Micelles of cleavable gemini surfactant induce fluorescence switching in novel probe: Industrial insight

Accepted Manuscript Title: Micelles of cleavable gemini surfactant induce fluorescence switching in novel probe: Industrial insight Authors: Imtiyaz Ahmad Bhat, Bibhisan Roy, Kabir-ud-Din PII: DOI: Reference:

S1226-086X(19)30196-0 https://doi.org/10.1016/j.jiec.2019.04.035 JIEC 4515

To appear in: Received date: Revised date: Accepted date:

18 February 2019 1 April 2019 20 April 2019

Please cite this article as: Ahmad Bhat I, Roy B, Kabir-ud-Din, Micelles of cleavable gemini surfactant induce fluorescence switching in novel probe: Industrial insight, Journal of Industrial and Engineering Chemistry (2019), https://doi.org/10.1016/j.jiec.2019.04.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

SHORT COMMUNICATION Micelles of cleavable gemini surfactant induce fluorescence switching in novel

Imtiyaz Ahmad Bhata*, Bibhisan Roya, Kabir-ud-Dinb E-mail address: [email protected] a

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probe: Industrial insight

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Department of Chemistry, Arba Minch University, Ethiopia

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Department of Chemistry, Indian Institute of Science Education and Research (IISER), Pune 411008, Maharashtra, India

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Graphical Abstract

Highlights

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Micelles of cleavable gemini surfactant induces fluorescence switching in novel probe

 Gemini surfactant was found to be efficient in color switching than

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conventional ones

Substantial yellow to blue color switch obtained can be utilized to devise an

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industrial sensing strategies

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ABSTRACT

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In this communication we have observed that cleavable gemini surfactant micelles

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induce fluorescence switching in novel probe (bound to lysozyme) at extremely lower concentrations (2.63mM) compared to conventional surfactants, which are

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known to induce such phenomenon at above 30mM. Fluorescence switching being important in sensing, binding, and probing the conformational states of proteins,

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therefore, in future this study could be significant to protein analytics and other

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relevant industrial domains.

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Keywords: Fluorescence switching, Docking, oxy-ester gemini

1. Introduction

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Owing to the complexity of conventional sensing strategies, supramolecular surfactant based sensing tactics are gaining interest among scientists [1-2]. Recent reports have shown that surfactant ensembles are being employed to sense and

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detect the analytes viz., metal ions/anions, amino acids, and explosives [1-3]. Further, emergence of protein recognition realms broaden its connotation to diagnostics (including Alzheimer’s, Huntington’s, Parkinson’s, and prion),

proteomics, bioterrorism, pathogen detection and other relevant fields [1-3]. The

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central crux governing the employment of supramolecular surfactant assemblies in

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these imperative domains is accredited to their inherent self-assemble behaviour.

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Surfactants self-assemble into dynamic, heterogeneous, supramolecular micelles, which offer the hydrophobic micro-domains to encapsulate guest molecules non-

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covalently, and this encapsulation ultimately tunes the photo physical

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characteristics of guest molecule. Further, micelles being thermodynamically

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stable are considered appropriate containers for potential application into domains

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of drug delivery, energy storage and opto-electronics [4-5]. Despite the intriguing properties of surfactants, higher CMCs, cleavability, and

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environmental compatibility are the main concerns prompting researchers to develop and design more proficient surfactant systems. To circumvent these flaws, scientists now-a-days have turned their interest to more advance surfactants, called cleavable-gemini-surfactants. These surfactants were found to be superior in 3

almost all physicochemical characteriscts than the conventional counterparts [6]. Keeping the advanced features in mind herein we have utilized the micelles of cleavable gemini surfactant, N-hexadecyl-N-[2-(N-hexadecyl-N,N-

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dimethylammonio)acetyloxyethyl]-N,N-dimethylammonium chloride (G) to

induce the fluorescence switching in novel fluorescence luminogen. This surfactant was chosen owing to its two interesting features (i) lower CMC and,(ii) cleavable ester-functionality and both these features are known to sophisticate the self-

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assembly phenomenon. Since self-assembling behaviour is considered as a central

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nerve to supramolecular sensing, therefore, we expect it to be a better candidate

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than the conventional counterparts. Further, its longer tail generates hydrophobic microdomains, significant to tune the overall photo-physical characteristic patterns

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of encapsulated luminogen [7].Moreover, luminogens have been extentesively

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explored owing to their potential applications in optical storage, mechanical

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sensors, security systems, optoelectronic devices, etc [8, 9]. In this context, we

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have explored the fluorescence switching property of the very efficient selfsynthesized luminogen, (Z)-5-(diphenylamino)-3-((phenylsulfonyl) methylene)

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isoindolin-1-one (P). It has very interesting photo-physical properties and its emission spectra exhibits dual emission peaks. The higher energy peak is ascribed to local excited states (LE), and lower energy peak is attributed to charge transfer

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(CT) states [10]. It is to be noted that for the sake of simplicity we have coded lysozyme, probe and gemini as L, P, and G, respectively.

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2. Materials and methods 2.1. Materials

The requisite gemini surfactant, N-hexadecyl-N-[2-(N0 -hexadecyl-N,N

dimethylammonio)acetyloxyethyl]-N,N-dimethylammonium dichloride (G) was

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synthesized as per the protocol given in our recent paper [6]. The enzyme, chicken

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egg white lysozyme (L) was purchased from Sigma–Aldrich with Lot No.

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BCBD8746V. Other requisite chemicals, N,N-dimethylhexadecylamine (>95%, Sigma– Aldrich), chloroacetyl chloride (98%, Spectrochem), 2-chloroethanol

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(98%, Spectrochem), acetone (99.5%, Rankem), ethanol (99.5%, Rankem), sodium

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monobasic phosphate (99.9%, SRL Chem), sodium dibasic phosphate (99.9%,

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SRL Chem), magnesium sulfate (99%, Rankem), diethyl ether (99.5%, Rankem)

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were utilized as received. The concerned probe (P) was synthesized and characterized as per protocol given in the literature [10].

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2.2. Methods

The fluorescence (steady-state) spectra were monitored on Fluoro Max-4 spectrofluorimeter (Horiba Scientific, USA). To gather the fluorescence spectra, we utilized the parameters as; ex = 360 nm, em-range = 370 –700 nm. For time 5

resolved fluorescence measurements we employed time correlated single photon counting (TCSPC) spectrometer (Horiba Jobin Yvon IBH, U.K.) and data were

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collected at the respective wavelengths given in table 1. The HEX 8.0 was utilized to obtain the probable docking conformations of probe with lysozyme. HEX 8.0 is a unique graphics program with Spherical Polar Fourier Correlations algorithm to explore and display feasible (lowest energy) docking

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modes for lysozyme–ligand complexes. The requisite crystal structure of lysozyme

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(PDB: 1azf) was obtained from protein data bank (http://www.rcsb. org./pdb) and

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the PDB files gemini surfactant and probe were modeled as per literature using

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chimera 1.9 (www.cgl.ucsf.edu/chimera). Then molecular docking program was

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3. Results and discussion

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run on Intel(R) core(TM) [email protected] GHz 64.bit operating system.

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3.1. Fluorescence analysis

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The main idea governing the construction of this system lies in the characteristic dual emission of P probe and its tunability in micro-heterogeneous cosmos [10].

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Keeping this notion in mind we have first dissolved the concerned probe into lysozyme solution and upon doing this, emission of P were observed at yellow regions (Scheme 1), indicating the complexation of L to P. However, addition of gemini micelles into Lyz-P solution induces disassembly of LP complex owing to 6

incorporation of probe into gemini hydrophobic domains, which consequently leads to gigantic fluorescence switching to blue regions (Scheme 1). We anticipate that this characteristic color-switch, in future could be significant to unravel the

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different conformational (folded/unfolded) states in proteins.

To authenticate our arguments experimentally, we have utilized the steady state fluorescence spectroscopy and the results are shown in Fig. 1(a). It can be observed

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that in the absence of gemini surfactant, fluorescence emission maximum lies at

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543 nm, signifying the localization of probe at the hydrophobic regions of

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lysozyme. However, upon addition of gemini surfactant, the fluorescence intensity

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not only boosts, but emission maxima shifts to lower wavelength regions (433 nm),

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suggesting stabilization of local excited state of probe, which in other words can be

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attributed to the binding of gemini surfactant to the hydrophobic regions of protein. Further in-depth examinination of the dynamics of P into heterogeneous

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microdomains was revealed by time resolved fluorescence, and the results are

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shown in Fig. 1(b-c) and Table 1. It can be observed that average lifetime was found to decreases upon incorporation of gemini surfactant into protein solution,

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inferring the perturbation of PL assembly by the binding of surfactant micelles [11, 12], and this observation is in harmony with other literature reports [11, 12]. Further, on careful observation of Table 1, decrements in amplitudes upon gemini additions suggest the incorporation of probe molecules into the gemini micelles. 7

This phenomenon induces the substantial color switching in the concerned probe molecules and hence arguments in harmony with steady state results.

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3.2. Docking analysis To confirm whether P really binds to lysozyme or not, we have utilized

molecular docking method. Molecular docking is a trustworthy tool to explore the probable ligand-protein interactions [13]. We have employed Hex 8.0 software for

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the task, reason being its unique algorithm to score the probable docked

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conformations in regard to their energy values. We have analyzed 2000 docked

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solutions and the best solution (having minimum energy) obtained are shown in

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Fig. 2(ab). Interestingly, P was found to be in the locality of Trp-108, Trp-63 and

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Trp-62 residues. This type of localization supplements the idea that when P is

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dissolved into lysozyme solution, it mostly locates itself at the hydrophobic

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patches of protein. Further, the negative interaction energy (289.49kJ/mol) obtained in PL system suggests that interaction is feasible and occurs via

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favourable surface contacts. Careful observation of Fig. 2(ac) reveals that the

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orientation of biphenyl ring of P lies towards the hydrophobic cavity and, the hydrophilic sulphone oxygen forms hydrogen bond with the ASP-48 (shown by green line) of lysozyme. This sort of hydrogen bonding further mitigates the hydrophilic characteristic of system and augments the hydrophobic contributions

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therefore, from these results it can be ascertained that hydrophobic interactions are playing the major role in binding of P to lysozyme. For comparative reasons, we have also docked the well known probe, ANS with lysozyme. ANS has been taken

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for comparison owing to its structural similarity to P [14]. It was observed that the binding pattern as well as interaction energies is different. Sulphone group was

found to face the interior of the hydrophobic cavity and no hydrogen bond between the ANS and lysozyme was found (Fig 2(b)). Further interaction energy obtained

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in ANS-L (234KJ/mol) system was found to be less than the PL. These results

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hint at the better potentiality of probe than the ANS. Moreover, on comparing the

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energy values with GL system, the order was found to be GL  PL  ANSL. This interesting observation suggests that G interacts more strongly than other two

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ligands. When P is added to lysozyme solution it binds near to aromatic patches of lysozyme, however, binding of surfactant micelles due to higher interaction

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capacity displaces P molecules and thus, generates a gigantic blue shift of 117nm.

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4. Conclusion

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In summary, we have shown that cleavable gemini surfactant induces substantial

fluorescence switching in P probe at very lower concentrations (2.63) of gemini surfactant. This observation was confirmed by steady- state and time resolved fluorescence. Molecular docking further advocates the involvement of hydrophobic

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forces. This feature could be utilized in future to develop and design the supramolecular sensing strategies for ultimate application in industrial and material science. On comparison our results with the literature, it was found that

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conventional surfactants (such as CTAB) induce fluorescence switching at higher concentrations [20]. Acknowledgements

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Authors are highly thankful to IISER Pune for instrumental facilities. IAB thanks

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to Science and Engineering Research Board (SERB), India for providing research

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grant (PDF/2016/002718).

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Fig. 1. Steady state fluorescence switching in P upon gemini micelle incorporation,

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(b) Lifetime decay transients of LP in the absence and presence of gemini micelles. ); [G] = 2.63 mM; [L] = 10M

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(b)

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(a)

(c)

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Fig. 2 Docking poses showing the localization of (a) ANS and, (b) P in the

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hydrophobic cavity of lysozyme

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(a)

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(b)

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Scheme 1 Structure of (a) probe, P and, (b) gemini surfactant, G

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Scheme 2 Fluorescence colour switching of P probe upon interaction with gemini

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surfactant micelles (G) ); [G] = 2.63 mM; [L] =10M

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Table 1 Lifetime measurements of LP in the absence and presence of gemini surfactant (G): [G] = 2.63 mM; [L] =10M α2(% )

α3 (%)

1 (ns) 2 (ns)

3 (ns)

 avg (ns)

2

CT Peak Water 560

0.44

0.34

0.23

0.23

2.23

6.9

2.43

LP

535

0.53

0.21

0.26

0.38

2.75

9.88

3.31

LP+G

544

0.66

0.23

0.1

0.1

0.11

1.55

1.6

1.1 2 0.9 6 1.0 9

LE Peak LP 422

0.72

0.23

0.05

0.093

5.06

4.43

LP+G

0.99

0.01

0

0.087 3.42

2.15

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0.32

1.1 9 0.8 6

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425

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α1(% )

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System coll(nm)

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