Spontaneous Formation of Vesicles by Self-Assembly of Nicotinyl

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Spontaneous Formation of Vesicles by Self-Assembly of Nicotinyl Amino Acid Amphiphiles: Application as “Turn-On” Fluorescent Sensors for the Selective Detection of Trace-Level Hg(II) in Water Aparna Roy, and Sumita Roy Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.6b02603 • Publication Date (Web): 05 Sep 2016 Downloaded from http://pubs.acs.org on September 8, 2016

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Industrial & Engineering Chemistry Research

Spontaneous Formation of Vesicles by Self-Assembly of Nicotinyl Amino Acid Amphiphiles: Application as “Turn-On” Fluorescent Sensors for the Selective Detection of Trace-Level Hg(II) in Water Aparna Roy and Sumita Roy* Department of Chemistry and Chemical Technology, Vidyasagar University, Paschim Medinipur-721 102, India. E-mail: [email protected] RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to) Abstract In this paper, the three nicotinic acid based amphiphiles, Sodium 6-dodecylnicotinic glycinate (SDDNAG), Sodium 6-dodecylnicotinic alaninate (SDDNAA) and Sodium 6-dodecylnicotinic valinate (SDDNAV) were synthesized and the effect of the head group on the self-assembly properties have investigated.

The interfacial properties were determined by tensiometry

measurement. Steady state fluorescence technique was used to investigate the microenvironment of the self-assemblies. The amphiphiles form vesicles in water and the size of the aggregates decreases with increase in hydrophobicity of the head group. GPC and kinetic studies were performed to establish the entrapping and releasing property of the vesicles. The arrangement of the hydrocarbon chains in the lamella was examined by XRD study. CD spectra suggested formation of chiral structure through aggregation of the chiral amphiphiles. It has been

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demonstrated that the amphiphiles have feasibility to develop the sensor for rapid determination of Hg+2 ions in aqueous biological and environmental samples. Introduction Self-assembly of amphiphiles into aggregates of different morphologies has been of interest across biological, chemical and physical sciences for several decades. Among them, Liposomes have been successfully used for the treatment of cancers, infectious and autoimmune diseases, as well as ocular inflammation.1-3 Liposomes, or vesicles, are nothing but self-closed spherical or ellipsoidal structures where one or more phospholipid bilayers entrap part of the solvent into their interior.4,5 Among the different types of driving forces hydrogen bonding is one of the most commonly used driving force for the formation of bilayer self-assemblies of single-chain surfactants.6,7 Recently Haldar et al. in 2014 established that the amide functionality in the hydrophobic tail enhanced the aggregation properties of the gemini surfactants compared to that of surfactants having no amide bonds, possibly by intermolecular hydrogen bonding.8 In an another report, Dey and Roy has established that the intermolecular hydrogen bonding interaction between the secondary amide groups of the neighboring molecules is the driving force for the bilayer formation which promote the formation of the linear array of two N-acyl amino acid surfactants.9 Also it is well-known that hydrogen bonds play very important roles in biological systems and most of the molecules of importance to living systems are chiral, e.g. amino acids, sugars, proteins and nucleic acids. Actually chiral surfactants are specialty molecules to be used in enantioseperation of pharmaceutical molecules and in stereoselective synthesis.10-14 Chiral vesicles are a special class of supramolecules that allow them to be used as enantiosensitive analytical reagent.15,16 One of the important effects of chirality in surfactant


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systems is the “chiral bilayer effect” where the hydrophobic aggregates have enhanced life time. Chiral vesicles are ideal candidates to be used as carriers for chiral drug molecules. Organic molecules which hold the electron donor nitrogen centre can acts as a mercury sensor. Lippard et

al. demonstrated that 2-{5-[(2-{[Bis-(2-ethylsulfanyl-ethyl)-amino]-methyl}-

phenylamino)-methyl]- 2-chloro-6-hydroxy-3-oxo-3H-xanthen-9-yl}-benzoic acid can detect environmentally relevant concentrations of Hg(II).17 J. Yoon and his co-workers observed that rhodamine 6G thiolactone derivative as a selective fluorescent and colorimetric sensor for Hg2+ in neutral aqueous solution.18 WHO (World Health Organization) reported that mercury poses a serious threat to human health, particularly to fetal development in utero and infants.19 In addition, it also affects the nervous, immune, heart and digestive systems and causes damage to the brain vision problems (constriction or narrowing of the visual field), tremors, deafness, and losses of muscle coordination, sensation, and memory20,21 as well as kidneys and lungs of human beings.22-25 According to the WHO and the U.S. Environmental Protection Agency (EPA) the maximum allowable levels of Hg in drinking water is 30 nM and 10 nM, respectively. Fluorescent chemosensors (small molecules whose fluorescence emission changes in response to a binding event) are widely appreciated tools for monitoring biological, biomedical, and environmental processes.26-28 With this background our current research focused on the selfassembly properties of three synthesized nicotinic acid derivatives having amide linkage in the head group (Figure 1) in water. O

R = H, SDDNAG CH3, SDDNAA CH(CH3)2, SDDNAV

R -

+

O Na N H

O

N

Figure 1. Molecular structure of synthesized compound.



We have examined the effect of hydrophobicity and chirality of the head group on the aggregation behavior of the amphiphiles. In our study we have established that all the three amphiphiles are fluorescent sensor for Hg2+ ions. Results and Discussion Interfacial Properties

70 2.0

60

1.8

A

50

I1/I3

-1

 (mNm )

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SDDPCA SDDPCG SDDPCV

40

1.4

30 -6

-5

-4 -3 log C (M)

1.6

-2

B SDDPCG SDDPCA SDDPCV

1.2 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 -2.0 log C (M)

Figure 2. (A) Plot of γ vs. log C and (B) Plot of polarity ratio (I1/I3) vs. log C of SDDNAG, SDDNAA and SDDNAV in aqueous solution. The minimum concentrations of aggregation i.e critical aggregation concentration (CAC) values of the amphiphiles were determined by most accurate tensiometry method. The change in surface tension value with logarithm of concentration of aqueous solutions of SDDNAG, SDDNAA and SDDNAV are depicted in the Figure 2A. Figure 2A showed progressive decrease of the surface tension with concentration and remained constant above the critical aggregation concentration. These plots confirmed the purity of the amphiphiles due to the absence of a minimum or shoulder around the CAC values of the respective amphiphiles. The CAC values are listed in Table 1. From the Table 1 it can be observed that CAC value decreases with increase in the hydrophobicity of the head group which is indicative that the process of aggregate formation


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becomes favorable with increased hydrophobic interaction of the head group. The very good surface activities of the amphiphiles are reflected from low γCAC (surface tension at the CAC) and high ΠCAC (γwater - γCAC) values (Table 1). The essential condition for tensiometry experiments is the evaluation of parameters regarding interfacial adsorption characteristics and spontaneity of aggregation process. Table 1. Physico-chemical properties of SDDNAG, SDDNAA and SDDNAV at 303K.

Properties CAC (mM)

SDDNAG

a

SDDNAA 0.80, 0.90a

1.90 , 2.00 33 39 γCAC (mN m ) -1 39.05 33.50 ΠCAC (mN m ) 0.46 0.73 α 6 -2 1.62 1.62 Г2 × 10 (mol m ) 1.02 a0 (nm2 molecule-1) 1.02 ° -1 -24.31 -30.18 ΔG a (kJ mol ) ° -1 -48.69 -50.86 ΔG ad (kJ mol ) 1.22 1.40 I1/I3 503.50 385.50 Rh (nm) 624.33 366 Nagg×10-4 12 2 -1 0.44 0.57 D×10 (m s ) 0.086 0.113 r -3 -1 4.50 × 10 1.36 × 10-3 kPerm (s ) 2.57 8.49 t1/2 (min) 5.36× 10-9 6.25 × 10-10 P (cm s-1) ‘a’ data are obtained from fluorescence measurement. -1

SDDNAV 0.26,0.28a 31.70 40.30 0.58 1.52 1.09 -48.18 -74.70 1.33 59.61 8.20 3.70 0.12 1.04 × 10-3 11.10 8.32 × 10-10

Therefore, the maximum surface excess concentration (Γ2) and minimum surface area per surfactant molecule at the air-water interface (a0) were obtained from Gibbs adsorption equation29 as given bellow Γ2 = − (1/2.303nRT)(dγ/(d log C))

(1)

a0= 1018 / (Γ2 NA )

(2)



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where T is the absolute temperature, dγ/(d log C) is the maximum slope, NA is Avogadro’s number and n is 2 for dilute solutions of monovalent ionic surfactants.30 The standard free energy change of adsorption (ΔGad°) at the air/water interface and spontaneity of the aggregate formation (ΔGa°) were also calculated (See SI for details). All the parameters describing interfacial property are summarized in Table 1. The range of a0 value (1.0 ≤ a0 ≤1.1) suggested formation of bilayer type of aggregates for all the amphiphiles.31 An important finding is that all the three amphiphiles showed more facile adsorption compared to the micellization process.32 The highest negative value of both ΔGad° and ΔGa° (Table 1) of SDDNAV proposed that the adsorption as well as aggregation process are most spontaneous for SDDNAV among all the amphiphiles due to the maximum hydrophobic interaction among the head groups. Micropolarity and rigidity of the Self-Assemblies The solvent dependence of vibronic band intensities in Pyrene fluorescence has captured great attention in the literature. More specifically, the I1/I3 ratio corresponding to the first (372 nm) and third (384 nm) vibronic bands in the fluorescence spectrum of Pyrene is very sensitive to solvent polarity.33 Kalyansundaram et al. studied and reported the polarity ratio values of pyrene in different organic solvents.33 Therefore, Pyrene molecule was used to investigate the micropolarity of the self-assemblies. The variation of the ratio of emission intensities (I1/I3) of the external fluoroprobe pyrene as a function of concentration of the amphiphiles SDDNAG, SDDNAA and SDDNAV solutions are depicted in Figure 2B. It is observed from the plot that at first when only water is present, I1/I3 value is maximum and when surfactant is solubilized in water the I1/I3 value decreases and finally becomes constant above CAC. This result revealed that the probe molecules are solubilized in more nonpolar region as the surfactant concentration increases. The obtained values of CAC from the plots (Figure 2B) of the three studied


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amphiphiles agreed very well within the limits of error with tensiometry measurements (Table 1). Analysis of the I1/I3 values, which have a direct correlation on the nonpolarity of the solublization site of pyrene, infers that all the amphiphiles provided a denser nonpolar microenvironment in comparison to simple surfactants such as SDS or CTAB. The steady state fluorescence anisotropy (r) value predicts the types of aggregates are formed by the amphiphiles in solutions.34,35 Here we have used 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescence probe molecule to detect the type of aggregates formed in aqueous solution by the three amphiphiles. It is well known that in bilayer aggregates the anisotropy is very high but in spherical or rod like micelles, the anisotropy value is lower comparatively to that of bilayer aggregates. In bilayer aggregates the hydrocarbon chains of the amphiphiles are tightly packed, and therefore the microenvironments are more rigid than normal micelles. The r values of the DPH probe in the self-assemblies formed by the three amphiphiles are listed in Table 1 and are greater than the spherical micelle of conventional anionic surfactant SDS (r ≈ 0.032)36 but less than that of sphingomyelin liposomes (r ≈ 0.247).37 This result concluded that the three amphiphiles form bilayer type of aggregates and the microenvironment of the aggregates are very rigid. In order to examine the concentration dependence of the anisotropy change and hence to get some information about the structural change due to concentration the steady-state fluorescence anisotropy (r) of DPH probe was measured at different concentrations above the respective CAC’s of the amphiphiles. The anisotropy value is relatively high at concentration just above the CAC and then increases steadily with increase in concentration and then decreases reaching a maximum (~ 3 times of the CAC). The plots of r value as a function of amphiphile concentration for the three amphiphiles are given in SI (Figure S8). The decrease of anisotropy value at higher amphiphile concentration recommended partial destruction of the



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rigidity of the aggregates. This is possible only if closed bilayer aggregates are transformed to open structures or micelle type of aggregates. Size and Shape of the organized assemblies To determine the mean hydrodynamic radius of the aggregates formed by the three amphiphiles dynamic light scattering (DLS) technique was emplyoed. The intensity average size distributions of the aggregates formed by the three studied amphiphiles in aqueous solutions are shown in Supporting Information (Figures S9). The very large z-average hydrodynamic radii (Rh) of the amphiphiles (Table 1) suggested existence of larger aggregates in solutions. The mean aggregation number (Nagg) of the aggregates (assuming a bilayer vesicle) was calculated by the equation 38 Nagg = 8πRh2 /a0

(3)

The apparent diffusion coefficient (Dapp) was also estimated by use of Stokes-Einstein equation (Dapp = KBT/6πηRh) considering spherical nature of the aggregates formed by the amphiphiles. The obtained Dapp and Nagg values of the amphiphiles are listed in Table 1. The very low Dapp (1/100 times of spherical micelles)39 and high Nagg values of the amphiphiles again are indicative of formation of very large aggregates in solutions. It can be observed from the Table 1 that the size of the aggregates decreases in the order SDDNAG>SDDNAA>SDDNAV. This observation suggests that the introduction of chiral amino acid as well as more hydrophobicity of head group moiety dismantles the aggregate formation. Dye Encapsulation Vesicular morphology can be confirmed by presence of confined aqueous compartment inside the assembly which can be tested by the capability of the aggregate to encapsulate hydrophilic guest molecules.40,41 The existence of an inner aqueous compartment of the aggregates, dye-


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trapping experiments were carried out by Gel Permeation Chromatography (GPC) experiment using a hydrophilic fluorescent dye, methylene blue (MB) as suggested by Walde and Namani42 as well as Dey et al.43 GPC experiment was carried out by loading 2 mL of amphiphile solution containing 0.1 mM methylene blue into a pre-equilabrated Sepharose 4B gel filtration column and the material was eluted with triply distilled water. The absorption of each 2 mL eluted material was recorded at 665 nm. In this experiment the translucent vesicular suspensions was eluted at first and then after void volume. When all the free MB was gel filtered the gel filtration was completed. A blank experimerint (GPC column was loaded with aqueous solution of 2 mL 0.1 mM MB having no amphiphile) was also performed to ensure the aqueous cavity of the aggregates. The presence of only large absorption peak (no initial small peaks) in the gel filtration profile for the blank experiment was observed (Figure S10 of SI) which suggested that the small initial peaks of the gel filtration profile of the amphiphiles are due to the MB entrapped into the large vesicles. The gel filtration profiles of the three aggregates are shown in Figure 3. 0.04

0.08 0.06

A

0.04 0.02

0.03

Absorbance (a. u.)

Absorbance (a. u.)

0.10

Absorbance (a. u.)

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0.03

B

0.02 0.01 0.00

0.02

C

0.01

0.00

0.00 0

60

Volume (mL)

120

0

60

120

Volume (mL)

0

70

140

Volume (mL)

Figure 3. Gel-filtration profile of the separation of the dye entrapped (small peaks) in vesicle of (A) SDDNAG, (B) SDDNAA and (C) SDDNAV from the corresponding free dye (large peak). The absorbance of methylene blue at 665(●) nm and turbidity at 850(○) nm due to scattering of light by the large vesicles were recorded as a function of the elution volume.



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The entrapment capacities of the aggregates are 5.8%, 4.7% and 0.75% of the total dye for SDDNAG, SDDNAA and SDDNAV respectively. The entrapment ability is highest for SDDNAG and lowest for SDDNAV which is in accordance with the order of the Rh value of the amphiphiles. Investigation of Membrane Permeability The permeable nature of the vesicular membrane was examined fluorometrically by performing a kinetic study of using riboflavin44,45 as a probe molecule. At first stock solution of the respective amphiphiles (above ~ 3 times of CAC) were prepared in a buffer solution of pH 6.8 containing 2×10-5 M riboflavin. The fluorescence emission intensity of the prepared solutions were measured at 514 nm (λex = 374nm). This is the fluorescence intensity of the probe molecules which were adsorbed on the outer membrane surfaces and were entrapped within the inner water pools. When the pH of the vesicle dispersion containing riboflavin was suddenly changed from 6.8 to 10.2, the fluorescence intensity of the riboflavin was abruptly decreased initially to about 72.5 ± 5% for SDDNAG, 32.30 ± 5% for SDDNAA and 42.15 ± 5% for SDDNAV from the original value at pH 6.8. The sudden decrease of the fluorescence intensity indicated that riboflavin molecules which are bound at the outer surfaces of the vesicles are deprotonated because probe molecules become nonfluorescent upon deprotonation (pKa of riboflavin ~10.2). This consequence implied that the aggregate formed by all the amphiphiles have an inner aqueous compartment. Again to further confirm the permeable nature of the bilayer aggregates we carried out the same experiment using only buffer solution of riboflavin at pH 6.8. For this case the fluorescence intensity dramatically reduces to very low constant value (1/67 times of initial value; Figure S11 of SI) without showing any decay after sudden increase of pH value from 6.8 to 10.2. This observation confirmed that the aggregates formed by SDDNAG,


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SDDNAA and SDDNAV have inner aqueous cavity and the walls of the closed bilayer aggregates are permeable in nature. The plots of decrease in the fluorescence intensity of probe molecule as a function of time of the three amphiphiles are given in Figure 4.

640

96 94

A

92 90 88

0

5 10 15 Time (min)

20

630

600

B

560 520 480

Intensity (a. u.)

Intensity (a. u.)

98 Intensity (a. u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 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 48 49 50 51 52 53 54 55 56 57 58 59 60


600 570

C

540 510

0

50 100 150 200 Time (min)

0

50 100 150 200 Time (min)

Figure 4. OH- permeability profile of riboflavin entrapped vesicular solution of (A) SDDNAG, (B) SDDNAA and (C) SDDNAV. The permeation constant values (P) were calculated assuming the time-dependent loss of fluorescence intensity by exovesicular OH− as permeation limited deprotonation of riboflavin entrapped in the internal aqueous compartment and determining the bilayer thickness of the amphiphile by XRD measurement (discussed later). The rate constant of permeation (kperm), halftime of the process (t½) and permeation constant (P) are summarized in Table 1. TEM To obtain a detailed view of the resulting morphology of all the amphiphiles, TEM imaging was performed. The TEM micrographs of 6 mM (~ 3 times of CAC) SDDNAG showed co-existence of vesicles, closed tubules, pentagons, hexagons etc. in aqueous solution (Figure 5B,E). Closer observation revealed that some of the vesicles are multilamellar in nature (Figure 5D). Probably the vesicles are joined together to form tubules, pentagons, hexagons etc. The sizes of the vesicles are in the range ~ 25-550 nm and the width of the tubules is 15-40 nm. At higher



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amphiphile concentration (10 mM, Figure 5F) the vesicles and other closed structures malformed to cylindrical micelles.

A

B

E

C

F

D

Figure 5. Negatively stained (with 1.5% aqueous Uranyl acetate solution) TEM micrographs of (A-E = 6 mM, F = 10 mM) SDDNAG in water.

Figure 6A-C demonstrated the morphology of the aggregates of SDDNAA in solution. Here also vesicular aggregates as well as nano rods were observed in TEM micrographs at the amphiphile concentration 3.0 mM. This observation assured the result obtained in DLS measurement (bimodal intensity average size distribution of SDDNAA, Figure S9B). The vesicles are in the range ~ 75-450 nm and the vesicles are fused together. The widths of the rods are 10-20 nm and are of length 60-135 nm. At higher concentration (5.5 mM), the vesicles of SDDNAA are transformed into cylindrical micelles.


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A

B

D

B

E

C

F

Figure 6. Negatively stained (with 1.5% aqueous Uranyl acetate solution) TEM micrographs of SDDNAA (A, B = 3 mM), (C = 5.5 mM) in water and SDDNAV (D, E = 1 mM), (F = 1.5 mM) in water. On the other hand, the size of the vesicles formed by the amphiphile SDDNAV (1.0 mM) suddenly decreased to 25-200 nm (Figure 6D-F). Interestingly unlike SDDNAG and SDDNAA, the vesicles of SDDNAV malformed to worm like micelles at higher amphiphile concentration. Optical Activity of the Bilayer Self-Assemblies Formation of chiral aggregates is normally manifested in the CD spectra and has been demonstrated by many authors.46-51 The aqueous solutions of SDDNAA and SDDNAV were therefore examined for chiral organization by CD spectroscopy. The CD spectra were recorded below and above CAC of the amphiphiles in water. The plots are shown in Figure 7.


b

2

a = 0.5 mM SDDPCA b = 9 mM SDDPCA

15 10 a

5

15

b

a = 0.05 mM SDDPCV b = 4 mM SDDPCV

-1

2

20

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B

20

A

 (mdeg. mol cm )

25

-1

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 (mdeg. mol cm )


0.8

10

0.4

5

0.0

a 0

200

250

300

0 200

250 300 Wavelength (nm)

350

200

250 300 Wavelength (nm)

350

Figure 7. Circular dichroism spectra of (A) SDDNAA and (B) SDDNAV in water. At the concentration below CAC the CD spectrum of SDDNAA showed positive bands at 239 nm and 266 nm. But the CD spectrum of SDDNAA above CAC exhibited appearance of two new positive bands at ~217 nm and 284 nm in addition to the other two bands obtained below CAC. This drastic change in the CD spectrum accompanied aggregate formation. The appearance of new bands at above CAC of SDDNAA is indicative formation of chiral structures through aggregation. The appearance of CD peak at 217 nm could be associated with the π→ π ⃰ transition of the amide bond, and the peak at ~284 nm is thought to have originated from the n→ π ⃰ transition. On the other hand, SDDNAV exhibited a new positive band at 286 nm which was absent below CAC. This new band at 286 nm is also attributed to the formation of helical or twisted ribbon type of aggregates above CAC of SDDNAV. XRD Patterns of cast films from the aggregates The thickness of the bilayer membrane of the vesicles can be estimated by XRD experiment. A typical diffraction curve for all the amphiphiles is shown in Figure S12 of SI. All the amphiphiles exhibited periodic peaks in the X-ray scattering plots. The wall thickness (d) can be


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calculated from the intensity corresponding to the peak of lowest 2θ value using Bragg equation (d = nλ/2sinθ, where d is the distance between scattering planes, n is an integer, λ is wavelength, and θ is the angle of incidence to the scattering planes). The obtained 2θ, corresponding planes and calculated ‘d’ values are listed in Table 2. Table 2. XRD parameters of the cast films of the amphiphiles. Amphiphiles 2θ 2.57 SDDNAG 5.07 7.69 2.20 SDDNAA 4.42 6.60 8.78 1.51 SDDNAV 3.11 5.15 6.37 7.72 8.93 9.97

d (Å) 34.48 17.48 11.53 40.31 20.07 13.44 10.11 58.74 28.52 17.22 13.93 11.49 9.94 8.90

(hkl) (001) (002) (003) (001) (002) (003) (004) (001) (002) (003) (004) (005) (006) (007)

lc (Å) 20.66 20.72

20.74

The wall thickness of SDDNAG and SDDNAA is slightly lower than two times of the hydrophobic chain length (lc, obtained from the energy-minimized structure via the Density Functional Theory by Gaussian 9.0) which indicates that the hydrocarbon chains of these two amphiphiles assemble into vesicles in interdigitated pattern. On the other hand, larger value of ‘d’, than twice of the lc suggested formation of non interdigited bilayer aggregates for SDDNAV. From the XRD data of all the amphiphiles it is clear that there exists only one type of morphology where the bilayers are untilted. Mercury Sensor One of the major demands of any kind of research work is its application in development of human beings. Fluorescence sensitivity with Hg2+ ion responsive chemosensors offers a



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promising approach for simple and rapid tracking of mercury ions in biological, toxicological, and environmental samples.17,52-57 Herein, we introduced the studied amphiphiles as a selective fluorescent sensor for Hg2+. Cd2+, Co2+, Cu2+, Fe2+, Hg2+, Mn2+, Ni2+, and Zn2+ ions were used to evaluate the sensing property of all the three amphiphiles. The fluorescence spectra were obtained by excitation of the three amphiphile-fluorophores at 268 nm and the emission spectra were recorded in between the range 300 nm to 600 nm. The amphiphiles (C = 5×10-5 M) showed large enhancement in fluorescence intensity only with Hg2+ (1×10-1M) among the various metal ions examined. The representative plot of SDDNAG is shown in Figure 8A. The plots for Hg2+ sensitivity for other two amphiphiles SDDNAA and SDDNAV are shown in Figure S14. Figure 8B shows when the mercury concentration increases from 1×10-9 to 1×10-1 M keeping SDDNAG concentration fixed (5×10-5 M), the intensity of the fluorescence spectra increases. The plots showing variation of concentrations of Hg2+ for other two amphiphiles (SDDNAA and SDDNAV) are given in SI (Figure S13). To ensure whether the three amphiphiles have any sensing property to other metals tested or not, the above experiment was carried out exciting the amphiphile-fluorophores at 224 nm (Figure S15) as the amphiphile fluorophores has two absorption maxima (224 nm and 268 nm; Figure S16). The Figure S15 again suggested that the amphiphiles are able to sense only Hg2+, not the other metals. Therefore, the amphiphiles act as sensor for Hg2+ ions but insensitive to other metal ions examined. However, the amphiphiles were unable to sense the Hg+2 ions above CAC. This is due to the fact that as the chelation to form the Hg-L (amphiphile) complex is occurring through the hetero atom (N) of the amide linkage and imine linkage (discussed later), above CAC the self-assembly formation by intermolecular hydrogen bonding interaction makes the N-atom of the amide linkage unavailable for chelation.


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In order to examine the detection limit of the amphiphiles, fluorescence titration method was adopted as suggested by Liu.58 The detection limit was calculated using the equation 3σbi / m, where σbi is the standard deviation of blank measurements, m is the slope between intensity versus sample concentration (Figure S17). The detection limit measured to be 2.76×10-9 M for SDDNAG, 3.20×10-9 M for SDDNAA and 6.2×10-8 M for SDDNAV. Hence, the detection affinity of the amphiphiles for the Hg2+ decreases in the order SDDNAG > SDDNAA > SDDNAV. This is due to the combined effect of the increased bulkiness of the amino acid side chain and the spatial orientation of the stereogenic centers which affect the extent of chelation to form the Hg-L (amphiphile) complex.

Fluorescence Intensity

100 75

I/I0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 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 48 49 50 51 52 53 54 55 56 57 58 59 60


A 50 25 0 Cu

Cd

Mn

Zn

Co

Ni

Fe

Hg

900

600 2+

Hg

B

300

0 530

540

550

Wavelength (nm)

Figure 8. (A) Fluorescence spectra of SDDNAG (5×10-5 M) with different metal ions (1×10-1 M) in water. (B) Fluorescence spectra of SDDNAG (5×10-5 M) as a function of Hg2+ (10-9-10-1 M) in water. Real Sample Assay To evaluate the applicability of the fluoremetric assay to real samples, various water samples namely environmental water samples (pond water and river water) were tested. As shown in Figure S18, the fluorescence emission spectra (λex = 268 nm) of all the amphiphiles remains



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Page 18 of 31

almost invariable in the presence of different water samples. Furthermore, for the water samples (pond water and river water), the recovery rates are 94.5−98.5% at 2 ppm Hg2+ (Table S1). Therefore, the excellent analytical performance (high sensitivity and selectivity) of the proposed system is competent for monitoring Hg2+ content in environmental water, even with regard to the standard of WHO and the U.S. Environmental Protection Agency (EPA). Binding Sites To gain a better understanding of the fluorescence source of the mercury sensor, 1H NMR study was performed in DMSO-d6 as shown in Figure S19. Upon addition of Hg2+, The –NH proton of amide linkage disappeared and the imine (–HC=N–) proton shifted to up field from 8.85 to 8.79 ppm. This observation strongly confirmed the involvement of heteroatom (N) of amide linkage as well as imine linkage into chelation to form the Hg-L complex in the sensing mechanism. Conclusion In conclusion, the self-assembly property of three nicotinic acid based amphiphiles having different amino acid moiety on the head group was investigated thoroughly.

Tensiometry

measurements suggest that all the amphiphiles are good surfactant and the adsorption as well as aggregation process is most facile for the amphiphile with highest hydrophobicity of the head group. The high rigidity and non polarity of the microenvironments of the self-assemblies was confirmed by steady state fluorescence measurements. TEM micrographs confirmed formation of closed bilayer vesicles in aqueous solutions of the amphiphiles. A decrease in the aggregate size with increase in the hydrophobicity of the head group was observed in DLS and TEM measurements. XRD study indicates formation of interdigited bilayer structure for SDDNAG and SDDNAA and formation of non interdigited lamellar structure for SDDNAV. The applicability of the vesicles was shown by encapsulation and release of water soluble molecules. The


Page 19 of 31

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entrapment capacity decreases in the order SDDNAG>SDDNAA>SDDNAV. All the amphiphiles can detect very low concentration of Hg2+. Fluorescence response suggests that the amphiphiles have practicability for further environmental and biological mecury ions detection. The entrapping and releasing properties of the amphiphiles could be used as potential carriers of pharmaceutical drugs and encapsulants for cosmetic products. The surface activity property of the amphiphiles can also be utilized in surfactant industries. Also the chirality of the surfactants can be exploited in investigation of chiral recognition. Experimental Section Materials.

2,5-Dibromopyridine,

Zinc

Chloride

(ZnCl2),

n-butyllithium,

Tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), N-hydroxysuccinimide (NHS), pyrene, 1,6-diphenyl-1,3,5-hexatriene (DPH), uranyl acetate, Sepharose 4B were purchased from Aldrich Chemicals. N,Nʹ-dicyclohexylcarbodiimide (DCC), 1-bromododecane, glycine, L-Alanine, LValine, riboflavin, methylene blue, Sodium bicarbonate, Sodium sulphate, Sodium dihydrogen phosphate, disodium hydrogen phosphate and Sodium hydroxide were purchased from SRL. All the probe molecules were used after recrystallization from ethanol. The metal salts Copper sulphate (CuSO4, 5H2O), Ferrous chloride (FeCl2), Cadmium chloride (CdCl2), Manganese chloride (MnCl2), Zinc acetate (Zn(OAc)2), Cobalt chloride (CoCl2), Nickel chloride (NiCl2) and Mercuric chloride (HgCl2) were also procured from SRL and were used without further purification. The Organic solvents used were purchased from Merck, of the highest purity commercially available and were purified as required. Triply distilled water was used for aqueous

solution

preparation. The amphiphiles

Sodium

6-dodecylnicotinic glycinate

(SDDNAG), Sodium 6-dodecylnicotinic alaninate (SDDNAA) and Sodium 6-dodecylnicotinic valinate (SDDNAV) were prepared according to the procedure described in our earlier



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Page 20 of 31

publications.59,60 The characterization data (such as melting point, FT-IR, 1H-NMR and LCMass) of the amphiphiles are given in the SI. Analytical Methods. 1H NMR spectra were taken in a Bruker 400 MHz instrument. LC-MS spectra were recorded in a Waters 2996 machine. Shimadzu (model 1601) spectrophotometer was used to obtain the UV-vis absorption spectra. The pH measurement was carried out with a EUTECH INSTRUMENTS pH-tutor (Cyber scan) using a glass electrode. Conductivity measurements were performed with a digital conductivity meter (Systronics, model 304). Tensiometry measurements were performed with a surface tensiometer (manual) using the Du Nüoy ring detachment method. The surface tension (γ) values of the synthesized amphiphiles were measured at different surfactant concentrations at 303 K. From the break point of the plot of γ versus log C critical aggregation concentration (CAC) values were obtained. The size of the aggregates was determined by dynamic light scattering technique using a Malvern Zeta size Instrument (Zetasizer Nano S90, Model No.: ZEN 1690) having Ar+ laser operated at 4 mW at λ = 633 nm having a detection angle of 900. All the surfactant solutions at the concentration of 3 times of their CAC were prepared in triply distilled water. Before each measurement the solution was filtered through sterile and endotoxin free 0.2 μm Whatman syringe filter. The measurements were carried out at scattering angle 90°and at 303 K after equilibration of 10 min. Hitachi F-7000 spectrophotometer optical system equipped with a 150 W Xe lamp was used for taking the all type of fluorescence spectra. For determination of micropolarity as well as CAC value of the amphiphiles, solutions of different concentrations were prepared in triply distilled water keeping pyrene concentration (~2×10-7 M) fixed. The excitation wavelength was kept at 335 nm with a band pass of 1/1. The steady state fluorescence anisotropy was measured in the


Page 21 of 31

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same instrument equipped with a polarization accessory which uses the L-format instrumental configuration. For anisotropy measurement the concentration of the amphiphile was three times of the CAC and the concentration of DPH was 2 µM. The Excitation wavelength of DPH was 350 nm and the emission intensity was recorded at 450 nm with band passes of 2.5 and 5 nm, respectively. The fluorescence anisotropy (r) of the probe molecule was calculated as follows r = (I║ - GI┴)/ (I║ + 2GI┴)

(4)

where I║ and I┴ are the fluorescence intensities polarized parallel ( Ex. and Em. Polarisers at 0°) and perpendicular (Ex. Pol. at 0° and Em. Pol. at 90°) to the excitation light. G is the instrumentation correction factor (G = i90-0 / i90-90). For dye encapsulation study methylene blue was taken as dye molecule. 2 mL of the pre equilibrated (~ 4 hours) stock solution of amphiphile ( 3 times of CAC) having 0.1 mM methylene blue was loaded into a column packed with a pre-equilibrated Sepharose 4B matrix (25 cm height and 1.2 cm diameter) and eluted with triply distilled water. The fraction of 2 mL was collected every time and the absorbance of all the fraction solutions was recorded at 665 nm. The obtained absorbance values of different fractions were plotted against the elution volume. Transmembrane Permeation study was performed using a Hitachi F-7000 spectrophotometer by running the kinetic study. For this measurement excitation wavelength was fixed at 374 nm with a band pass of 5/10. OH− permeation across the bilayer vesicles was obtained by measuring the fluorescence intensity of the amphiphilic solution containing riboflavin at 514 nm upon adjustment of the pH from 6.8 to 10.2. For XRD measurement, a thin film of the aggregate was prepared by placing the aqueous aggregates of each amphiphile on a precleaned glass plate and air dried to afford a thin film of the aggregate on the glass plate. X-ray diffraction of an individual cast film was performed on a



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Page 22 of 31

Pan analytica X′Pert pro X–Ray diffractometer using a Cu target (Cu Kα) and Ni filter at a scanning rate of 0.001/s between 1 to 10, operating at a voltage of 40 kV and current 30 mA. The morphology and structure of the amphiphiles were characterized by transmission electron microscope ((JEOL-JEM 2100, Japan) operating at 200 keV. One drop of the stock solution of the amphiphile was placed on a carbon coated grid of mesh size 300 and stained with 1.5% aqueous uranyl acetate solution. The specimens were dried in a vacuum desicator for 24 hours before mounting in the transmission electron microscope. The

Circular

Dichroism

(CD)

spectra

were

recorded

on

a

Jasco

J-810-150-S

spectropolarimeter using quartz cell of 0.1 cm path length. Average of four scan of each spectrum was taken under the conditions of 1 nm bandwidth, 2-second response time, and 50 nm min-1 scan speeds. Each spectrum was corrected by subtracting the appropriate reference blank. All measurements were carried out at 303 K. To investigate the sensing property of the synthesized amphiphiles towards metal ions, eight different kinds of metal ions Hg2+, Co2+, Mn2+, Cd2+, Cu2+, Ni2+, Zn2+, Fe+2 were tested separately using a fluorometer (Hitachi, Model No. F-7000). The Cu2+ solution was prepared from copper sulphate. The solutions of Hg2+,Co2+, Mn2+, Cd2+, Ni2+ and Fe2+ were prepared from their corresponding chloride salts. Zn2+ was obtained from zinc acetate salt. All the solutions of metal salts were freshly prepared in triply distilled water just before performing the experiment. Acknowledgement We gratefully acknowledge CSIR, New Delhi (grant no. 02(0195)/14/EMR-II) for financial support. A.R. acknowledges CSIR, New Delhi for her fellowship. We would like to acknowledge the University Scientific Instrumentation Centre (USIC), Vidyasagar University, and Indian Institute of Technology, Kharagpur, for providing instrumental facilities.


Page 23 of 31

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Author Information Corresponding author E-mail: [email protected] Notes The authors declare no competing financial interest. Supporting information (SI) available LC-MS and 1H NMR spectra, FT-IR data, conductivity plots, size distribution plots, steady state anisotropy plot, XRD spectra, UV spectra SDDNAG with different metal ions, fluorescence spectra of the amphiphiles in presence of mercury. This material is available free of charge via the Internet at http://pubs.acs.org.

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“For Table of Contents Only” R=H

9 O

2 I X 10 (a.u.)

Page 31 of 31

R

2+

6

-

Hg

+

O Na N H

3

n

O

R = CH3

N

n = 11 0 530

540

 (nm)

550 R = CH(CH3)2


Spontaneous Formation of Vesicles by Self-Assembly of Nicotinyl

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