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Hydrophobically Tailored Carbon Dots towards Modulating Microstructure of Reverse Micelle and Amplification of Lipase Catalytic Response Saheli Sarkar, Krishnendu Das, and Prasanta Kumar Das Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04750 • Publication Date (Web): 01 Apr 2016 Downloaded from http://pubs.acs.org on April 5, 2016
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Hydrophobically Tailored Carbon Dots towards Modulating Microstructure of Reverse Micelle and Amplification of Lipase Catalytic Response Saheli Sarkar, Krishnendu Das and Prasanta Kumar Das* Department of Biological Chemistry, Indian Association for the Cultivation of Science Jadavpur, Kolkata – 700 032, India
*To whom correspondence should be addressed. E-mail:
[email protected] 1
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ABSTRACT This article delineates the modulation of microstructure of cationic reverse micelle utilizing hydrophobically modified carbon dots (CDs) with varying surface functionalizations. Citric acid was used as the source of the carbon core and Na-salt of glycine, glycine, Na-salt of 11aminoundecanoic acid, 11-aminoundecanoic acid and n-hexadecylamine were used for the surface fabrication of CDs to produce CD 1s, CD 1a, CD 2s, CD 2a, and CD 3, respectively. All these CDs having dimension of 5-7 nm were characterized by spectroscopic and microscopic techniques. The hydrodynamic diameter of cetyltrimethylammonium bromide (CTAB) reverse micelle (CTAB/isooctane/n-hexanol/water) at z ([co-surfactant]/[surfactant]) = 6.4 and W0 ([water]/[surfactant]) = 44 is around 15-20 nm. Interestingly, the size of the water-in-oil (w/o) microemulsions dramatically increased up to 120-200 nm upon doping hydrophobic surface functionalized CD 2a and CD 3. This is possibly due to change in the micellar exchange dynamics and reorganization of the micellar aggregates via hydrophobic interaction between surfactant (CTAB) tail and hydrophobic surface modifier of the carbon dots. However, no alteration in the size of reverse micelles was noted in presence of carbon dots CD 1s, CD 1a and CD 2s. Spectroscopic and microscopic investigations confirmed that the hydrophobic CD 2a and CD 3 were localized at the interface of reverse micelles whereas CD 1s, CD 1a and CD 2s were possibly located in the water pool (away from interface). The activity of Chromobacterium viscosum lipase encapsulated within CD 3 and CD 2a doped significantly large CTAB reverse micelles showed remarkable improvement (3.7 fold and 3.4 fold) in its catalytic response. However, hydrophilic carbon dots CD 1s, CD 2s as well as moderately hydrophobic CD 1a and had no significant effect on the microstructure of reverse micelles as well as on the lipase activity.
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1. INTRODUCTION The water-in-oil (w/o) microemulsion, also known as reverse micelle is the self-organization of surfactants in non-polar organic solvent with water pool in their polar core.1-3 It is a macroscopically homogenous nanometer-scale colloidal system that comprises of distinct interfacial area and has the ability to compartmentalize molecules of varying polarities. The interactions between the solvent molecules and the polar head groups of surfactants offer a distinctive environment at the microscopic water/oil interface that is dramatically different from that of the bulk solvent.4 Applications of thermodynamically stable w/o microemulsions are wide-ranging that includes synthesis of nanocrystals with regulated shape and size, modulating enzyme activity, drug delivery etc.5-15 In addition, w/o microemulsion has been extensively used in biochemistry and biocatalysis because of their resemblance to biological membrane and the capability of hosting intracellular enzymes and proteins.16-17 Different biomolecules have been solubilized in the w/o microemulsions without the loss of their biological activities. Formation of w/o microemulsions is primarily dictated by the structure of surfactants and
also
by
the
presence
of
co-surfactant
in
case
of few surfactants
like
cetyltrimethylammonium bromide (CTAB). The important microstructural parameter of reverse micelle is W0 ([water]/[surfactant]).4 The other parameter, z (z = [cosurfactant]/[surfactant]) also has very much relevance on the stability and formation of reverse micelles where co-surfactant becomes integral part of w/o microemulsion. On several accounts, these microstructural parameters have been modulated towards preparing reverse micelles of varying sizes as well as for tuning the activity of encapsulated enzymes.18-21 In addition, alteration in head groups, counterion of surfactants, incorporation of nanomaterials like surface functionalized carbon nanotube, graphene oxide within w/o microemulsions have resulted in improving the activity of interfacially solubilized enzymes like lipase, peroxidases
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possibly through augmenting the ‘space’ at the interface of reverse micelles.13,22-26 Ionic liquids, colloidal gold nanoparticles have been utilized to tune the microenvironment of the water pool to modulate the activity of water pool solubilized enzymes like trypsin.20,27 Till date hydrophilic or amphiphilic nanomaterials were chosen for inclusion within w/o microemulsions to modulate the ‘space’ in vicinity of the enzyme/protein located at the interface or the water pool. In most of the cases the inclusion of different nanomaterials modestly altered the area at interface or the dimension of water pool of reverse micelle depending on the size of the nanomaterials. However, the influence of a hydrophobic nanoparticle over the microstructure of a reverse micelle and consequently on the enzyme activity has not been explored. At this point, we intrigued to explore the effect of surface modified carbon dot (CD) towards the microstructure of cetyltrimethylammonium bromide (CTAB) reverse micelle (CTAB/isooctane/n-hexanol/water) at z = 6.4. Carbon dot, a zero dimensional spherical allotrope of carbon have been widely explored in different disciplines of advanced materials to biomedicine because of its unique physical and chemical properties like nanoscale dimension, intrinsic fluorescence, high thermal stability, superior biocompatibility etc.28-34 Herein, we have synthesized different hydrophilic and hydrophobic surface functionalized carbon dots from citric acid. Na-salt of glycine (CD 1s), glycine (CD 1a), Na-salt of 11aminoundecanoic acid (CD 2s), 11-aminoundecanoic acid (CD 2a) and n-hexadecylamine (CD 3) have been used as the surface functionalities of carbon dots (Figure 1). Sizes of the synthesized carbon dots are found to be in the range of 5-7 nm. The dimension of CTAB reverse micelle at z = 6.4 and W0 = 44 is around 20 nm. Interestingly, the size of the w/o microemulsions dramatically increased up to 120-200 nm upon doping hydrophobic surface functionalized carbon dots whereas no alteration in the size of reverse micelles was noted in presence of hydrophilic carbon dots. Influence of hydrophobically tailored carbon dots on the
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interfacial properties of the aggregates as well as on the activity of Chromobacterium viscosum (CV) lipase was investigated. Lipase showed remarkable improvement (3.7 fold) in its activity within significantly large CTAB reverse micelle doped with hydrophobically functionalized carbon dots, CD 3. 2. EXPERIMENTAL SECTION Materials and Methods. Chromobacterium viscosum lipase (EC 3.1.1.3) was purchased from Millipore, India. Glycine, 11-amino undecanoic acid, n-hexadecylamine, fluorescein sodium salt (Na-fl) were purchased from SRL (India). Analytical-grade CTAB and citric acid were procured from Spectrochem (India). Dialysis bag (MWCO 2000) was procured from Sigma-Aldrich Chemical Pvt. Ltd. CTAB was crystallized three times from methanol/diethyl ether before use. The recrystallized CTAB was without minima in its surface tension plot. Milli-Q water was used throughout the study. IR-grade KBr, HPLC-grade isooctane, methanol, n-hexanol, all other reagents and solvents were purchased from SRL (India). pNitrophenyl-n-octanoate was synthesized following the procedure reported earlier.35 Transmission electron microscopy (TEM) images were taken on a JEOL JEM 2100F UHR microscope. X-ray photoelectron spectroscopy (XPS) was performed on Omicron (series 0571) X-ray photoelectron spectrometer. Dynamic light scattering (DLS) of reverse micelle and carbon dot doped reverse micelle was measured in a zetasizer Nano-ZS of Malvern Instruments Limited. The UV-vis absorption spectra were recorded on a Perkin Elmer Lambda 25 spectrophotometer. Fluorescence spectra were performed using Varian Cary Eclipse luminescence spectrometer. FTIR spectra were performed using a Perkin Elmer Spectrum 100 spectrometer. 1H NMR spectra were recorded on a Bruker Avance DPX-300 spectrophotometer. Lyophilization was done using a Virtis 4KBTXL-75 freeze-drier and Thermo Scientific Espresso centrifuge was used for centrifugation.
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Synthesis of CD 1s. For the synthesis of CD 1s, 1.1 g of glycine (14 mmol) was converted to its carboxylate salt by the addition of an equivalent amount of NaOH solution (2 mL). To this, 2 mL of aqueous citric acid solution (3 g, 14 mmol) was added by maintaining a 1:1 molar ratio. This water-soluble mixture was evaporated to dryness at 100 °C. The sticky mass was collected and dried in hot oven at 80 °C for 3 days. The solid was crushed into a fine powder and was heated in a furnace at 200 °C for 2 h in a porcelain crucible and then cooled to room temperature. The brownish-black product was extracted with 25 mL of hot water. The solution was centrifuged at 12000 rpm for 30 min to remove the insoluble pellet. The supernatant was collected and dialyzed against water using a dialysis bag (MWCO 2000) for further purification. The resultant solution was lyophilized to get the carbon dots. The yield was 62%. Synthesis of CD 1a. CD 1s (200 mg) was dissolved in 1 mL water and to that 1 M HCl (1 mL) was added. A black mass was precipitated out from the solution. It was centrifuged at 12000 rpm for 30 min. The supernatant was discarded and the black residue was collected and dried by lyophilization to get the desired carbon dot, CD 1a. Synthesis of CD 2s. 11-aminoundecanoic acid (2.8 g, 14 mmol) was converted to its carboxylate salt by the addition of an equivalent amount of NaOH solution (2 mL). To this, 2 mL of citric acid solution (3 g, 14 mmol) was added by maintaining a 1:1 molar ratio. This water-soluble mixture was evaporated to dryness at 100 °C. The sticky mass was collected and dried in hot oven at 80 °C for 3 days. The solid was crushed into a fine powder and was heated in a furnace at 200 °C for 2 h in a porcelain crucible and then cooled to room temperature. The brownish-black product was extracted with 25 mL of hot water. The solution was centrifuged at 12000 rpm for 30 min to remove the insoluble pellet. The supernatant was collected and dialyzed against water using a dialysis bag (MWCO 2000) for
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further purification. The resultant solution was lyophilized to get the carbon dots. The yield was 55%. Synthesis of CD 2a. CD 2s (200 mg) was dissolved in 1 mL water and to that 1 M HCl (1 mL) was added. A black mass was precipitated out from the solution. It was centrifuged at 12000 rpm for 30 min. The supernatant was discarded and the black residue was collected and dried by lyophilization to get the desired carbon dot, CD 2a. Synthesis of CD 3. For the synthesis of CD 3, 100 mg of CD 1a, n-hexadecylamine (300 mg), EDCI (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (300 mg) and DMAP (4dimethylaminopyridine) (300 mg) were taken in a round bottom flask. It was dissolved in 5 mL dry DMF (dimethyl formamide) and was stirred for 12 h. The DMF was evaporated under vacuum and the black residue was dissolved in dichloromethane. Then the solution was washed with 1M HCl, NaHCO3 solution and brine solution successively. The mass obtained after evaporation of dichoromethane gave the desired CD 3. The yield was 63%. Synthesis of Free Carbon Dot (FCD, carbon dot derived from citric acid only). For the synthesis of FCD, 1 g of citric acid was heated in a furnace at 200 °C for 2 h in a porcelain crucible and then cooled to room temperature. The brownish-black product was insoluble in water. The mass was extracted with DMSO and dried under vacuum to get the carbon dots. The yield was 67%. Preparation of Carbon Dot Doped Reverse Micelles. CTAB (36.4 mg) was dispersed in isooctane in a 2 mL volumetric flask to which a calculated amount of n-hexanol (80 µL) was added to attain z = 6.4, and shaken vigorously. Finally aqueous buffer (20 mM phosphate buffer, pH = 6.0) was added to attain the W0 = 44 and the whole solution was vortexed to prepare transparent solution of CTAB reverse micelle. Carbon dots stock solutions were prepared using water or DMSO depending on their solubility and doped into reverse micelle 7
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to achieve the desired carbon dot (CD) concentration. The whole solution was vortexed to obtain a clear homogeneous solution of CD-doped reverse micelle. The maximum CD concentration was maintained depending on the stability of reverse micelles. High concentration stock solution (8 mg mL-1) of carbon dots were prepared in DMSO or water in such a way that maximum 1-2 µL of carbon dot solution has to be added to attain the desired concentration of CDs in 2 mL of reverse micelle. The rest of the volume (78-79 µL) including the aqueous solution of enzyme (4.5 µL)) was adjusted using aqueous buffer to attain W0=44. Hence the W0 was thoroughly maintained at 44 throughout the experiment. Low DMSO content was used to avoid any denaturing effect on lipase. FTIR Spectroscopy. Equal amounts of the nano-constructs (CD 1s, CD 1a, CD 2s, CD 2a, CD 3 and FCD) were mixed with IR-grade KBr, and subsequently KBr pellets were prepared for FTIR studies. This pellet was further dried by storing in vacuum desiccators. Spectra of these pellets were recorded using a Perkin Elmer Spectrum 100 spectrometer. Each time background correction was performed to eliminate interference from air (or any other parameters). X-ray photoelectron spectroscopy (XPS). Two drops of prepared carbon dot solution was put on a rectangular Cu plate and it was dried under vacuum for 8 h before the experiment which was performed using an Omicron (series 0571) X-ray photoelectron spectrometer. Transmission Electron Microscopy (TEM). Aliquots of CD solutions (4 µL, either in water or DMSO or reverse micelles) were placed on a 300 mesh copper-coated transmission electron microscopy (TEM) grid and dried under vacuum for 4 h before acquiring TEM images. TEM measurements were performed on a JEOL JEM 2100F UHR microscope. TEM images of reverse micelle and carbon dot doped reverse micelle were taken through reverse phase contrast imaging technique.
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Dynamic Light Scattering (DLS). Mean hydrodynamic diameters of non-doped reverse micelle and CD-doped reverse micelles were determined by dynamic laser light scattering using zetasizer Nano-ZS of Malvern Instruments Limited. Concentrations of hydrophilic and hydrophobic carbon dots were 12 µgmL-1 and 8 µgmL-1, respectively. Phase Behavior Study. Microemulsions were prepared by titrating the mixtures of surfactants, n-hexanol, and water with isooctane. A constant mass ratio (1:2) of the surfactant and n-hexanol was dissolved in water, forming solutions of different concentrations taken in different screw-topped test tubes and stirred until the solutions became clear. These solutions were then titrated with isooctane from a micro-burette at 25oC until just turbid or phase separation. The isotropy/turbidity of the solutions was checked by naked eye and thus measured phase boundaries are of fair accuracy. Fluorescence Study. Fluorescence spectra of Na-fl doped reverse micelle (λex = 490 nm) were recorded using Varian Cary Eclipse luminescence spectrometer. Fluorescence spectra of lipase within reverse micelle in presence or absence of CDs were measured by exciting lipase at 280 nm. The excitation and emission slits were kept at 5 nm. Lipase Activity in w/o Microemulsions. The second-order rate constant (k2) in lipasecatalyzed hydrolysis of p-nitrophenyl-n-octanoate in reverse micelles was determined following a similar protocol as reported earlier.13 4.5 µL of aqueous enzyme stock solution (0.34 mg mL-1) was added to 1.5 mL of reverse micelle (in presence and absence CD) having 50 mM CTAB and desired pH (pH refers to the pH of the aqueous buffer used for preparing the w/o microemulsions; pH within the water pool of the reverse micelles might not vary significantly (
