Supramolecular-Assisted RNA-Templated Fluorescing Colloidal CdSe

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C: Physical Processes in Nanomaterials and Nanostructures

Supramolecular Assisted RNA-Templated Fluorescing Colloidal CdSe QDs Organized in Porous Morphology in the Presence of 1,3-Diaminopropane – Study of Their Multifunctional Behavior Anil Kumar, and Komal Gupta J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b10863 • Publication Date (Web): 07 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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The Journal of Physical Chemistry

Supramolecular Assisted RNA-Templated Fluorescing Colloidal CdSe QDs Organized in Porous Morphology in the Presence of 1,3Diaminopropane – Study of their Multifunctional Behavior Anil Kumar* and Komal Gupta Department of Chemistry Indian Institute of Technology Roorkee, Roorkee - 247667, India *

E-mail: [email protected]; Tel.. +91-1332-285799; Fax. +91-1332-273560

ABSTRACT

The synthesis of colloidal CdSe nanostructures mediated by RNA in the presence of 1,3diaminopropane (DAP) and excess Cd2+ produces CdSe QDs (av. dia 4.5 nm) forming a chain

ACS Paragon Plus Environment

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like morphology (SP1-DAP) with fairly high fluorescence efficiency and average lifetime (): 36 % and 121 ns (λflu,sh=650 nm), respectively. The addition of DAP induces its transformation to porous morphology in the process of self-assembly (SP1A-DAP) as evidenced by AFM and TEM analyses, and increase in rotational time constant from 14.7 to 31.5 ns. These changes are associated with a blue shift in emission maxima from 545 to 530 nm and an increase in from 102 to 111 ns (λmax,flu=545 nm). For SP1A-DAP, the development of distinct bands (cm-1) in IR analysis (1240 (s) and 1183 (sh)); blue shift in positive peak at 271.1 nm in CD spectra, and changes in 1H and 31P NMR spectra indicating RNA configuration change from C2'-endo to C3'endo, clearly demonstrating the interaction of excess Cd2+ on RNA strand through –NH2 in process of self-assembly lead to a change in configuration from B to A form. These nanostructures could selectively detect Hg2+ ions at ≥ 0.5 nM, as was followed by quenching of fluorescence, obeying both dynamic (kq=1.5×1012 mol-1 dm3 s-1) and static mechanisms. Hg2+ ions get bound to SP1-DAP (Ka = 1.4 ×108 mol-1 dm3) and SP1A-DAP (Ka = 6.8 ×107 mol-1 dm3) involving -NH2. I–V studies of the drop casted film of SP1-DAP and SP1A-DAP exhibited the rectifying behavior from which ILight/IDark have been computed to be 10 and 12.5, respectively under forward bias condition. Interestingly, the synthesis of RNA mediated CdSe nanohybrids in the presence of DAP produces fluorescing porous nanostructures displaying novel electronic and electrical behavior, suggesting their future technological potential for fluorescence imaging, sensing and optoelectronic devices.

Introduction During the past two decades quantized CdSe QDs have been extensively investigated for their novel optical, fluorescing and electrical properties, which have found tremendous usage for multidisciplinary applications.1-5 In a number of previous reports, the properties of the CdSe QDs


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have been modified by changing their dimensionalities to 1D (nanowires, nanorods, nanoneedles, nanotubes), 2D (nanosheets, nanodiscs) and 3D superstructures.6-10 In recent years porous nanomaterials are being synthesized and explored for their advanced scientific and technological importance such as in solar energy conversion and storage devices, catalysis, drug delivery, adsorption and sensing of hazardous metal ions and biological molecules.11-17 Several synthetic strategies involving hard and soft templates have been adopted to achieve this objective.18,19 But the soft template assisted synthesis is becoming more popular as it could provide a convenient tool to control the porosity by manipulating specific interactions between template and inorganic(s). In this context biomolecules/biopolymers could serve as an effective capping agent for CdSe NPs because of their specific structural characteristics and large functionalities, which might be exploited for interaction with NPs through non-covalent bonds.2022

In our previous study we have succeeded in stabilizing CdSe NPs by using torula yeast RNA

as a template, which produced biotemplated fluorescing QDs (SP1) and could be converted into nanoneedles in the process of self-assembly.23 These particles demonstrated a fluorescence quantum efficiency (ɸfl) of 22%. In biological systems, biopolymers along with polyamines are among important constituent(s) in living organism. Polyamines, being present in relatively high concentration (mM), have been suggested to involve polyamines – RNA complex24 in wide ranging biological processes, and are known to play a critical role in the cell cycles for their development by interacting with different functionalities of nucleic acids through PO2- anion and nucleotides.25,26 As such the interaction of CdSe QDs, synthesized using trioctylphosphine oxide (TOPO) in non-aqueous medium, has been investigated earlier extensively in the excited state with several


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amines,27-33 in which some interesting observations about their complex interactions with amine(s) have been reported following fluorescence due to these QDs. In view of the enormous significance of polyamine(s) in biological processes, in the present work we have synthesized RNA-mediated CdSe NPs in the presence of polyamine, namely 1,3diaminopropane (DAP) in aqueous medium. Its interaction with CdSe in the ground as well as excited state has been analyzed. The presence of 1,3-diaminopropane induces supramolecular interactions through Cd2+ ions present in CdSe as well as free Cd2+ ions bound to RNA strand, resulting in its folding associated with a change in its configuration from B to A form, and produces a chain like structure of fluorescing CdSe QDs with a fairly high ɸfl of 0.36 ± 0.02. Self-assembly of these NPs increased the crystallinity and creates fluorescing honeycomb like porous network of building blocks. An understanding of such system might reveal important mechanistic aspects involved in the biological process(es)24 and, specifically in biosynthesis of CdSe nanohybrids.34, 35 These nanohybrids displayed enhanced multifunctional properties like optical, fluorescence, electronic and electrical having vast potential for making devices. Their fluorescing behavior has been explored for Hg2+ sensing in the presence of interfering ions like Pb2+, Mn2+, Co2+, Ni2+ at 5µM of each. Experimental Section 2.1 Reagents. Ribonucleic acid (RNA) derived from Torula yeast type VI, cadmium perchlorate, mercuric acetate, Se powder and fluorescein dye (Sigma-Aldrich); sodium hydroxide (BDH); perchloric acid (Qualigens); nitrogen gas (Grade 1, purity >99.99%) (Sigma, India); 1,3-diaminopropane (Avra); manganese acetate (Himedia); sodium borohydride, nitrobenzene (Merck); cobalt


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acetate, nickel acetate (Thomas baker). All chemicals used were of analytical grade and were not purified any further. RNA used was a heterogeneous mixture of RNA molecules of varied molecular weight(s) and length(s) without any particular sequence. Freshly prepared millipore water, having the conductivity of 18.2 MΩ at 25 °C, was used for preparing the solutions. 2.2 Equipment Optical absorption spectra and steady state fluorescence in the UV-visible range (200–800 nm) were recorded on a Shimadzu UV2100S and FluoromaxR-4 Spectrofluorometer, respectively. Zeta potential measurements were performed on a Malvern Zetasizer Nano ZS90. X-Ray diffraction patterns were analyzed on a Bruker AXS D8 Advance X-ray diffractometer (XRD) using the Cu-Kα line (1.5418 A˚) of the X-ray source. X-ray photoelectron spectroscopy (XPS) measurements were made on a PHI 5000 Versa Probe II supplied by ULVAC-PHI Inc., Japan equipped with a monochromatic Al Kα 945 radiation source. Different morphologies were analyzed on Carl Zeiss ultra plus field emission scanning electron microscopes (FE-SEM). The surface topography and the depth profile in porous structure were examined by an atomic force microscope (AFM) supplied by NTEGRE (NT-MDT). Transmission electron micrographs (TEM), high resolution transmission electron micrographs (HRTEM), selected area electron diffraction (SAED) and EDS measurements were made on a FEI-Tecnai G2 20 S-TWIN equipped with a CCD camera. A Thermo Nicolet Nexus Fourier transform infrared (FTIR) spectrophotometer was used for recording the spectra in the mid IR range (4000–400 cm-1). Bruker Advance (500 MHz) and JEOL 400 MHz NMR (Model ECX 400 II) spectrometers were used for recording of 1H and

31

P NMR spectra, respectively. Circular dichroism (CD) spectra

were recorded on a Chirascan spectropolarimeter supplied by M/s Applied Photophysics. Current–voltage (I–V) experiments were performed on I–V Test system equipped with Solar


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Simulator Mode II CT50AAA from Photo Emission Tech. Inc., USA and I-V –Tracer auxillary unit from Keithley. The photostablity of as synthesized sample was examined on an AM1.5G Solar Simulator (one sun intensity level) equipped with an optical cut filter (330 nm). Fluorescence lifetimes, time resolved emission spectra (TRES), and time resolved anisotropy measurements were carried out on a Horiba Jobin Yvon “FluoroCube Fluorescence Lifetime System” using NanoLEDs and LDs as excitation source and TBX-PS detector. Steady state anisotropic measurements were made on an Edinburgh FLS-980 spectrofluorophotometer equipped with excitation and emission polarizers.

2.3 Synthesis RNA mediated CdSe QDs were synthesized using a previously reported method.18 The solution of cadmium perchlorate (100 µL of 0.1 mol dm-3) was added to the deaerated solution of RNA (0.015g/100 mL) followed by the addition of 265 µL of freshly prepared HSe- (38 × 10-3 mol dm3

), prepared by reacting NaBH4 (0.02 g) with Se powder (0.015 g) in 5 ml water, under nitrogen

environment at 4°C under vigorous stirring. The pH at each step was maintained at 9.2 using dil. NaOH and perchloric acid. The resulting yellow solution was purged with nitrogen strongly for about 10 min followed by the drop-wise addition of excess Cd2+ solution (2 ×10−4 mol dm−3). Thereafter, the varied amount of 1,3-diaminopropane (DAP) ((0.5−2.0) × 10−3 mol dm−3) were added and then the pH was adjusted to 9.2. In the presence of DAP, once the pH is adjusted at 9.2, thereafter it remained fairly constant. Even the aging of the as synthesized nanohybrids did not show any appreciable change in pH. The resulting solution was further purged with N2 for about 10 min. As synthesized nanoparticles were fairly photostable as was examined by their illumination under Solar Simulator equipped with optical cut filter (330 nm).


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Diamines are known to catalyze hydrolysis of RNA under stringent condition of 1 mol dm-3 of DAP, at 50 ᴼC and pH 8.36 However, in a control experiment under the used experimental conditions ([DAP] = 1×10-3 mol dm-3; pH = 9.2 at room temperature), we did not observed any hydrolysis as was measured by following the absorption due to RNA in the entire spectral range of 200 nm to 350 nm. 2.4 Methodology The colloidal CdSe samples were used as such for recording optical absorption, steady-state fluorescence, zeta potential, fluorescence lifetime, CD, and anisotropy. For the determination of quantum yield (QY) of fluorescence, the fluorescein dye (QY = 0.90) was used as a reference. The solid samples were obtained by removing water on a rotatory evaporator at 40°C and were employed for recording XRD, XPS and IR spectra. Samples for FESEM and AFM analyses were prepared by applying them on a glass slide, which were dried overnight in dark in a desiccator. In these analyses the sample size were kept at about 10 µL, taken from the three times diluted as synthesized sample(s). AFM images (2D and 3D) were recorded in the semi-contact mode. For these images, the average surface roughness (surface roughness distribution) and for porous structure the depth profile were analyzed using NOVA software. The FE-SEM images were recorded by applying an acceleration voltage of 15 - 20 kV. Samples for TEM analysis were prepared by applying 3- times diluted colloidal sample on a carbon-coated copper grid 200 mesh. The excess of solution was removed with the help of a tissue paper and the coated grid was then dried overnight in dark. Electron micrographs of these samples were recorded at different magnifications. ImageJ software was used for calculating the d spacing in HRTEM images, diameter of different rings in the SAED patterns, analysis of the pore size and performing the 3D interactive surface plotting of TEM images. Indexing of selected area electron diffraction


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patterns was carried out using ratio method, and Miller indices/ different planes were then assigned corresponding to different rings. The IR spectra were recorded in the mid IR range of 4000–400 cm-1 in KBr medium and OMNIC v6.1 software was used for the data analysis. For NMR spectroscopy, mixture of H2O and D2O (9:1) was used for making the deuterated samples and the measurements were done using BBO probe. MestReNova software was used for analyzing 1H and 31P NMR spectra. For I-V measurements, a drop of concentrated liquid sample was applied on a transparent conducting indium tin oxide substrate and then dried overnight in desiccator at room temperature to form a thin film, on this, silver paste was applied for making the electrical contacts. The decay curves obtained by fluorescence lifetime measurements were analyzed by iterative reconvolution technique using multi-exponential fitting program provided from IBH. The fitting of these curves was carried out by DAS 6.3 software. The goodness-of-fit was determined by observing the value of χ2 and then the average lifetime () was calculated from different fluorescence lifetime components.23 Surface area was calculated by carrying out the adsorption studies in which the dye molecule (Rhodamine B) of known radii was selected for adsorption on their surface. The extent of adsorption for SP1-DAP and SP1A-DAP was measured spectrophotometrically by recording the change in absorbance. In both the cases, it follows the Type IV adsorption isotherm behavior. However, in the initial concentration range of dye (1×10-7 to 2×10-6 mol dm-3), it followed Langmuir adsorption isotherm. From these curves, the adsorption capacity, binding constant and surface area has been estimated (Figure S1, Table S1). The adsorption experiments and sensing measurements were designed at pH 9.2 using borate buffer for maintaining the pH throughout.


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3. Results 3.1 Optical Study CdSe QDs in the presence of DAP were optimized by performing their absorption and fluorescence measurements in the presence of varied concentration of (0.5 × 10-4 to 2 × 10-3 mol dm-3) and pH (10.5 – 7.0). An increase in the concentration of DAP does not show any significant change in the absorbance. These particles exhibited a broad excitonic peak at 430 nm. The excitation of these particles by 430 nm (2.88 eV) showed the broad fluorescence band having fluorescence maximum at 545 nm (2.27 eV) with a weak shoulder at 640 nm (1.94 eV) (Figure 1a). The emission due to these though showed a regular increase in intensity up to 1× 103

mol dm-3 DAP, thereafter it starts showing a decrease in intensity at higher concentrations of

DAP, i.e. beyond 1.5 × 10-3 mol dm-3 (Figure 1b; Figure S2a and S2b). A very similar variation was observed in fluorescence lifetime measurements due to these, from which the average lifetime () was found to increase with an increase in [DAP] up to 1×10-3 mol dm-3, thereafter a slight decrease was observed (Figure 1b; Figure S3a and S3b, ; Tables S2a and S2b). The pH of the CdSe solution containing 1×10-3 mol dm-3 of DAP was optimized by varying the initial pH from 10.5 to 7.0. A decrease in the pH from 10.5 up to 7.0 did not show any change in the absorption spectrum but the fluorescence intensity was observed to increase up to pH 9.2, thereafter it started showing a decrease in intensity without exhibiting any shift in fluorescence band (Figure S2c and S2d). Thus the optimized sample as regard to the highest fluorescing intensity contained 0.015 g/100 ml of RNA, 1×10-3 mol dm-3 DAP and a pH of 9.2, and this sample has been abbreviated as SP1-DAP. It exhibited an absorption onset at around 580 nm and a broad absorption band at 430 nm, which has been assigned as the excitonic absorption. The fluorescence spectra exhibited λmax,flu at 545 nm with a shoulder at 640 nm (Figure 1a). The


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quantum efficiency corresponding to this band was estimated to be 0.36 ± 0.02 using fluorescein as reference dye (ϕflu = 0.90). The optimized particles were further characterized by lifetime measurements. The lifetime decay curves, recorded at: 545 nm (λflu,max) and at 650 nm (λflu,sh) are shown in Figure 1c, both the decay curves were found to best fit in three-exponential kinetic program, from which the average lifetime () were calculated to be 102 ns and 121 ns (Table S2a and S2b) respectively. The aging of these particles (SP1A-DAP) show a significant blue shift in the fluorescence band to 530 nm (λflu,max) and 635 nm (λflu,sh) (Figure 1a ) associated with an increase in lifetime to 111 ns and 126 ns at 545 nm and 650 nm respectively (Figure 1c and Table S2a and S2b).

Figure 1. (a) Absorption and fluorescence spectra of SP1-DAP and SP1A-DAP. (b) I/I0 and / vs. [DAP]. (c) Fluorescence lifetime decay curves of SP1-DAP and SP1A-DAP at different emission wavelengths. From the lifetime data recorded at 650 nm, the depth of the traps for SP1-DAP has been estimated to be 367 meV, which is slightly higher for the SP1A-DAP (370 meV).


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It may be mentioned that in case of DAP, the enhancement in fluorescence was more prominent as compared to those of other amines such as ethylenediamine (ED) and 1,4- diaminobutane (DAB). The enhancement and reactivity of diamines appears to be controlled by their pKa values (pKa1 and pKa2: 6.9 and 9.9 (ED); 8.9 and 10.6 (DAP); and 9.6 and 10.8 (DAB), respectively)37 and redox potential(s). Among the used amines, it is mainly the basic species of these amine(s) and that too corresponding to the pKa1 exhibits the highest surface passivation. At higher pH the used diamine(s) might lead to strong chelation with the surface of CdSe,38 which might be reducing the fluorescence intensity. Thus, DAB having the highest pKa will have its more fraction in the protonated form at the used pH of 9.2, and showed the least enhancement. In case of DAP a change in pH brings a complex change in the surface stability of nanostructures as well as the speciation of amine.39 Both SP1-DAP and SP1A-DAP show the highest stability at pH 9.2 having the zeta potential value of about -33 ± 1 mV. A change in pH to lower (7.5) and higher (10.5) values exhibited a significantly lower zeta potential values of -12 mV and -14 mV, respectively, suggesting these nanostructures to be better stabilized at pH 9.2. 3.2 XRD Analysis The XRD patterns of SP1-DAP and SP1A-DAP are presented in Figure 2. The XRD Pattern for SP1-DAP exhibits two fairly broad peaks at 25.12 and 44.60 masked by some sharp as well as weak reflections. The reflections at 23.65, 24.73, 26.03 and 44.60 matched with the reflections corresponding to CdSe in the wurtzite phase (JCPDS File no. 77-2307). The XRD Pattern for SP1A-DAP again shows relatively better resolved reflections at: 23.82, 24.94, 25.94, 34.25 and 45.19 again matching to CdSe in the wurtzite phase. Besides these, some other reflections have been

assigned

to

Cd(OH)2/Cd2+

(JCPDS

file

monoclinic/hexagonal phase for both SP1-DAP and

no.

020-0179,

SP1A-DAP.

14-0024)

in

the

A comparison of XRD


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patterns due to SP1-DAP and SP1A-DAP shows that the peaks due to SP1A-DAP are relatively more intense and better resolved. Specifically, the ratio of the peaks corresponding to (100) and (002) plane, was found to be 3.55, which is only 1.12 for the bulk sample (JCPDS File no. 772307) having the same phase. It clearly shows that there is a significant growth of these nanostructure along (100) plane upon aging in the presence of DAP.

Figure 2. XRD patterns of SP1-DAP and SP1A-DAP. 3.3 XPS analysis The surface of SP1-DAP was further analyzed to gather the knowledge about the elements and their environment in the as synthesized nanohybrids by performing XPS (Figure 3). The survey scan spectrum of this sample was recorded in the energy range of 0 to 800 eV (Panel 3a). The high resolution spectra (HRS) corresponding to various elements were also analyzed in different energy range and are presented in panels b to g. Panel 3b shows the HRS due to Cd, it exhibited two peaks at the binding energies (eV) of: 404.2 and 411.0 with the spin orbital coupling of 6.8 eV matching very well with the literature data40 and have been assigned to its energy states 3d5/2 and 3d3/2. Panel 3c shows the appearance of a broad peak, the deconvolution of which showed the presence of two peaks at 52.6 eV and 53.9 eV corresponding to the Se 3d5/2 and 3d3/2 energy


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states respectively due to Se2- species.41 The peaks observed due to other elements in various narrow energy scan range presented in different panels gives different components upon deconvolution at binding energies (eV) (corresponding to functional group): C1s (Panel 3d) 282.7 (C=C graphitic C), 283.35 (C-C), 284.8 ((C-N/ CH2 contributed by DAP) and 286.6 (C=O)/C-O/C=N)42; N1s (Panel 3e) 397.7 (pyridinic) and 398.95 (pyrrolic) and 400.5 (H-bonded -NH2 of DAP);43 O1s (Panel 3f) - 531.27 (C-O/ metal hydroxides) and 534.90 eV (C=O /adsorbed water),40 and P2p (Panel 3g) - 132.2 (P2p1/2), 133.1 (P2p3/2) and 136.727 (contributed by the electrostatic interaction between PO22- with Cd2+).44 The C1s, N1s and O1s spectra show significant changes as compared to the SP1,23 which have been considered to arise due to interactions of CdSe nanostructures with DAP and has been discussed vide infra.


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o1s

30000

15000

Se 3d

10000

C1s

Cd3p3/2

Counts

20000

SP1-DAP Cd 3d5/2, Cd3d3/2

a

25000

5000 0 800

700

600 500 400 300 200 Binding Energy (eV)

100

0

110

1000

1200

52.6 (3d5/2)

53.9 (3d3/2)

100

Counts

105 411 eV (Cd 3d3/2 )

1200

1000 800

405

410 Binding Energy (eV)

415

51

52

53 54 55 Binding Energy (eV)

56

280

57

4500 398.95 eV Pyrrolic N

N1s

4000

O1s

f

3500

800 397.7 eV Pyridinic N

700

Counts

400.49 eV -NH2

900

3000 2500

534.90 C=O

398 399 400 Binding Energy (eV)

401

402

1000 530


290

P2p

g 132.2 eV 2P1/2

250 133.1 eV 2P3/2

200

1500 397


300

2000

600

350

531.27 C-O

Counts

e

1000

500 396

286.58 eV C-O/C=O/C=N

282.7eV C=C

400

600

1100

284.75 eV (CH2)n/ C-N

283.43 eV C-C

600

95

800

1200

C1s

d 1400

404.2 eV (Cd 3d5/2 )

1400 Counts

1600

Se 3d

c

Cd (3d5)

b

Counts

1600

Counts

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

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137.1 eV

150 132


138

Figure 3. XPS survey scans of: SP1-DAP (a); high resolution spectra: Cd3d (b) Se3d (c); C1s (d) N1s ( e) O1s (f) and P2p (g). 3.4 AFM Analysis SP1-DAP shows mainly the organized chains of spherical particles with average surface roughness (roughness distribution) of 6.7 nm (2-13 nm) (Figure 4, Panel a, b, c), which are folded partially at some locations. The aging of these particles generated porous honeycomb like nanostructures with an average surface roughness (roughness


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distribution) of 6.0 nm (2-13 nm) (Figure 4, Panel, a', b', c'). The pore size of these nanostructures was estimated to be 3.5 ±1 nm (Figure 4, Panel d').

Figure 4. 2D, 3D and roughness histograms for SP1-DAP (a, b and c) and SP1A-DAP (a',b' and c'), depth profile of SP1A-DAP (d'). 3.5 TEM analysis TEM, HRTEM and SAED analyses for SP1-DAP and SP1A-DAP are shown in Figure 5. TEM image of SP1-DAP shows the presence of organized particles, which are partially folded at places (Figure 5, Panel a). The average size (size distribution) of these particles has been


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estimated from their size histogram (Figure S4a) to be 4.5 nm (3 - 6.5 nm). Whereas, TEM image for SP1A-DAP (Figure 5, Panel a') shows the porous morphology, the porosity has also been examined by its 3D image (Figure S4b). The SAED analyses for both of the TEM images corresponding to SP1-DAP and SP1A-DAP (given in Panels a and a') exhibit the concentric ring patterns masked with relatively light and bright spots, respectively (Figure 5, Panel b and b'). The indexing of each of these patterns corresponded to (100), (101), (002), (102), (103) and (110) planes matching with CdSe having wurtzite structure (JCPDS file No. 77-2307). The appearance of brighter spots due to SP1A-DAP as compared to SP1-DAP is considered to possibly arise due to the increased crystallinity. The HRTEM images for SP1-DAP and SP1ADAP show the lattice fringes, from which the ‘d’ spacing value(s) has been found to be 0.374 ± 0.003 nm, which correspond to the (100) plane of wurtzite CdSe in line with XRD data. EDS analysis for these nanostructures shows the presence of elements Cd and Se along with the C, N, P, O (contributed by RNA) (Figures S5a and S5b), matching to those of XPS analysis.


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Figure 5. TEM images of SP1-DAP and SP1A-DAP (a and a'), SAED patterns (b and b') and HRTEM images (c and c') respectively. 3.6 FTIR Analysis The FTIR spectra of SP1-DAP and SP1A-DAP were monitored in mid IR range from 4000 – 400 cm-1 (Figure 6). The spectrum for SP1-DAP exhibited different IR bands (cm-1) at: 1649, 1600, 1488, 1415, 1392, 1338, 1238, 1147, 1087, 979 and 825 and have been assigned to A&C, G (C=N ring vib of G), U, In plane C2′-OH, purine in anti-conform., purine in syn conform., PO2- (assym. stretch), ribose (C1' C2' OC3'), PO2- (sym stretch), RNA backbone and coupled furanose-phosphodiester chain vib respectively.45 A comparision of the IR bands in SP1-DAP with that of CdSe prepared in the absence of DAP (SP1)23 (Figure 6; Figure S6a and Table 1)


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shows a change in vibrational frequencies. The peaks due to A&C, U, purine anti conform, purine syn conform, PO2- asymm. stretch and RNA backbone, at (cm-1): 1646, 1388, 1335, 1234 and 974 in CdSe prepared in the absence of DAP get shifted to higher frequencies at (cm-1): 1649, 1488, 1392, 1338, 1238 and 979 in SP1-DAP respectively and the peak due to C=N ring vib of G, and in plane C2'-OH at (cm-1): 1603 and 1420 get blue shifted to 1600 and 1415 cm-1, respectively. The peak at 1269 cm-1 due to cytosine get vanished in SP1-DAP shows that there is a very strong interaction of amine with cytosine. A prominent band at 979 cm-1 is contributed by the puckering of ribose sugar moiety. It is difficult to predict the influence in other regions, as the presence of DAP masked other peaks. In the process of self-assembly in SP1A-DAP upon aging the peaks due to: coupled furanosephosphodiester vib, assym. stretch PO2- , U, in plane C2’-OH and, purine in anti-conform (cm-1 ): at 825, 1238, 1488, 1415 and 1388 get shifted to higher frequency at 827, 1240, 1491, 1435 and 1398 and became sharper. The peaks due to A&C, C=N ring vib of G, purine in syn conform and RNA backbone get shifted to lower frequency at (cm-1): 1646, 1575, 1331 and 971 respectively. The shift to higher wavenumber as compared to SP1 can be assigned to increased interaction of these components with Cd2+ in the presence of DAP and the blue shift due to the weaker interactions. DAP gets bound possibly due to excess Cd2+ on RNA strand, weakening the G-Cd2+ due to C=N ring vib of G which result into the shift from 1603 to 1575 cm-1. The peak at 1240 cm-1 along with the development of a smaller peak at 1183 cm-1 evidently indicates the conversion of RNA from B to A form in SP1A-DAP. Whereas, the peak at 825 cm-1 in case of SP1-DAP becomes sharper and gets shifted to higher frequency in SP1A-DAP, suggesting the coupling of furanose phosphodiester chain to antisymmetric O-P-O starts taking place upon aging.


18

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Apart from these, there are some vibrational bands observed at about 3280, 3030 and 793 cm-1 due to N-H str, -CH2, and N-H wagging, of DAP (Figure S6b and Figure S6c). Intrestingly, in SP1-DAP these peaks observed in SB1-DAP (Cd2+- RNA -DAP) disapperaed possibly due to the interaction of Cd2+ present in CdSe interacting through –NH2 of DAP rather than with excess Cd2+ present on RNA strand. Unlike SP1-DAP, in SP1A-DAP in all the peaks present in SB1DAP apperaed again at a very similar frequency, clearly suggesting the interaction of excess Cd2+ present on RNA strand with DAP become more prominent apart from its other interactions shown in Figure S6b and Figure S6c, Table S3.

.

The above noted interaction of excess Cd2+ with amine in SP1-DAP was also verified by designing a control experiment with 1:1 CdSe in which there was no excess Cd2+ present (SPDAP). It exhibited more prominent peaks 825 cm-1, 975 cm-1 and a broad peak at 1235 cm-1 suggesting stronger interaction of Cd2+ bound to CdSe with DAP. It supports that RNA is mainly present in B Form in the absence of excess Cd2+ (Figure S6d).

Figure 6. FTIR spectra of SP1-DAP and SP1A-DAP.


19


Page 20 of 51

Table 1. FTIR spectral peaks for SP1, SP1-DAP and SP1A-DAP Peak assigned

SP1 (cm-1 )

SP1-DAP (cm-1 )

SP1A-DAP (cm-1 )

A&C

1646

1649

1646

G (C=N ring vib of G)

1603

1600

1575

G

1535

Disappeared

Disappeared

U

1484

1488

1491(Sharp)

In plane C2'-OH

1420

1415

1435

Purine in anti confm.

1384

1392

1398

Purine in syn confm.

1335

1338

1331(Sharp)

C

1269

Disappeared

Disappeared

Assym. Stretch PO2 -

1234

1238(sharp)

1240 (sharp)

Ribose C1' C2' OC3'

1147

1147

1144(sharp)

Sugar backbone vib. (from sugar in C3'-endo confm.)

-

1183

CO stretch of backbone

1066

-

-

Sym. PO2 2-Stretch

1088

1087

1087

RNA backbone

974

979

971

Coupled furanose- 820 phosphodiester chain vib.

825

827 (Sharp)

3.7 NMR studies 1

H and

31

P NMR spectra of the SP1-DAP and SP1A-DAP have been recorded to further

gain knowledge of interaction of CdSe with different functionalities of RNA (Figure 7a and Figure 7b; Figures S7a - S7d; Table S4). 1H NMR of SP1-DAP and SP1A-DAP showed all the peaks corresponding to protons of sugar 2'-OH, sugar protons (H2′, H3′,


20

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H4′, H5′, H5′′), purine and sugar base (H1′, H5), aromatic protons of purine and pyrimidine bases (H2, H8, H6) similar to that reported for CdSe in the absence of DAP23,46 except most of these were either shielded or deshielded. Apart from these, the peaks due to DAP were also observed in SP1-DAP and SP1A-DAP (Figure S7b and S7b′). The spectral changes observed in Figure 7 have been summarized in Table S3. Interestingly, in SP1-DAP the peaks due to sugar protons (H2′, H3′, H4′, H5′, H5′′) get shielded while the peaks due to sugar base (H1′, H5), and aromatic protons of purine and pyrimidine bases (H2, H8, H6) shows deshielding in comparison to those of in CdSe synthesized in the absence of DAP (SP1)23 (Figure S7a and Table S4). As regard to the interaction through amino group in DAP in SP1-DAP with -OH of RNA back bone and other nucleotides; it leads to slightly shielding of DAP protons as compared to those of reported for free DAP47 base. Interestingly, in the process of self-assembly the peaks due to DAP in SP1A-DAP show deshielding. Whereas, the peak due to 2'-OH proton for CdSe prepared in the absence of DAP (SP1) disappeared in SP1-DAP, which suggests its interaction through amino group. However, this peak reappears in the SP1A-DAP with a slight shielding, which now clearly indicates the increasing interaction of excess Cd2+, bound to RNA strand, through amino group. This very well supports the above observation of deshielding of the peaks due to DAP in SP1A-DAP. These findings are also in line with the changes observed due to in plane C2'-OH frequencies due to its H-bonding through –NH2 of DAP in SP1-DAP and SP1A-DAP in IR spectral data (Table 1). The

31

P NMR spectra for SP1-DAP and SP1A-DAP are shown in Figure 7a' and

7b' and the data has been summarized in Table S5. As per literature data the chemical


21


Page 22 of 51

shift in the upfield region can be assigned to phosphorus of the phosphodiester bond and the

downfield

chemical

shifted

peak

may

be

assigned

to

the

terminal

monophosphoester.48 Upon interaction with DAP in SP1-DAP both of these peaks are shielded in comparison to CdSe in the absence of DAP23 (Figure S7a' and Table S5), whereas upon aging in SP1A-DAP, the peak due to phosphodiester bond is shielded while the peak due to terminal monophosphoester gets deshielded. The shielding may arise due to increased interaction of DAP with PO2- of RNA backbone, whereas the deshielding may arise due to the binding of excess Cd2+ bound to PO2- of sugar, now interacting with amine of DAP in the process of folding.

Figure 7. 1H and 31 P NMR spectra of SP1-DAP (a and a′) and SP1A-DAP (b and b′).


22

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An increased intensity of the splitted peak due to terminal monophosphoester in downfield region suggests the interaction with the neighbouring phosphate nuclei which gets closer in C3'-endo ribose conformation in A-form of RNA. All the changed interactions in SP1A-DAP support the folding of RNA from B to A configuration. 3.8 Circular Dichroism study The circular dichroism measurments were performed to investigate the change in the conformation of RNA upon interaction with CdSe particles containing DAP, i.e. SP1-DAP as well as in its self assembly obtained upon aging (SP1A-DAP) (Figure 8a, Table S6). DAP as such is optically inactive, however, it influences the structure of RNA-Cd2+ (SB1) significantly, i.e. in SB1-DAP. SP1-DAP exhibited peaks (nm) (ellipticity) at: 209.4 (-4.82) (Peak 1), 241.75 (1.05) (Peak 2) and 273.1 (4.34) (Peak 3), which are observed to shift with a change in both the wavelength as well as ellipticity to: 208.4 (-5.33), 238.36 (-1.38) and 271.1 (5.80) respectively upon aging (SP1A-DAP). The observed shift in the peaks suggests the change in RNA conformation from B form (in the absence of DAP) to its A form.49 The formation of A form of RNA in the presence of polyamine has earlier been reported to have the characterstic positive peaks in the vicinity of 271 nm and 264 nm corresponding to polynucleotides, polyC.polyG and polyA.polyU, respectively.50 The deconvolution of the observed positive peaks in SP1-DAP and SP1A-DAP exhibited three peaks each at 263.4, 272.1, 281.9 and 258.8, 269.3, 279.7, respectively (Figure 8b) suggesting to match with the A form of RNA. An examination of CD spectra corresponding to the deconvoluted peak in SP1-DAP and SP1A-DAP clearly shows that the contribution corresponding to these component exhibited blue shifted peak associated with an increase in ellipticity. It suggested an increased transformation of B-form of RNA to A-form upon aging. Besides this, the 2nd component is increased significantly to that of the third


23


component by about more than 25 % in SP1A-DAP as comapred to that of

SP1-DAP,

suggesting increased interaction of the DAP, i.e. with poly C and G component in the former.

6

SB1-DAP SP1-DAP SP1A-DAP

271.1 272.3

5

a

273.1

4 2 0 -2 -4 -6 200

240.6 241.75

4

Ellipticity (mdeg)

8

Ellipticity (mdeg)

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

Page 24 of 51

238.36

209.3 209.4 208.4

2 1 0 6 5 4 3 2 1 0

250 250 Wavelength (nm)

b

SP1-DAP

3

272.1

281.9

263.4

SP1A-DAP

269.3

279.7

258.8

260

300


290

300

Figure 8. (a) CD spectra of SB1-DAP, SP1-DAP and SP1A-DAP, (b) deconvolution of the positive peaks of SP1-DAP and SP1A-DAP. 3.9 Analysis of Photophysical Changes - Time Resolved Emission Spectroscopy Time resolved emission spectroscopy (TRES) measurements were performed for analyzing the nature of fluorescing species (Figure 9). For the sample SP1-DAP and SP1A-DAP we noted three species in both cases having λmax (nm) (after time interval (ns)): SP1-DAP -532 (3.2); 535 (8); 547 (44) (Figure 9a and a'); SP1A-DAP- 530 (2.3); 536 (8); 542 (44) (Figure 9b and b'). A comparison of the TRES spectra of SP1-DAP with SP1A-DAP clearly shows that the emission maxima for the latter is blue shifted with a decrease in intensity. At longer period of time in both the cases the emission was found to decay without any spectral changes. These observations were very well corroborated with decrease in intensity in the steady state emission measurements (Figure 1a).


24

Page 25 of 51

6000

532 nm

a

SP1-DAP Delays

5000

Counts

3.2 ns 8 ns 44 ns

535 nm

4000 3000

547 nm

2000 1000 0 500

1800 1600

550


530.5 nm

b

SP1A-DAP

700

1200

750

Delays 2.3 ns 8 ns 44 ns

1400

Counts

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


536 nm

1000 800

542 nm

600 400 200 0 500

550

600

650

700

750

Wavelength (nm)

Figure 9. TRES spectra along with the 3D image of SP1-DAP (a and a′) and SP1A-DAP (b and b′). 3.10 Anisotropy Steady state anisotropy measurements for the SP1-DAP and SP1A-DAP show the anisotropy values to be 1.48 × 10-2 and 2.78 × 10-2, respectively, indicating that the aging of the sample for about 1 month enhances the anisotropy by about a factor of about 1.9. The observed change in anisotropy was further monitored by recording rotational correlation time of these samples (Figure. 10a and Figure. 10b). The anisotropy decay for both the samples, SP1-DAP and SP1ADAP was found to follow bi-exponential decay having the time constants for the two processes to be: 0.2 ns and 14.7 ns and 0.6 ns and 31.5 ns, respectively. In both the systems the first component has a very small lifetime of less than a ns and the second component exhibited


25


significantly higher lifetime. However, the value of the second component for the aged sample is about two times higher to that of the fresh sample. The higher value of the anisotropy of the second component, matching with the observation made by using steady state measurements, as well as decay constant can be attributed to the formation of porous nanostructures, which is expected to have slower rotational diffusion, thereby expected to rotate relatively slowly. A comparison of the anisotropy data with those of observed earlier with RNA templated CdSe in the absence of DAP reveals two major differences. Firstly the anisotropy of the fresh sample was slightly lower by about a factor of 1.5 times and followed single exponential decay, whereas in the present case both the sample showed bi-exponential decay having the first time constant to be less than 1ns and the second time constant about more than an order of magnitude higher. It clearly indicates that even the fresh sample has some contribution of porous morphology exhibiting higher time constant of anisotropy value for the second component. The aged sample having longer decay time constant suggesting the increased folding contributing to the rotational diffusion. This finding is very well supported by the observed morphological changes by AFM and TEM analysis. 0.6

SP1-DAP

a

0.4

SP1A-DAP

b

0.3

0.4

0.2 Anisotropy

Anisotropy

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

Page 26 of 51

0.2 0.0

0.1 0.0 -0.1

-0.2

-0.2

-0.4 200

400

600

800

100

200

Channels

300 400 Channels

500

600

Figure 10. Time resolved anisotropy decay curves for SP1-DAP (a) and SP1A-DAP (b).


26

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3.11 Hg2+ Sensing The fluorescence spectra of SP1-DAP and SP1A-DAP in the presence of [Hg2+] solutions (1 × 10-10 – 1 × 10-6 mol dm-3) alone (Figure 11a and Figure 11a' ) as well as in its mixture containing other possible toxic metal bivalent ions (Pb2+, Mn2+, Co2+, Ni2+ at 5µM each) (Figure S8a and Figure S8a') were recorded in order to examine its effect on fluorescence behavior. Other bivalent metal ions like Zn2+ and Cu2+, which play an important role in metabolism and are relatively nontoxic in the used concentration range, were not included in this mixture.51-53 The addition of Hg2+ to SP1-DAP from 1 × 10-10 mol dm-3 to 1 × 10-6 mol dm-3 did not influence the absorption spectrum (not shown), but it influences the fluorescence behavior significantly (Figure 11a). The plot of I0/I vs concentration of quencher (Hg2+) shows a non-linear SternVolmer behavior with a downward curvature at higher concentrations, suggesting to involve some complex behavior, which cannot be explained by the combined dynamic and static quenching alone. In lifetime measurements, an increase in the [Hg2+] also resulted in a decrease in the average fluorescence lifetime due to SP1-DAP. The plot of / vs [Hg2+] (1 × 10-10 – 1 × 10-6 mol dm-3) shows a linear variation with concentration (Figure 11b). From this plot, the dynamic quenching rate constant (kq) was calculated to be 1.5 × 1012 mol-1 dm3 s-1. In a similar analysis for the quenching of emission due to SP1A-DAP by Hg2+, (the quenching constant was slightly higher (2.0 × 1012 mol-1 dm3 s-1) (Figure 11b'), suggesting a possibility of relatively higher fraction of fluorophore to be available for the dynamic quenching with the aged sample. This, however needs further analysis in terms of the initial possibility of binding of Hg2+ to the RNA strand which might be influencing the quenching behaviour of CdSe and has been discussed vide infra. It may be mentioned that


27


Page 28 of 51

a very similar values of kq were observed due to Hg2+ ions for both of these samples in the presence of interfering ions (Figure S8b and Figure S8b'; Table 2).

Figure 11. Fluorescence spectra of SP1-DAP and SP1A-DAP with varied concentration of [Hg2+] (mol dm-3) (0 – 1 × 10-6) (a and a' respectively); Stern-Volmer plots: I0/I and / vs [Hg2+] for SP1-DAP and SP1A-DAP (b and b' respectively). 3.12 Electrical Properties Electrical behavior of the as synthesized sample were also analyzed both in dark and under visible light illumination in order to analyze the charge dynamics by drop casting their film on ITO plate. The I–V curves obtained in the dark and under light illumination are shown in Figure 12. The fresh sample (SP1-DAP) in dark shows poor current suggesting large surface resistant between the samples and silver metal coating. The electrical current is significantly increased under visible light illumination in the forward bias direction beyond 1 volt which attains the


28

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maximum current (1.05 mA) at about 3.6 volt. Whereas, relatively much less current flow is observed in the reverse bias. A careful examination of this curve in the voltage range of - 4 to + 4 volt clearly shows the nonlinear asymmetric I-V features, (Figure 12a) suggesting the rectifying behavior of CdSe nanostructures. The I-V characteristics, however, show a significant change for the aged sample (SP1A-DAP) in dark as well as under light illumination, retaining the very similar asymmetric nonlinear nature of these curves as observed with SP1-DAP. In this case the increase in the current upon illumination has been observed to start in the forward bias direction at about 2.2 volt with a significant increase of IL/ID to 12.5, recorded at about 3.6 volt (Figure 12b). In a difference with SP1-DAP, the nature of curve though remain very similar under reverse bias upon illumination to that observed in dark, which indicates an increased rectification behavior for the aged sample. An examination of I–V data shows that the current increases by a factor of about 10 (IL/ID = 10), after illuminating the sample for a period of about 10 min and, thereafter it starts taking a plateau value for SP1-DAP (Figure S9 a). Whereas, for the aged sample (SP1A-DAP), this value is further increased to about 12.5 times (IL/ID = 12.5) upon irradiation (Figure 12). In contrast to the fresh sample, this value is though attained only after 1 min of illumination (Figure S9 a'). These observations clearly suggest a much better charge separation upon illumination in SP1ADAP (Figure S9 a'). Any prolonged illumination, thereafter, however showed a slight decrease in current and starts taking a plateau value (Figure S9 a'). Such behavior possibly arises due to the presence of more defects in this sample. The time constant for the stored charge was also examined in two cases by monitoring the decay of current in different time intervals and the scan was recorded after every 1 min of irradiation from the voltage range of -4 to +4 volt. The current was found to decay by about 90%


29


within the time constant of 10 and 30 min for SP1-DAP and SP1A-DAP, respectively (Figures S9b and S9b'; Figures: S9c and S9c'). Fairly high values of decay time constant(s) observed for the drop-casted films of these samples suggest the occurrence of effective charge separation upon illumination. A comparison of charge separation behavior of solid CdSe sample to that of liquid sample however exhibits that in both the cases there is an increase in the separation of charge but the magnitude of increase in lifetime in solution was relatively much smaller upon illumination but the separate charge shows the prolonged decay time constant in the drop cast film. It is due to fact that the charge carrier mobility in solid is relatively low as compared to the solution.54 Fairly high time constant decay value(s) observed in the present case suggests the effective charge separation upon illumination. It suggests the delocalization of separated charge on the film, which is further enhanced in the aged sample. An increase in the effective charge separation in SP1A-DAP is understood to arise possibly because of the change in morphology and increased networking through excess Cd2+ bound to DAP in CdSe nanostrtuctures.

0.0012

0.0012

a

SP1-DAP

Dark Light

0.0008 0.0006

IL/ID=12.5

Dark Light

0.0008

IL/ID = 10

0.0004

b

0.0010 Current (Amp)

0.0010

Current (Amp)

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

Page 30 of 51

0.0002 0.0000 -0.0002

SP1A-DAP

0.0006 0.0004 0.0002 0.0000

-0.0004

-0.0002

-0.0006

-0.0004

-0.0008 -5

-4

-3

-2

-1

0

1

2

3

4

-5

-4

Voltage (Volt)

-3

-2

-1 0 1 Voltage(Volt)

2

3

4

Figure 12. I-V curves for SP1-DAP (a) and SP1A-DAP (b).


30

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4. Discussion In the presence of DAP, freshly prepared SP1-DAP produces a chain like morphology consisting of CdSe QDs in hexagonal structure, the formation of which is supported by, XRD (Figure 2), and SAED analysis using TEM (Figure 5b and b'). The self-assembly of these nanostructures, however, results in their prominent growth along (100) plane in contrast to that of the bulk sample in which the growth is reported along (002) plane, while retaining the same hexagonal structure (Figure 2). The observed growth along (100) plane is associated with a change in morphology from chain like to porous network producing honeycomb like structure (Figure 4a'). FESEM analysis for SP1A-DAP also showed the formation of similar honeycomb like morphology upon aging (Fig S10). Notably, in the absence of DAP the needle like morphology consisting of CdSe QDs has been observed.23 The participation of DAP in interaction between SP1 and DAP, causing a change in morphology is also manifested by CD spectroscopy. In SP1A-DAP, it resulted in increased interactions with poly C and G components of RNA as compared to those of Poly A and Poly U.51 An analysis of CD data for SP1-DAP, specifically the characteristic 3rd peak (256 - 296 nm) with positive ellipticity, exhibiting the deconvoluted peak(s) (nm) at 263.4, 272.1, 281.9 corresponded to the mixture containing both B and A configurations with relatively more of B form. Whereas, in SP1A-DAP, relatively more prominent conversion to A configuration in which is considered as all deconvoluted peaks were relatively more blue shifted with an increased ellipticity (Figure 8). These features have earlier been assigned to A form of RNA.50 This finding is also supported by the FTIR spectrum of SP1A-DAP, which showed the appearance of a sharp peak at 1240 cm-1 along with a shoulder at 1183 cm-1, which are clear indication of increased transformation of B form to A form involving the interaction of PO2- with


31


Page 32 of 51

Cd2+ (Figure 6). An electrostatic interaction between PO22- with Cd2+ is also indicated clearly by XPS analysis of P element (Figure 3). The interaction between different functional groups of RNA with CdSe and Cd2+ present on RNA strand and DAP has also been arrived by 1H as well as

31

P NMR studies. In 1H NMR spectrum, the absence of interaction of –OH through –NH2

group in SP1A-DAP, which caused –NH2 away from –OH of sugar resulted in the reappearance of 2'-OH peak unlike in SP1-DAP, suggesting the folding of RNA upon aging (Figure 7). Similarly,

31

P NMR spectrum of SP1A-DAP exhibited an increase in intensity due to terminal

phosphomonoester along with the increased splitting as compared to that of contribution of

31

P

phosphodiester peak observed in up-field range (Figure 7; Table S5). These changes are understood because of the transformation of the ribose conformation in RNA from B form to A form, exhibiting C2'-endo to C3'-endo conformational change. Thus, the changes noted in the CD, IR and NMR spectroscopy clearly indicate the folding of RNA resulting in its increasing conversion from B to A form. The non-covalent interactions involving various functionalities of RNA, excess Cd2+ and CdSe QDs in SP1-DAP (in the nucleation stage, II) and SP1A-DAP nanohybrids (III), have been considered to be responsible for the folding of RNA (Scheme 1) and, thereof inducing the noted morphological transformation.


32

Page 33 of 51 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


Scheme 1. Showing different interactions for - I (SP1): Cd2+ - A/G/C ( CdSe -A/G/C ( DAP (

), CdSe-OH (

), OH-DAP

(

), Cd2+-PO2- (

)

); II (SP1-DAP) (in nucleation stage): CdSe - -NH2 in

), PO2-—DAP (

); III (SP1A-DAP): Porous honeycomb like

structure, (D) SP1A-DAP: building block of porous honeycomb like structure, indicating the presence of DAP in the center having interaction with excess Cd2+. Cd2+-NH2 ( (

), NH2-G/C

). From the above discussion it is arrived that SP1-DAP produces a metastable state, which

involve partial folding of RNA upon interacting with CdSe QDs and is eventually transformed into relatively more thermodynamically stable porous morphology in the process of selfassembly in SP1A-DAP. It evidently suggests that it is the interactions of mainly DAP with SP1,


33


Page 34 of 51

which contributes to the observed change. A change in the morphology upon aging is also evidenced by an increase in anisotropy and rotational correlation time, suggesting a slower rotational diffusion of porous honeycomb like nanostructures as an increase networking in the structure makes it more difficult to rotate (Figure 10 a and b). The observation about an increase in the intensity of the fluorescence band (545 nm) in the presence of DAP (SP1-DAP) by a factor of more than 1.5 times to that in the absence of DAP (SP1) has been attributed to the interaction of these particles through biotemplate with DAP. In particular, a regular increase in the emission intensity with an increase in concentration of DAP up to 1× 10-3 mol dm-3 without bringing any change in the emission wavelength (Figure S2a), is interpreted by the increasing surface passivation upon incremental addition of DAP up to this concentration, which essentially enhances the radiative recombination. An increase in from 92 to 102 ns (at 545 nm) also supports this hypothesis. A more prominent increase in lifetime to 126 ns at longer wavelengths (650 nm) in SP1A-DAP suggests relatively more trapping of charge carriers in the deeper traps upon aging, thus bringing an increased charge separation. Such a high value of fluorescence lifetime is hitherto unreported for CdSe nanosystem in aqueous medium. The CdSe surface stabilized by RNA in SP1 is observed to consist of both the shallow and deeper trap states located at varied energies (Table S2c). The addition of DAP is understood to influence the emission behavior of CdSe in two steps. The DAP at its lower concentrations (≤ 1×10-3 mol dm-3) passivates the surface of CdSe QDs by binding to the trap states through RNA functionalities involving non-covalent interactions, as arrived by IR spectroscopy (Figures 6 and Table 1); 1H NMR and

31

P NMR (Figures 7; Table S4 and S5) to generate a new surface

structure. It enhances the emission by minimizing the non-radiative recombination pathways


34

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(Figure S2b). Under these conditions, the trapping of holes by the amine upon illumination of CdSe QDs seems to be inefficient process as increasing [DAP] caused a regular increase in the emission intensity (Figure S2b). Similar effect of passivation of CdSe surface by amine(s) has been reported earlier.29-33 In the present system, the observed photophysical changes at lower DAP possibly arises on account of the increased networking through excess Cd2+ bound to DAP in CdSe nanostructures, which might be acting as heterojunctions and are involved in the trapping of charge carriers. But, at higher [DAP], it might have bound to the hole trapping surface states lying in the band gap, which quenches the fluorescence by scavenging the photogenerated holes. The effect of higher [DAP] (> 1× 10-3 mol dm-3) has been further probed by designing the fluorescence lifetime measurements in which its concentration was doubled, it resulted in the reduction of lifetime due to CdSe QDs from 102 ns to 97 ns, thereby suggesting a possibility of scavenging of photogenerated holes by amines at its higher concentration(s).55 It, however, requires further investigation to probe this aspect in details. The quenching of emission by Hg2+ is more complex as the binding of Hg2+ with Cd2+-RNACdSe might involve some complexation29,56 with DAP in the following equilibrium (Scheme 2): 2+

xCd2+- RNA-CdSe-DAP + yHg Static quenching hv

xCd2+- RNA-CdSe-DAP - yHg (Non-fluorescent)

2+

yHg2+

hv'

(x-y) Hg2+ / DAP-CdSe-RNA-Cd2+ + yCd2+

xCd2+- RNA-CdSe-DAP*

Dynamic quenching

Scheme 2: Mechanism for quenching of fluorescence This possibility was examined by using the following modified Stern-Volmer equation:

=

[]

+


35


Where I0 is the intensity of fluorophore in the absence of quencher, I is the intensity of fluorophore in the presence of quencher, fa is the fraction of fluorescence accessible to the quencher (Hg2+) and Ka is its apparent binding constant with the SP1-DAP / SP1A-DAP. Interestingly, for both the systems plots of I0/I0-I vs 1/[Q] were found to be linear (Figure 13a and 13a'), suggesting the above relation to be valid under the used experimental conditions. From which the value of fa and Ka have been worked out (Table 2). Similarly, the values of kq, fa and Ka, were observed due to Hg2+ ions for both of these samples in the presence of interfering

ions (Table 2; Figure S8b and S8b'; S11a and S11b). The remarkably high value of kq (Figure 11b and 11b', Table 2) for both SP1-DAP and SP1A-DAP nanohybrids is perceived to arise because of the availability of Hg2+ on RNA strand bound to DAP, being in the close proximity of excited CdSe. At higher concentration of Hg2+ (>1×10-6 mol dm3

) the direct interaction of Hg2+ with Se2- cannot be ruled out because of its very low

solubility product57 (~ 4 ×10-59 mol4 dm-12). Notably, the value of fa is higher for SP1A-DAP and Ka is accordingly less, suggesting increased binding of Hg2+ bound to RNA strand with DAP through –NH2 group. 50 40

60

a

SP1-DAP

SP1A-DAP

40 I0/I0-I

20 Intercept = 5.4 Slope = 4.02E-8 R-Square = 0.96

10

30 20

Intercept = 3.83 10

Slope = 5.6E-8 R-Square = 0.99

0

0 0.0

a'

50

30 I0/I0- I

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

Page 36 of 51

2.0x10

8

8

4.0x10 6.0x10 1/[Hg2+ ]

8

8.0x10

8

1.0x10

9

0.0

3.0x10

8

6.0x10 1/[Hg2+ ]

8

9.0x10

8

Figure 13. I0/I0-I vs 1/[Hg2+] plots for SP1-DAP (a) and SP1-DAP (a').


36

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Table. 2 Values of fa, Ka and kq for different samples. Samples

fa

Ka(mol-1 dm3)

kq ( mol-1 dm3 s-1)

SP1-DAP

0.18

1.4 × 108

1.5 × 10

SP1A-DAP

0.26

6.8 × 107

2.0 × 10

SP1-DAP + IM

0.16

1.3 × 108

1.6 × 10

SP1A-DAP + IM

0.21

8.4 × 107

2.0 × 10

12 12 12 12

The dynamics of charge carriers upon illumination in SP1-DAP and SP1A-DAP was also examined in solution by adding efficient electron acceptor nitrobenzene (NB). An increase in the amount of NB resulted in increasing quenching of emission in steady state (Figure S12a and S12a') and in fluorescence lifetime decay experiments (Figure S12b and S12b'). A plot of I0/I vs. [NB] shows a nonlinear curve, which becomes exponential at its higher concentrations (Figure S12c and Figure S12c'). It suggests that the excess DAP present in this system might be acting as a hole scavenger. The contribution of dynamic quenching was also worked out by recording the fluorescence lifetime as a function of [NB]. Interestingly, the plot of / vs [NB] shows a linear Stern-Volmer plot (Figure S12c and Figure S12c') from which the rate constant of this process was evaluated to be 6.3 × 109 mol-1 dm3 s-1, suggesting increased scavenging of free electrons and hole by the redox couple. However, in case of SP1A-DAP the quenching constant (1.8 × 109 mol-1 dm3 s-1) (Figure S12 c and Figure S12c') was observed to be about 3.5 times less as compared to fresh sample (SP1-DAP). It is probably due to the poor reactivity of the trapped charge carriers due to a change in morphology. In this case the trapped electrons might be taking a little longer to reach to the surface to react with adsorbed nitrobenzene.


37


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A comparison of the as synthesized nanohybrids as regard to their synthetic protocol used medium, pH, photophysical data and detection limit for toxic Hg2+ ions have been presented in Table 3.

Table 3. A comparison of different features of biotemplated fluorescent CdSe nanostructures.

Biotemplate used for stabilization of CdSe nanostructures RNA in presence of Polyamine

Method Steps

One step synthesis Fresh

pH

Aqueous

9.2

One step synthesis Fresh

Aqueous

One step synthesis

QY (%)

Nanochains

545

36

Porous network

530 (Anisotropic)

28

Spherical

545

22

fibrous Nanoneedles Spherical

530 (Anisotropic) 630

19

Aqueous

DNA

3-Step Synthesis (DNA-capped CdSe/ZnS (T- rich) and CdSe/ZnS (C- rich)

Step 1Nonaqueous Step 2Aqueous

Commercial CdSe/ZnS QDs in toluene followed by phase transfer into aqueous

Ref.

121 (650)/ 126 (650)

0.5*

This work

106 (650)/ 116 (650)

1

23

Not reported

4.5

58

Not Reported

Not reported

6.0

59

Not Reported

Not reported

10

60

25

8.5-9.0

Two step synthesis

Av. Lifetime (ns) (λem in nm)

Detection limit of Hg2+ (nM)

9.2

Self-Assembly

Cysteine

Fluorescence λmax (nm)

Medium

Self-Assembly

RNA

MorphoLogy

8.5-9.0

Nanoclusters

530

Spherical 7.4

560 620

*[Hg2+] (nM) estimated in the presence of 5 µM each of other heavy metal ions: Pb2+, Ni2+, Co2+ and Mn2+. Table 3 clearly shows that this nanosystem exhibit relatively higher fluorescence efficiency, better charge separation and fairly high sensing capability for Hg2+ ions as compared to other


38

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biotemplated CdSe nanohybrids. Moreover, the present synthetic protocol is much simpler involving one step only. For the drop casted film consisting of CdSe QDs, the observed increase in current with increasing irradiation time (Figure S9a and S9a'), suggests progressively more trapping of charge carriers possibly into the different existing trap states. An initial increase in the current (up to 10 min) and, thereafter a decrease with increasing irradiation time suggest all the traps possibly get occupied up to 10 min and, thereafter annihilation of charge carriers takes place. An initial increase in current can also be understood in terms of the difference in mobility of electrons and holes in CdSe. The former being several order of magnitudes higher to that of holes,54 evidently explains an increase in current. In case of SP1A-DAP, the higher generation of current at relatively lower timing of irradiation (1 min) suggests more number of traps available on the surface of these nanohybrids in contrast to that of SP1-DAP. A similar conclusion about the presence of more heterojunctions for these nanostructures has been arrived by fluorescence lifetime measurements (Figure S3a and S3b and Tables S2a, S2b and S2c). Such a behavior in the presence of amine(s) has also been noted earlier enhancing the separation of charge in several other reports.61 The generation of particles on porous matrix in other nanosystem has also been observed to contribute to the enhanced optical and electrical properties.62,63 The higher time constant for the stored electrons for the drop casted film of SP1A-DAP (Figure S9 b' and c') suggest the trapped electron to stay longer on the aged particles. The observation of rectification behavior of these nanostructures along with a fairly high value of IL/ID evidently suggest the potential of these nanostructures for optoelectronic measurements. To the best of our knowledge this is the first report analyzing the involvement of RNA-polyamine complex in passivating the


39


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surface of CdSe nanoparticles, and might reveal mechanistic steps involved in green biological synthesis 34,35 of such nanosystems.

5. Conclusions We have successfully synthesized RNA mediated CdSe QDs in the presence of DAP which initially forms a chains of QDs in metastable state followed by their growth along (100) plane to produce thermodynamically more stable honey-comb like porous morphology in the process of self-assembly. The DAP initially binds nucleotide(s) of RNA through alcoholic group of sugar and PO2- to produce QDs, which eventually causes a change in the configuration of RNA from B form to thermodynamically more stable A form involving the prominent binding of excess Cd2+ on RNA strand with DAP and its different moieties such as guanine, cytosine, uracil, –OH group of sugar, and PO2-; and as evidenced by IR, 1H NMR and 31P NMR, XPS and CD spectroscopy. These interactions have also been analyzed by a change in emission behavior of CdSe QDs involving the passivation of their surface by donation of electron(s) from amine group to the different surface states and at their higher concentrations scavenge the holes, thereby causing the quenching of CdSe fluorescence. The passivation of the surface associated with enhanced fluorescence lifetime has been understood by trapping of charge carriers into different heterojunctions formed by Cd2+-amine interaction. The generation of heterojunctions is also evidenced by I-V data, which demonstrated enhanced IL/ID value(s) for these nanostructures under forward bias condition only. As synthesized nanostructures demonstrated fairly high selective sensing of Hg2+ ions even at their low concentration of ≥ 0.5 nM in the presence of other interfering heavy metal ions. The observed enhanced properties of Q-CdSe: optical, fluorescence and I-V behavior evidently suggest their future multifunctional potential for bio imaging and the development of


40

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optoelectronic devices. The novelty of the present manuscript lies in exhibiting the tRNApolyamine interaction, envisaged earlier in cell growth, for enhancing the photophysical and electrical features of colloidal CdSe nanohybrids.

Supporting Information. – Figures: Adsorption isotherms (S1), effect of DAP (a and b) and pH (c and d) on absorbance and fluorescence behavior of CdSe NPs ( S2), fluorescence lifetime decay curve of SP1-DAP having different amount of DAP and depth of traps (S3a-S3c), histograms for average diameter of QDs, 3D TEM image for SP1A-DAP (S4), EDS analysis of SP1-DAP and for SP1A-DAP (S5), IR spectra supporting the interaction of DAP with Cd2+ of CdSe (S6), 1H and

31

P NMR of SP1(S7), quenching due to [Hg2+] in SP1-DAP and SP1A-DAP

containing ionic mixture (5 µM) (S8) current at different illumination time for SP1-DAP and SP1A-DAP, current vs time plots for SP1-DAP and SP1A-DAP (S9), FESEM analysis (S10), modified Stern-Volmer plots for SP1-DAP and SP1A-DAP (S11), quenching of SP1-DAP and SP1A-DAP by nitrobenzene (S12), Tables:- Surface characteristics data (S1) fluorescence lifetime data of SP1 at different [DAP] (S2a, S2b and S2c), FTIR spectral peaks in amine region (S3), 1H NMR and

31

P NMR spectral data (S4 and S5), circular dichroism spectral data (S6).

AUTHOR INFORMATION Corresponding author’s E-mail: [email protected]; Tel.: +91-1332-285799; Fax: +911332-273560;

Author Contributions The manuscript was written through equal contributions of both authors. Both authors have given approval to the final version of the manuscript.


41


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ACKNOWLEDGEMENTS Thanks are due to the Head, IIC, IIT Roorkee for providing the facilities of FESEM, AFM, TEM, XRD, TCSPC. K.G. is thankful to CSIR, New Delhi for the award of SRF.

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31

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Supramolecular-Assisted RNA-Templated Fluorescing Colloidal CdSe

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