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Aggregation Induced Fabrication of Fluorescent Organic Nanorings: Selective Bio-Sensing of Cysteine and Application to Molecular Logic Gate Soumya Sundar Mati, Sayantani Chall, and Subhash Chandra Bhattacharya Langmuir, Just Accepted Manuscript • Publication Date (Web): 20 Apr 2015 Downloaded from http://pubs.acs.org on April 21, 2015
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Aggregation Induced Fabrication of Fluorescent Organic Nanorings: Selective Bio-Sensing of Cysteine and Application to Molecular Logic Gate
Soumya Sundar Mati, Sayantani Chall, Subhash Chandra Bhattacharya*
Department of Chemistry, Jadavpur University, Kolkata-700032, India. *e-mail:
[email protected],
[email protected] Phone No: 033 2414 6223 / Fax: 91(033) 24146584
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ABSTRACT Self-aggregation behavior in aqueous medium of four naphthalimide derivatives has shown substitution dependent, unusual, aggregation induced emission enhancement (AIEE) phenomena. Absorption, emission and time resolved study initially indicated the formation of J-type fluorescent organic nanoaggregates (FONs). Simultaneous applications of infrared spectroscopy, theoretical studies and Dynamic Light Scattering (DLS) measurements explored the underlying mechanism of such substitution selective aggregation of a chloro-naphthalimide organic molecule. Furthermore, Transmission Electron Microscopy (TEM) visually confirmed the formation of ring like FONs with average size of 7.5-9.5 nm. Additionally, naphthalimide FONs also exhibited selective and specific cysteine amino acid sensing property. The specific behavior of NPCl aggregation toward amino acids was also employed as molecular logic gate in information technology (IT).
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Introduction Fluorescence organic nanoaggregates (FONs), owing to their smart and stimulating applications particularly in optoelectronic devices, OLEDs (organic light emitting diods), fluorescent biological labels or optical sensors, are now considered as an attractive fluorescent benign in the recent research.1-5 Their unique properties were thought to be originated from quantum-size effects. Moreover, self-assembled organic molecules can lead to some new photophysical properties that are not commonly available to its monomer structure since orientation of monomeric molecules in the self-assembly may have significant role in controlling physical characteristics.6 Nakanishi and his co-workers were known to initiate researches on FONs.5,7 Literature reviews of previous investigations also exposed the shape and photophysical behaviors of solvent dependent aggregation behavior of different naphthalimide derivatives as well as other organic molecules.2-4,8-10 Compared to the previous literature reports, in the present work, we have made an endeavour to establish mechanistically substitution controlled aggregation behaviour of 1,8Naphthalamide derivative. Naphthalamide derivatives are already proven as a promising candidate for organic electronic devices, biosensing, cellular staining etc.11,12 Soni et. al.13 in their work reported the formation of rod like and spherical naphthalamide FONs along with their photophysical characteristics. However, till date, there is lack of sufficient information for understanding proper mechanism of aggregate formation from the molecular viewpoint. From this consideration, the main focus of the work is to study mechanistically, how a chlorosubstituted naphthalamide (NPCl) monomers (Abbreviations in Scheme 1) is engaged in electrostatic interaction to build up ring type nanostructures via J-type stacking. In principle, on
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aggregation the band placed at higher energies is defined as the H-band, while that at lower energies is defined as the J-band. For H-aggregates, the molecules are in edge-face orientation, while in J-aggregate they are in line.14,15 Whitten et. al. reported different structural basis for aggregation mode of oligo-p-Phenylene Ethynylenes.15,16 We herein report the circular J aggregation of organic molecules (here, NPCl) following the Kasha's rule17 of angle restriction. In accordance with steady state and time resolved studies, infra red and Zeta potential measurements established the possibility of NPCl aggregation. Additionally, DFT calculation, dynamic light scattering (DLS) and transmission electron microscopy (TEM) analysis were also carried out to confirm formation of molecular J aggregation with ultimate, stable ring type morphology. Furthermore, aggregated NPCl FONs are able to specific cysteine (amino acid) sensing thereby demonstrating themselves as noteworthy in the field of bio-sensing applications as amino acids have drawn much attention because of their many physiological importances.18 Eventually amino acids detection by NPCl aggregation via OFF-ON fluorescence property shows NOR molecular logic gate behavior which can be miniaturized in information technology.
Materials and Methods Material: Naphthalimide derivatives, UnNP, NPNH2, Br-NPCl and NPCl (Scheme 1) were synthesized using described methods elsewhere19,20 and they were purified by column chromatography. The compounds were re-crystallized using ethyl acetate-pet ether (1:1) before use. The spectral grade solvents ethanol (EtOH), methanol (MeOH), acetonitrile (ACN), ethylene glycol (EG), were purchased from E. Merck, India. Amino acids and N-[2hydroxyethyl] piperazine-N-[2-ethanesulphonic acid] (HEPES) buffer were obtained from SRL, India. D2O was purchesed from Cambridge isotope laboratory (CIL). Spectroscopic grade 1,44 ACS Paragon Plus Environment
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dioxane (SRL, India) were used as received. Millipore water was used throughout the experiment. The stock solution of the compounds (1×10-3 M) was prepared in 1:1 dioxan-water solvent mixture.
Scheme 1. Structure of the four naphthalimide derivatives of our investigation.
Methods: A Shimadzu (model UV1700) UV-VIS spectrophotometer and a Shimadzu spectrofluorimeter (model RF 5301) were used to collect absorption and fluorescence spectra, respectively Fluorescence lifetimes were determined from time-resolved intensity decay by the 5 ACS Paragon Plus Environment
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method of time correlated single-photon counting using a nanosecond diode LED at 370 nm (IBH, nanoLED-03) as light source. The data stored in a multichannel analyzer were routinely transferred to IBH DAS-6 decay analysis software. For all the lifetime measurements, the fluorescence decay curves were analyzed by single and bi-exponential iterative fitting program provided by IBH as in Eq. 121
F(t) = ∑i ai exp (−t/τi )
(1)
Where ai is the pre exponential factor representing the fractional contribution to the time resolved decay of the component with lifetime τi. . All measurements were done repeatedly and reproducible results were obtained. All fluorescence spectra were corrected with respect to instrumental response. The FTIR spectrum of the powdered sample (using KBr pellets of the sample at a KBr–sample ratio of 100:1) was recorded with Perkin Elmer, Spectrum RXI equipment with a resolution of 2 cm-1. The spectrum was recorded from 400 cm-1 to 3000 cm-1. The surface pressure (π)–area (A) isotherm of NPCl at 298±1K was measured in a Teflon-barbarrier type surface balance (Langmuir Blodgett type, model 2004C, Apex Inst. Co., India). Size and Zeta potential of aggregation were measured by using Dynamic Light Scattering (DLS) instrument (Model: ZS 90, Malvern). The morphology of NPCL was examined by transmission electron microscopy (Model: JEOL-JEM-2100) at an accelerating voltage of 200 kV. The concentration of NPCl was maintained at 15 µM and 50 µM and these solutions were directly employed during the grid preparation for TEM analysis. Ground state geometries were optimized employing Density Functional Theory22,23 using the B3LYP24,25 functional with the standard basis set, 6-31G(d,p), for all atoms in Gaussian 03 program26.
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Result and Discussion Analysis of Aggregation formation from Spectroscopic viewpoint: In addition to spectral characteristics, photophysical behaviors of three naphthalimide derivatives i.e., NPCl, NPNH2 and UnNP, are also governed by the polarity of the medium.27,28 In aqueous solutions absorption spectra of three naphthalimide derivatives (0.2 µM to ~ 50µM) are characterized by broad intense absorption, in the range 300–500 nm (Figure 1). Examination of Figure 1 reveals that with increasing concentration, the absorption of the two derivatives, NPNH2 and UnNP are very regular in enhancement. In comparison, the anomaly in spectral behavior was
Figure 1. Absorption spectra of (a) UnNP (b) NPNH2 and (c) NPCl in the concentration range 0.2 to 50 × 10-6 M in aqueous medium. noticed for NPCl. It has been observed that when NPCl concentration was below 10µM, a broad absorption peak persisted with the maxima centered at 354 nm. However, with increasing concentration of NPCl, another peak was found to develop at 377 nm because of aggregation of NPCl. Even more, enhanced intensity of the new peak at 377 nm was also noticed compared to the peak at 354 nm caused by monomer (Figure 2a). As dye aggregates are usually classified on the basis of the observed spectral shift of the absorption maximum relative to the respective absorption maximum of the monomer,13,29 consequently lower energy i.e. bathochromic absorption maxima (here the peak at 377 nm), relative to the monomer band (354 nm) corresponds to J-type aggregates. 7 ACS Paragon Plus Environment
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Figure 2. In aqueous medium [NPCl] was varied to monitor the (a) absorption spectra (0.2 to 50 µM); Inset: Absorption peak ratio OD373 /OD354 (b) emission spectra (0.2 to 48 µM) (λex=350 nm) (c) lifetime and (d) DLS measurement.
The changes in fluorescence spectra of NPNH2 and UnNP in the aqueous concentration range from 0.2 to ~50 µM are shown in Figure S1. In comparison to absorption spectra the apparent fluorescence spectral changes of NPCl was also observed above 10 µM (Figure 2b), primarily from the monomer spectra (below 10 µM). The regular enhancement of fluorescence intensities of NPNH2 and UnNP with increasing concentration in different non-aqueous solvents is evidenced to their non-aggregated states. At low concentrations (upto 10 µM), the fluorescence emission spectra of NPCl in aqueous medium showed a well-resolved structure at 410 nm, which
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is characteristic of the monomer fluorescence of NPCl. Above this concentration, a new broad red-shifted emission band is developed from wavelength 438 to 449 nm and become stronger upon aggregation. The spectral property of the monomer NPCl is little influenced by the other bulk solvent environments (Figure S2) as observed in water. Hence only in aqueous medium there occurs a switch over from monomer to aggregate above concentration of ̴ 10 µM. The pronounced spectral changes with increasing concentration in solvents provide clear evidence for the aggregation indicating strong electronic interactions between the aggregated chromophores.30 The interesting fluorescence behavior of NPCl can be well monitored by measuring the lifetime. As there is a tendency of monomer to aggregate at about ̴ 10 µM, so in lifetime value of NPCl a reflection should be noticed. On account of the single lifetime value at concentration 3 µm (0.90 ns, λems= 400 nm) evinces the existence of only monomer species of NPCl at that concentration. Furthermore, when the concentration was above 10 µm (λems= 460 nm) the bi-exponential decay can be explained due to the existence of both monomer and aggregated form of NPCl (Figure 2c). As expected for aggregation, the emission lifetime is increased with respect to the monomer; hence the formation of aggregate enhanced the lifetime from 0.90 ns for monomer to 6.33 ns to aggregated NPCl (Table 1). A closer look to Table 1, indicates that on increasing the probe concentration, percentage composition of monomer decreases as well as that of the aggregated species increases.
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Table 1. Lifetime of NPCl in aqueous medium at different concentration.
Conc. of NPCl
τ 1 / ns (a1)
τ 2 / ns (a2)
χ2
3 µM
0.90 (100)
-
1.00
10 µM
0.90 (41)
6.15 (59)
1.02
15 µM
1.03 (32)
6.28 (68)
1.05
25 µM
1.08 (24)
6.36 (76)
1.11
35 µM
1.10 (22)
6.35 (78)
1.07
For the aggregated NPCl in H2O drastic enhancement of absorption and emission intensity was noted with additional features compared to monomer emission. In solvents other than water, no such solvent-dependent change in the spectral property was observed for the control molecule NPCl under identical conditions (Figure S2) clearly suggesting specific aggregation in case of aqueous solution only. Interestingly, the pH of the medium has a significant contribution in the aggregation behavior of NPCl such that in between pH 1 to pH 10 the effect is very similar but at higher pH (≥12) the aggregation does not occur at all. So there is an optimum concentration of OH- responsible for the formation or annihilation of aggregation. When NPCl aggregate was titrated by adding NaOH, monomeric NPCl was formed. From Figure 3a-d, it can be clearly observed that this conversion of aggregated NPCl to NPCl monomer was occurred at and above optimum pH 11. This information regarding the destruction of aggregation in presence of optimum basic concentration leads to a competition between the aggregation stability and base concentration. As J aggregate is the result of electrostatic interaction,31 the repulsive interaction between the negatively charged aggregated NPCl moiety and OH- predominates above pH=11.
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Figure 3. Effect of pH on aggregation: Formation of monomer from aggregation with increase in pH (a) fluorescence intensity enhancement ratio of monomer (monomer/aggrgation) (b) absorption (c) emission (d) no aggregation at pH 13.
The aggregation of a molecule leads to a strong coupling of the molecular transition dipoles causing the splitting of energy levels defining the band placed at higher energies as the H-band, while that at lower energies as the J-band.31 According to Kasha's theory,14 J- or H-aggregates can be formed depending on the angle (φ) between the longitudinal axis of the molecules (monomer transition moments) and the adsorption surface (Scheme 2). The in-plane oblique angle configuration exists when φ >54.7º, H-aggregates are formed and when φ
