNA and ONPG as the substrate, respectively. The activities were normalized to that of only ChT and β-gal.

zwitterionic MoS2. This behavior can be attributed to the ligand, which has a sulfonate group externally that is responsible for inhibition of ChT and hyperactivity of β-gal. These observations unambiguously suggest that only functionalized MoS2 can be used to efficiently recognize the proteins and other biomolecular surfaces for real-life applications. The comparative efficiency of functionalized MoS2 with the reported synthetic receptor for both ChT (3.2 μm) and β-gal (0.5 nM) are also done and plotted in Figure 5.10,31,37,38,47−52 Compared to other reported macromolecules and nanomaterials, the efficacy of functionalized MoS2 is close to the other 2D materials, that is GO. This again established the importance of 2D materials in biomolecular binding. Hence, the functionalization enables and enhances the ability to recognize two different charged proteins selectively. Further, not only the effectiveness can be tuned but also the stability of ce-MoS2 can be enhanced through functionalization. Nature of Interaction and Effect on the Protein Structure. On the basis of the earlier reports, we have 17534

DOI: 10.1021/acsomega.8b02598 ACS Omega 2018, 3, 17532−17539

ACS Omega

Article

Figure 5. (a) Comparison of efficiency of negative MoS2 vs the existing synthetic receptor for ChT inhibition and (b) positive MoS2 vs the existing synthetic receptor for β-gal inhibition.

Figure 6. (a) Effect of ionic strength on binding (preincubation) and reversal (postincubation) of ChT by negative MoS2. [ChT] = 2.5 μM in 5 mM PB, pH 7.4. (b) CD spectra of ChT (2.5 μM), denatured ChT, and with negatively charged MoS2 (3 μg/mL). (c) Fluorescence of ChT (2.5 μM), denatured ChT, and complexed with negative MoS2 in 5 mM PB, pH = 7.4. (d) Estimation of the percentage of denaturation at different time intervals by using fluorescence spectra.

electrostatic complexation, which is attenuated at the higher ionic strength of the solution, whereas in the case of postincubation, the restoration of activity by increasing the salt concentration clearly indicates that binding between negative MoS2 and ChT is reversible. The maximum recovery of 80% was found at 100 mM NaCl solution (Figure 6a). However, we fail to execute the salt study for β-gal because higher salt concentration itself affects the β-gal activity because of the salt bridge formation and leads to the aggregation of the protein54 (Supporting Information, Figure S6). Along with the reversible interaction, the secondary structure of proteins should be preserved by an efficient artificial receptor. To investigate the effect on the protein structure upon complexation, we used circular dichroism (CD) and fluorescence study. The CD spectra of native ChT show two characteristic peaks at 230 and 204 nm. Denaturation of ChT can be assessed by the loss of peak at 230 nm and a blue shift for the peak at 204 nm, as shown in the CD spectra of thermally denatured ChT. The complex of negative MoS2 and ChT shows a small deviation from native ChT, with increment

assumed that the inhibition is mainly because of the complementary electrostatic interaction, and hence it can be reversed by increasing the ionic strength of the medium that is traditionally used in the technique. Also, modulating the enzymatic activity by varying the ionic strength of the medium is biologically important because the salt concentrations in biological systems can vary from 5 mM (bile) to 250 mM (red blood cells).53 To test this hypothesis, we have done two experiments. In the first experiment, we have incubated 2.5 μM ChT with negative MoS2 for 30 min (postincubation) and then added various concentrations of NaCl solutions so that the final concentration of NaCl varies from 0 to 200 mM. In the second experiment, similar concentrations of negative MoS2 and ChT were added simultaneously in the presence of NaCl without the 30 min incubation (preincubation). These two studies will suggest the nature of interaction and possible reversibility. The activity of ChT was monitored with respect to the hydrolysis of the SPNA substrate. It was noted that in both the cases, the activity of ChT increases with increasing salt concentration. The preincubation study confirms the 17535

DOI: 10.1021/acsomega.8b02598 ACS Omega 2018, 3, 17532−17539

ACS Omega

Article

Figure 7. (a,b) Enzyme velocity of ChT (2.5 μM) (a) and β-gal (0.25 nM) (b) as a function of substrate concentration at various fixed concentrations of inhibitors. (c,d) Lineweaver−Burk plot for ChT and β-gal. Both plots exhibit a similar pattern, which indicates the noncompetitive inhibition.

ChT and positive MoS2 for β-gal (Figure 7). The data were fitted by nonlinear regression by using Graph-Pad Prism 5 on the basis of the most general equation for the velocity (V) of an enzymatic reaction in the presence of an inhibitor (I)55

in the intensity of 204 nm and a slight decrease in the peak intensity of 230 nm (Figure 6b), which indicates a small percentage of denaturing. The CD spectra does not give complete information regarding the extent of denaturation. To determine the denaturation quantitatively, we used fluorescence spectroscopy (Figure 6c). This can be estimated by measuring fluorescence (Figure 6d) of native ChT at 340 nm, considered as 0% denaturation, and thermally denatured ChT shows fluorescence at 360 nm with a red shift due to the exposure of tryptophan to aqueous environment considered as 100% denaturation. ChT incubated with negatively charged MoS2 shows a red shift from native ChT over time, and from there, we estimated the denaturation percentage by peak shift between 0 and 100%. The normalized curve in Figure 6d was obtained after subtracting the aging effect of ChT over 24 h. The analysis reveals that ∼17% of denaturation of the protein occurs after normalization, with only ChT denaturation over a period of 24 h (Figure 6d). Even though a small deviation of the protein structure is observed, that effect is reversible in nature. When we incubated the complex in 100 mM salt solution, it regains its native conformation. However, the effect of the inhibitor on these secondary structures of β-gal was unable to be determined by both CD and fluorescence spectroscopies because for CD and fluorescence, the minimum concentration of β-gal is 25 nM (Supporting Information, Figures S7 and S8), which is a hundred times higher than being used for inhibition study (0.25 nm). For fluorescence study, when we used 25 nM β-gal with 10 μg/mL positive MoS2, the inhibitor itself quenches the fluorescence of β-gal (Supporting Information, Figure S7). In the case of CD, 10 μg/mL positive MoS2 causes a very high voltage (700 V), which crosses more than the instrumental limit. Mode of Inhibition. Understanding the mode of inhibition is also important to determine the mechanism of synthetic receptor binding with the protein. Hence, we have determined the enzyme velocity as a function of the substrate (S) concentration at different concentrations of negative MoS2 for

V=

Vmax[S]

(

[S] 1 +

[I] αK i

) + K (1 + ) m

[I] Ki

The above equation was used to calculate the α value and the inhibition constant (ki). α value can be described as competitive, noncompetitive, or uncompetitive depending on its values.55 Specifically, an α value close to unity indicates that the inhibitor does not bind to the active site but nearby the active site and prevents the substrate binding, which is noncompetitive. When α ≪ 1, inhibitor binding increases the enzymatic substrate binding, which represents uncompetitive inhibition. When α ≫ 1, binding of the inhibitor was prevented by binding of the substrate, which is competitive inhibition. For ChT with negative MoS2, we found that the value of α is close to unity (α = 1.67). Lineweaver−Burk plot shows that the y-intercept at 0 is not the same, but in x-axis, the starting point is the same (Figure 7a,b). These two observations indicate mostly noncompetitive inhibition for ChT by negative MoS2. Similarly, for β-gal, α = 1.2, and Lineweaver−Burk plot shows a similar pattern (Figure 7c,d), which again indicates the noncompetitive type of inhibition. Both the proteins with functionalized MoS2 exhibit noncompetitive inhibition, which could be due to the nonspecific binding of negative MoS2 to positively charged residues on ChT and alternatively for β-gal (Figure 1b). From the above analysis, we observed the inhibition constants, ki, for ChT and β-gal to be 0.312 and 1.382 μg/mL, respectively. Most interestingly, a similar class of material, that is, GO, exhibits competitive inhibition in the case of ChT. 17536

DOI: 10.1021/acsomega.8b02598 ACS Omega 2018, 3, 17532−17539

ACS Omega

■

Article

CONCLUSIONS In summary, we have explored functionalized 2D MoS2 as a synthetic 2D nanomaterial for protein surface recognition and assessed the suitability of these materials for biological applications. We have found that functionalized MoS2 can efficiently be used for protein surface recognition. Negative MoS2 recognizes the positively charged protein, ChT, and positive MoS2 inhibits the enzymatic activity of the negatively charged protein, β-gal. Neutral MoS2 does not interact with any proteins, but zwitterionic MoS2, albeit overall neutral in nature, inhibits ChT and enhances the activity in β-gal. Nonfunctionalized ce-MoS2 is not suitable for biological applications as it does not effectively interact with the protein at a relevant concentration as well as not stable in biological media. The mode of inhibition reveals that both proteins are inhibited in a noncompetitive manner. We also observed during the protein−receptor complexation that there is no significant change in the secondary structure of proteins, which indicates the biocompatible nature of the receptors. This study demonstrated the possibility and potency of functionalized 2D materials in future biomedical applications.

with the corresponding substrate was carried out. Enzyme activity was monitored over a period of 3 h by estimating the production of p-nitroaniline for SPNA and o-nitroaniline for ONPG formation at 405 nm with a microplate reader. The assays were performed in triplicate and in sodium phosphate buffer (5 mM, pH 7.4) at 25 °C. For studying the salt effect, ChT (2.5 μM) was incubated with sodium chloride (NaCl) before the addition of 3 μg/mL negative MoS2 (preincubation) or after the 30 min incubation with 3 μg/mL negative MoS2 (postincubation). The final NaCl solution concentration was varied from 0 to 200 mM in 5 mM sodium phosphate buffer (pH 7.4). Finally, SPNA (0.5 mM) was added, and the activity was monitored by measuring the formation of p-nitroaniline at 405 nm. CD. Far-ultraviolet CD spectra were measured by using a quartz cuvette of 1 mm path length from 190 to 250 nm at 25 °C. The concentration of ChT was maintained at 2.5 μM for all measurements. CD spectra was recorded for only ChT, thermally denatured ChT, and ChT incubated with 3 μg/mL negative MoS2 at different time intervals. The CD spectra of negative MoS2 and buffer were subtracted from the complex spectra to eliminate any background effects. Fluorescence Spectroscopy. The fluorescence spectra were measured for ChT incubated with 3 μg/mL negative MoS2, only native ChT, and thermally denatured ChT with excitation at 295 nm, and the emission spectra were recorded from 300 to 450 nm. The emission maxima for native ChT is 334 nm, and by complete thermal denaturation, the maxima shifted to 352 nm. The extent of denaturation of ChT incubated with negative MoS2 is reflected by the different values of the emission maxima shift. According to our calculation, we have considered 0% denaturation at 334 nm and 100% denaturation at 352 nm. The percentage shift in the emission maxima of the ChT−negative MoS2 complex indicates the extent of denaturation. As a control, only native ChT was measured for estimating the aging effect and subtracted from the corresponding measurement. Enzyme Kinetics. For the enzyme kinetic studies for both proteins, the rate of hydrolysis of the corresponding substrate at varying concentrations was measured at a fixed concentration of negative MoS2 for ChT (2.5 μM) and positive MoS2 for β-gal (0.25 nM). Enzymatic velocity for each functionalized MoS2/substrate combination was obtained by linear fittings of nitroaniline (hydrolysis byproduct) production over time interval of 30 min. The initial velocity was determined by the linear fitting obtained by enzyme hydrolysis without any inhibitor. All fittings were performed using GraphPad Prism 5 by using the mixed-model inhibition equation, which is a general velocity equation that includes competitive, uncompetitive, and noncompetitive inhibition as special cases. Lineweaver−Burk plot was used to understand the mode of inhibition, which was estimated by plotting the reciprocals of enzymatic velocity and substrate concentrations.

■

EXPERIMENTAL SECTION Characterization. All characterization and studies were done by using the following instruments: JPK instruments (AFM), FEI Sirion XL30 FEG scanning electron microscope (SEM), Malvern Zetasizer Nano UK (zeta potential), Thermo Scientific Varioskan Flash Multimode Reader (ultraviolet− visible measurement), CD spectrometer (JASCO, J-815), and fluorescence spectrophotometer (Varian Cary Eclipse). Chemical Exfoliation of MoS2. The exfoliation of MoS2 was done according to the literature procedure.56 Briefly, under a nitrogen atmosphere, 400 mg of bulk MoS2 was taken in a glass vial and placed inside the glove box. n-Butyllithium solution (5 mL, 1.6 M) in hexane was added and stirred for 48 h. The intercalated bulk MoS2 with lithium was removed from the glovebox, filtered (Whatman #1), and washed with hexane to remove excess n-butyl lithium. The lithium-intercalated MoS2 powder was then suspended in ice-cold water and sonicated for 30 min. The obtained solutions were centrifuged at 10 000 rpm three times, and the precipitate was collected. The precipitate was redispersed in water and centrifuged at 2000 rpm. The obtained supernatant solution was collected and directly utilized for further functionalization. Functionalization of MoS2. The positive, negative, and zwitterionic thiol ligands (15 mg) were dissolved in 8 mL of water. To this solution, 2 mL of ce-MoS2 (∼2 mg/mL) was added and stirred for 1 day. To remove the excess ligand, the functionalized solution was subjected to dialysis for 1 day using a snakeskin dialysis membrane with 10 000 molecular weight cutoff (Thermo Scientific). A similar method was followed for the neutral ligand, but the ligand was dissolved in 1:1 water− ethanol system. Activity Assays. The activity assay was performed by incubating ChT (2.5 μM) and β-gal (0.25 nM) with various concentrations of functionalized MoS2. The enzymatic hydrolysis reaction was estimated by adding SPNA for ChT (0.5 mM) (dissolved in 9:1 ethanol−dimethylsulfoxide) and ONPG (dissolved in the buffer) for β-gal (0.5 mM) as the chromogenic substrate. The activity of each combination was measured after 30 min of incubation in a 96-well plate. For the control study, the enzymatic activity of only ChT and β-gal

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02598. Synthesis and characterization of ligands, size distribution plot of ce-MoS2, AFM of functionalized MoS2, and related CD, fluorescence, and salt study of β-gal (PDF) 17537

DOI: 10.1021/acsomega.8b02598 ACS Omega 2018, 3, 17532−17539

ACS Omega

■

Article

Interactions Based on Graphene−Peptide Complex. Anal. Chem. 2011, 83, 7276−7282. (15) Ryoo, S.-R.; Lee, J.; Yeo, J.; Na, H.-K.; Kim, Y.-K.; Jang, H.; Lee, J. H.; Han, S. W.; Lee, Y.; Kim, V. N.; Min, D.-H. Quantitative and Multiplexed MicroRNA Sensing in Living Cells Based on Peptide Nucleic Acid and Nano Graphene Oxide (PANGO). ACS Nano 2013, 7, 5882−5891. (16) Wang, Z.; Huang, P.; Bhirde, A.; Jin, A.; Ma, Y.; Niu, G.; Neamati, N.; Chen, X. A nanoscale graphene oxide−peptide biosensor for real-time specific biomarker detection on the cell surface. Chem. Commun. 2012, 48, 9768. (17) Ou, L.; Luo, Y.; Wei, G. Atomic-Level Study of Adsorption, Conformational Change, and Dimerization of an α-Helical Peptide at Graphene Surface. J. Phys. Chem. B 2011, 115, 9813−9822. (18) Nicolosi, V.; Chhowalla, M.; Kanatzidis, M. G.; Strano, M. S.; Coleman, J. N. Liquid Exfoliation of Layered Materials. Science 2013, 340, 1226419. (19) Chou, S. S.; De, M.; Kim, J.; Byun, S.; Dykstra, C.; Yu, J.; Huang, J.; Dravid, V. P. Ligand Conjugation of Chemically Exfoliated MoS2. J. Am. Chem. Soc. 2013, 135, 4584−4587. (20) Jeong, M.; Kim, S.; Ju, S.-Y. Preparation and characterization of a covalent edge-functionalized lipoic acid−MoS2 conjugate. RSC Adv. 2016, 6, 36248−36255. (21) Presolski, S.; Pumera, M. Covalent functionalization of MoS2. Mater. Today 2016, 19, 140−145. (22) Cheng, Z.; He, B.; Zhou, L. A general one-step approach for in situ decoration of MoS2 nanosheets with inorganic nanoparticles. J. Mater. Chem. A 2015, 3, 1042−1048. (23) Wang, X.; Kalali, E. N.; Wang, D.-Y. An in situ polymerization approach for functionalized MoS2/nylon-6 nanocomposites with enhanced mechanical properties and thermal stability. J. Mater. Chem. A 2015, 3, 24112−24120. (24) Zhou, L.; He, B.; Yang, Y.; He, Y. Facile approach to surface functionalized MoS2nanosheets. RSC Adv. 2014, 4, 32570. (25) Presolski, S.; Wang, L.; Loo, A. H.; Ambrosi, A.; Lazar, P.; Ranc, V.; Otyepka, M.; Zboril, R.; Tomanec, O.; Ugolotti, J.; Sofer, Z.; Pumera, M. Functional Nanosheet Synthons by Covalent Modification of Transition-Metal Dichalcogenides. Chem. Mater. 2017, 29, 2066−2073. (26) Yin, W.; Yu, J.; Lv, F.; Yan, L.; Zheng, L. R.; Gu, Z.; Zhao, Y. Functionalized Nano-MoS2 with Peroxidase Catalytic and NearInfrared Photothermal Activities for Safe and Synergetic Wound Antibacterial Applications. ACS Nano 2016, 10, 11000−11011. (27) Li, Z.; Wong, S. L. Functionalization of 2D transition metal dichalcogenides for biomedical applications. Mater. Sci. Eng., C 2017, 70, 1095−1106. (28) Liu, T.; Wang, C.; Gu, X.; Gong, H.; Cheng, L.; Shi, X.; Feng, L.; Sun, B.; Liu, Z. Drug Delivery with PEGylated MoS2Nano-sheets for Combined Photothermal and Chemotherapy of Cancer. Adv. Mater. 2014, 26, 3433−3440. (29) Sim, D. M.; Han, H. J.; Yim, S.; Choi, M.-J.; Jeon, J.; Jung, Y. S. Long-Term Stable 2H-MoS2 Dispersion: Critical Role of Solvent for Simultaneous Phase Restoration and Surface Functionalization of Liquid-Exfoliated MoS2. ACS Omega 2017, 2, 4678−4687. (30) Yin, W.; Yan, L.; Yu, J.; Tian, G.; Zhou, L.; Zheng, X.; Zhang, X.; Yong, Y.; Li, J.; Gu, Z.; Zhao, Y. High-Throughput Synthesis of Single-Layer MoS2 Nanosheets as a Near-Infrared PhotothermalTriggered Drug Delivery for Effective Cancer Therapy. ACS Nano 2014, 8, 6922−6933. (31) Li, J.; Wu, L.-J.; Guo, S.-S.; Fu, H.-E.; Chen, G.-N.; Yang, H.-H. Simple colorimetric bacterial detection and high-throughput drug screening based on a graphene−enzyme complex. Nanoscale 2013, 5, 619−623. (32) Holleran, W. M.; Ginns, E. I.; Menon, G. K.; Grundmann, J. U.; Fartasch, M.; McKinney, C. E.; Elias, P. M.; Sidransky, E. Consequences of beta-glucocerebrosidase deficiency in epidermis. Ultrastructure and permeability barrier alterations in Gaucher disease. J. Clin. Invest. 1994, 93, 1756−1764.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (M.D.). ORCID

Subbaraj Karunakaran: 0000-0001-5093-490X Subhendu Pandit: 0000-0002-4542-2069 Mrinmoy De: 0000-0001-8394-9059 Present Address †

Department of Chemistry, University of Illinois at UrbanaChampaign, 509 W University Ave., Urbana, Illinois 61801, United States. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors would like to thank the DST-SERB (SB/FT/CS139/2014) for the financial support. S.K. thanks the DSTINSPIRE for the doctoral fellowships.

■

REFERENCES

(1) Maitra, U.; Gupta, U.; De, M.; Datta, R.; Govindaraj, A.; Rao, C. N. R. Highly Effective Visible-Light-Induced H2Generation by SingleLayer 1T-MoS2and a Nanocomposite of Few-Layer 2H-MoS2with Heavily Nitrogenated Graphene. Angew. Chem., Int. Ed. 2013, 52, 13057−13061. (2) Agarwal, V.; Chatterjee, K. Recent advances in the field of transition metal dichalcogenides for biomedical applications. Nanoscale 2018, 10, 16365−16397. (3) Pandit, S.; De, M. Interaction of Amino Acids and Graphene Oxide: Trends in Thermodynamic Properties. J. Phys. Chem. C 2017, 121, 600−608. (4) Bang, G. S.; Cho, S.; Son, N.; Shim, G. W.; Cho, B.-K.; Choi, S.Y. DNA-Assisted Exfoliation of Tungsten Dichalcogenides and Their Antibacterial Effect. ACS Appl. Mater. Interfaces 2016, 8, 1943−1950. (5) Girish, Y. R.; Biswas, R.; De, M. Mixed-Phase 2D-MoS2 as an Effective Photocatalyst for Selective Aerobic Oxidative Coupling of Amines under Visible-Light Irradiation. Chem.Eur. J. 2018, 24, 13871−13878. (6) Goda, T.; Miyahara, Y. Engineered zwitterionic phosphorylcholine monolayers for elucidating multivalent binding kinetics of Creactive protein. Acta Biomater. 2016, 40, 46−53. (7) Ivan, T.; Enkvist, E.; Viira, B.; Manoharan, G. b.; Raidaru, G.; Pflug, A.; Alam, K. A.; Zaccolo, M.; Engh, R. A.; Uri, A. Bifunctional Ligands for Inhibition of Tight-Binding Protein−Protein Interactions. Bioconjugate Chem. 2016, 27, 1900−1910. (8) Wang, T.; Zhu, R.; Zhuo, J.; Zhu, Z.; Shao, Y.; Li, M. Direct Detection of DNA below ppb Level Based on Thionin-Functionalized Layered MoS2 Electrochemical Sensors. Anal. Chem. 2014, 86, 12064−12069. (9) Huang, P.-J. J.; Pautler, R.; Shanmugaraj, J.; Labbé, G.; Liu, J. Inhibiting the VIM-2 Metallo-β-Lactamase by Graphene Oxide and Carbon Nanotubes. ACS Appl. Mater. Interfaces 2015, 7, 9898−9903. (10) De, M.; Chou, S. S.; Dravid, V. P. Graphene Oxide as an Enzyme Inhibitor: Modulation of Activity of α-Chymotrypsin. J. Am. Chem. Soc. 2011, 133, 17524−17527. (11) Zou, X.; Zhang, L.; Wang, Z.; Luo, Y. Mechanisms of the Antimicrobial Activities of Graphene Materials. J. Am. Chem. Soc. 2016, 138, 2064−2077. (12) Kenry, K.; Lim, Y. B.; Nai, M. H.; Cao, J.; Loh, K. P.; Lim, C. T. Graphene oxide inhibits malaria parasite invasion and delays parasitic growth in vitro. Nanoscale 2017, 9, 14065−14073. (13) Hu, W.; Peng, C.; Luo, W.; Lv, M.; Li, X.; Li, D.; Huang, Q.; Fan, C. Graphene-Based Antibacterial Paper. ACS Nano 2010, 4, 4317−4323. (14) Lu, C.-H.; Li, J.; Zhang, X.-L.; Zheng, A.-X.; Yang, H.-H.; Chen, X.; Chen, G.-N. General Approach for Monitoring Peptide−Protein 17538

DOI: 10.1021/acsomega.8b02598 ACS Omega 2018, 3, 17532−17539

ACS Omega

Article

(33) Ali, S. R.; Pandit, S.; De, M. 2D-MoS2-Based β-Lactamase Inhibitor for Combination Therapy against Drug-Resistant Bacteria. ACS Appl. Bio Mater. 2018, 1, 967−974. (34) Hutchison, D. C. S. Natural history of alpha-1-protease inhibitor deficiency. Am. J. Med. 1988, 84, 3−12. (35) Janoff, A. Elastases and Emphysema - Current Assessment of the Protease-Antiprotease Hypothesis. Am. Rev. Respir. Dis. 1985, 132, 417−433. (36) Selkoe, D. J. Alzheimer’s Disease: Genes, Proteins, and Therapy. Physiol. Rev. 2001, 81, 741−766. (37) Sandanaraj, B. S.; Vutukuri, D. R.; Simard, J. M.; Klaikherd, A.; Hong, R.; Rotello, V. M.; Thayumanavan, S. Noncovalent modification of chymotrypsin surface using an amphiphilic polymer scaffold: Implications in modulating protein function. J. Am. Chem. Soc. 2005, 127, 10693−10698. (38) Verma, A.; Simard, J. M.; Worrall, J. W. E.; Rotello, V. M. Tunable Reactivation of Nanoparticle-Inhibited β-Galactosidase by Glutathione at Intracellular Concentrations. J. Am. Chem. Soc. 2004, 126, 13987−13991. (39) Chou, S. S.; Kaehr, B.; Kim, J.; Foley, B. M.; De, M.; Hopkins, P. E.; Huang, J.; Brinker, C. J.; Dravid, V. P. Chemically Exfoliated MoS2as Near-Infrared Photothermal Agents. Angew. Chem., Int. Ed. 2013, 52, 4160−4164. (40) Sangsuwan, A.; Kawasaki, H.; Matsumura, Y.; Iwasaki, Y. Antimicrobial Silver Nanoclusters Bearing Biocompatible Phosphorylcholine-Based Zwitterionic Protection. Bioconjugate Chem. 2016, 27, 2527−2533. (41) Huo, S.; Jiang, Y.; Gupta, A.; Jiang, Z.; Landis, R. F.; Hou, S.; Liang, X.-J.; Rotello, V. M. Fully Zwitterionic Nanoparticle Antimicrobial Agents through Tuning of Core Size and Ligand Structure. ACS Nano 2016, 10, 8732−8737. (42) Venault, A.; Chang, Y. Designs of Zwitterionic Interfaces and Membranes. Langmuir. 2018. DOI: 10.1021/acs.langmuir.8b00562 (43) Liu, Q.; Liu, L. Novel Light-Responsive Hydrogels with Antimicrobial and Antifouling Capabilities. Langmuir 2018, DOI: 10.1021/acs.langmuir.8b01663. (44) Xin, X.; Li, P.; Zhu, Y.; Shi, L.; Yuan, J.; Shen, J. MusselInspired Surface Functionalization of PET with Zwitterions and Silver Nanoparticles for the Dual-Enhanced Antifouling and Antibacterial Properties. Langmuir 2018, DOI: 10.1021/acs.langmuir.8b01603. (45) Kisley, L.; Serrano, K. A.; Davis, C. M.; Guin, D.; Murphy, E. A.; Gruebele, M.; Leckband, D. E. Soluble Zwitterionic Poly(sulfobetaine) Destabilizes Proteins. Biomacromolecules 2018, 19, 3894−3901. (46) Ivanov, I.; Tsacheva, I.; Stoyanova, V.; Nikolov, M.; Tchorbadjieva, M. I.; Petrova, S.; Christov, L.; Georgieva, V.; Georgiev, G. Chaperone-Like Effect of Polyzwitterions on the Interaction of C1q with IgG. Z. Naturforsch., C: J. Biosci. 2009, 64, 149−154. (47) De, M.; You, C.-C.; Srivastava, S.; Rotello, V. M. Biomimetic Interactions of Proteins with Functionalized Nanoparticles: A Thermodynamic Study. J. Am. Chem. Soc. 2007, 129, 10747−10753. (48) Chiba, F.; Hu, T.-C.; Twyman, L. J.; Wagstaff, M. Dendrimers as size selective inhibitors to protein−protein binding. Chem. Commun. 2008, 4351. (49) Kano, K.; Ishida, Y. Regulation of α-Chymotrypsin Catalysis by Ferric Porphyrins and Cyclodextrins. Chem.Asian J. 2008, 3, 678− 686. (50) Park, H. S.; Lin, Q.; Hamilton, A. D. Protein Surface Recognition by Synthetic Receptors: A Route to Novel Submicromolar Inhibitors for α-Chymotrypsin. J. Am. Chem. Soc. 1999, 121, 8− 13. (51) Zhang, B.; Xing, Y.; Li, Z.; Zhou, H.; Mu, Q.; Yan, B. Functionalized Carbon Nanotubes Specifically Bind to α-Chymotrypsin’s Catalytic Site and Regulate Its Enzymatic Function. Nano Lett. 2009, 9, 2280−2284. (52) Cha, S.-H.; Hong, J.; McGuffie, M.; Yeom, B.; VanEpps, J. S.; Kotov, N. A. Shape-Dependent Biomimetic Inhibition of Enzyme by

Nanoparticles and Their Antibacterial Activity. ACS Nano 2015, 9, 9097−9105. (53) Costa, F. V.; Ambrosioni, E.; Montebugnoli, L.; Paccaloni, L.; Vasconi, L.; Magnani, B. Effects of a Low-Salt Diet and of Acute Salt Loading on Blood Pressure and Intralymphocytic Sodium Concentration in Young Subjects with Borderline Hypertension. Clin. Sci. 1981, 61, 21s−23s. (54) Yang, S.-T.; Marchio, J. L.; Yen, J.-W. A Dynamic LightScattering Study of Beta-Galactosidase - Environmental-Effects on Protein Conformation and Enzyme-Activity. Biotechnol. Prog. 1994, 10, 525−531. (55) Copeland, R. A. Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis; John Wiley & Sons, 2004. (56) Joensen, P.; Frindt, R. F.; Morrison, S. R. Single-layer MoS2. Mater. Res. Bull. 1986, 21, 457−461.

17539

DOI: 10.1021/acsomega.8b02598 ACS Omega 2018, 3, 17532−17539