FRET on Surface of Silica Nanoparticle: Effect of Chromophore

Subscriber access provided by United Arab Emirates University | Libraries Deanship

Article

FRET on Surface of Silica Nanoparticle: Effect of Chromophore Concentration on Dynamics and Efficiency Anjali Dhir, and Anindya Datta J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b05242 • Publication Date (Web): 26 Aug 2016 Downloaded from http://pubs.acs.org on August 28, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry C is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23

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

The Journal of Physical Chemistry

FRET on Surface of Silica Nanoparticle: Effect of Chromophore Concentration on Dynamics and Efficiency Anjali Dhir, Anindya Datta* Department of Chemistry, Indian Institute of Technology Bombay, Mumbai-400 076, India. E-mail: [email protected] Ph: +91 22 25767149

ACS Paragon Plus Environment

1


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 2 of 23

ABSTRACT. Förster resonance energy transfer (FRET) has been studied between fluorescein (donor, D) and rhodamine B (acceptor, A) bound covalently to the surface of silica nanoparticle (SNP-dye). This is a part of an ongoing effort towards development of light harvesting nanoantennae. The role of the D:A ratio and the total chromophore content on the efficiency of FRET has been investigated. At low number density (ca. 75± 15 dye molecules/ particle) efficiency is dependent on the D:A ratio. It appears that the distribution of the dyes on the surface is inhomogeneous and that FRET occurs only between D and A molecules in very close proximity. The effect of self-quenching of the donor is not very significant for this number density. At higher number densities (ca. 700-1100 dye molecules/ particle), a prominent rise in the acceptor emission is observed, perhaps indicating a more homogeneous distribution of the dye molecules and D emission is quenched completely. However, D-D quenching is an issue here. So, it appears that the lower number density is a more favorable condition for FRET in these systems.


2

Page 3 of 23

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


Introduction Artificial photosynthesis is one of the holy grails of contemporary research. Light harvesting antennae have an important role to play in this area. The potential of a myriad of molecular, supramolecular and nanomolecular systems are being explored in the quest for ideal light harvesting antennae. Some examples of such systems are porphyrin arrays,1,2 dendrimers,3 nanostructures and nanoconjugates.4,

5, 6

involves elaborate chemical synthesis.3,

7

The preparation of light harvesting antennae often This requirement can be circumvented by employing

matrices8, 9, 10, 11 that can act as scaffolds supporting ordered arrangements of molecules and thus leading to efficient light harvesting.12 Our interest in this context is to design light harvesting antennae that utilize a silica nanostructures13–15 as scaffolds and study the excited state dynamics in these systems. Chromophores can be attached covalently to silica by simple silanol chemistry, making it possible to generate a high local concentration of donors and acceptors within the nanostructure.16 Silica – bound fluorophores have been shown to be endowed with increased brightness of fluorescence. Besides, they are usually more photostable than free chromophores.18, 19

This can prove to be the solution to the problem of photobleacing, which often poses a major

hindrance to the operation of light harvesting antennae.19 Additional stability to the system is rendered by the inherent inert nature of silica towards external factors like pH, temperature etc.20 Our approach differs from previous studies of FRET in dye-silica nanoconjugates36,

37

in the

sense that the emphasis in the present study is to adopt a time resolved, rather than steady state fluorescence approach to elucidate the excited state dynamics in our systems. Very recently, we have achieved Förster resonance energy transfer (FRET) between fluorescein (energy donor, D) and rhodamine B (energy acceptor, A, Figure S1),23,

24

cocondensed in nanoparticles and nanoshells of silica.25 Very high concentrations of


3


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 4 of 23

chromophores have been used in that study, leading to competing pathways that deactivate the excited state of the donor nonradiatively. Clear risetimes have not been observed for the acceptor emission. The efficiency of energy transfer has been found to depend on the total dye concentration rather than the ratio of the number of donor and acceptor molecules. Also, since the chromophore molecules are embedded within the matrix, it is difficult to further utilize the energy transferred from the donor to the acceptor molecules. So, in the present study, a different approach has been adopted. Instead of cocondensing the chromophores with the silica precursor to generate silica-dye nanoconjugates in situ, pristine silica nanostructures have first been prepared. Then, their surface has been modified by incorporating amine groups, which have subsequently been made to react with isothiocyanate derivatives of rhodamine B and fluorescein, to prepare nanoconjugates whose surfaces are decorated with the donor and acceptor. This leads to the consumption of lesser quantities of the chromophores. More importantly, since FRET takes place entirely at the surface, further utilization of the energy transferred to the acceptor is potentially more feasible. From prior knowledge of FRET involving fluorescein-rhodamine B pair,10, 23, 24, 26, 27 it is well understood that direct excitation of acceptor needs to be avoided to the maximum extent possible, in order to minimize the effect of introducing an error therefrom. So, an optimal excitation wavelength of 440 nm is chosen. Besides, the D and A emission spectra have very significant overlap. This needs to be kept in mind during analysis of the lifetime data. As has been shown in our earlier study, high concentration of fluorophores can affect the quality of data. Two different total chromophore number densities have been used in the present work: < 100 and >700 molecules per particle, in an attempt to understand the role of dye-dye interaction that affects the dynamics and efficiency of FRET on the surface of silica.


4

Page 5 of 23

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


Experimental Section Tetraethyl orthosilicate (TEOS, ≥ 99%, Sigma Aldrich), 2-propanol (HPLC, Merck), cyclohexane (HPLC, Merck), ammonium hydroxide (25 % v/v), toluene (HPLC, Merck), N,Ndimethyl formamide (DMF; HPLC, Merck), Fluorescein isothiocyanate (FITC, 90% Sigma Aldrich) and Rhodamine B isothiocyanate (RITC mixed isomer, Sigma Aldrich) have been used without further purification. 3-Aminopropyltrimethoxysilane (APTS, 98%, Sigma Aldrich), is used as the silane coupling agent for covalent binding of fluorophores with silanol groups of silica network.28 Sodium hydroxide pellets (NaOH, Merck) have been used for leaching silica for calculation of fluorophore concentration. Ethanol (spectroscopy grade, Spectrochem, Mumbai, India) is used as the dispersion medium for all spectroscopic study. The nanostructures have been characterized by Field emission Transmission electron microscopes (FEG-TEM, JEOL JEM – 2100F). Steady state spectra have been recorded on JASCO V530 spectrophotometer and Varian Cary Eclipse fluorimeter. λex = 440 nm and bandwidth = 5 nm for steady state fluorescence studies. A Time Correlated Single Photon Counting (TCSPC) system, from IBH, UK, with λex = 440 nm, has been used to record the fluorescence decays. The full width at half maximum (FWHM) of the instrument response function is ~ 270 ps. The decays have been collected with emission polarizer at a magic angle 54.7º with respect to excitation pulse and have been fitted to a sum of exponentials (eq. 1):

I (t ) = I (0)∑ Ai exp ( −t / τ i )

(1)

i

using IBH DAS v6.229 using the iterative re-convolution method. I(0) and I(t) are the fluorescence intensity at time zero and t after excitation, respectively. Ai is the amplitude of the ith decay component.


5


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 6 of 23

The ultrafast fluorescence transients have been recorded using the femtosecond optical gating technique (FOG) described in detail elsewhere.25 A brief description is as follows: λex = 435 nm has been generated by frequency doubling the 870 nm output of a mode-locked Ti: Sapphire laser (Tsunami, pumped by a 4.6 W Millennia, both from Spectra Physics). In the FOG setup from CDP Corporation,.30 the upconversion is performed in a β-BBO crystal with a thickness of 1 mm, θ = 38° and φ = 90°. Detection of the upconverted light has been done by a double grating monochromator –photon counting photomultiplier combination. The full width at half maximum (FWHM) of cross-correlation function ~ 230 fs. λem = 520 nm and 630 nm. The data have been analyzed by iterative re-convolution method by using IGOR software. The nanoconjugates have been prepared by surface functionalization31,

32

First, silica

nanoparticles (SNP) of diameter ∼29 nm are prepared by template based synthesis using Brij58 as the soft template.33 3.4 g of Brij58 is dissolved in 15 mL of cyclohexane at 50˚C and then cooled to RT with stirring. 0.45 mL of MilliQ water is added and stirred until a homogeneous solution is obtained. To this, 1.2 mL of aq. ammonia is added dropwise and allowed to homogenize by stirring. In the reaction mixture, 1070 µL of TEOS is added and stirred for 3-4 h at RT. The resultant solution is washed with 2-propanol to remove Brij58 and unreacted molecules. The sample is finally washed with ethanol and dried at 60˚C. In the next step, the surface of the nanoparticles is modified by incorporation of amine groups. 100 mg of SNP were dispersed in 25 mL toluene and sonicated for 15 minutes. N2 gas is bubbled through the dispersion at 95˚C for 45 min. Then, 50 µL of APTS is added and stirred for 4 hrs. The mixture is removed and washed thoroughly with ethanol and dried at 60 ˚C in an oven. The resultant sample is referred as SNP-NH2. For surface functionalization, 50 mg of SNP-NH2 is dispersed in


6

Page 7 of 23

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


10 mL of DMF. Aliquots of solutions of FITC/RITC (1mg /mL in DMF) are added and stirred at RT for 24 hrs. The sample is washed thoroughly with ethanol to remove unreacted dyes. Table 1. Surface functionalized SNP with varying D:A at low concentration (< 100 molecules/ nanoparticle) and high concentration (850 ±150 molecules/ nanoparticle) D:A calculated

FITC/µL

RITC/µL

SNP-NH2 /mg

Low Concentration: < 100 molecules/ nanoparticle D 0.6:1 1.1:1 1.7:1 2.2:1 4.6:1 7.6:1 D 2.3:1 3.4:1 4:1 5.3:1 13.6:1

300 0 50 500 100 500 300 500 500 500 500 400 300 400 High Concentration: 850 ±150 molecules/ nanoparticle 2000 2000 2000 2000 1800 2000 1600 2000 1400 2000 800

50 50 50 50 50 50 50 50 50 50 50 50 50

Efficiency of Energy Transfer The efficiency of FRET has been calculated from the fluorescence lifetimes using equation 2:

η ET = 1 −

τ DA τD

..(2)

where τD and τDA are the fluorescence lifetime of D in the absence and presence of A, respectively. The fluorescence decays being multi-exponential, amplitude–average lifetimes (eq. 3) of the samples have been used to calculate the energy transfer efficiency, ηET, as amplitude, rather than intensity averaging is more appropriate for FRET.34, 35


7


τ avg = ∑

…(3)

Aiτ i

∑A

i

Results and Discussion

70 60 # Particles

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 8 of 23

50 40 30 20 10 0

20

25 30 35 Diam eter /nm

40

Figure 1. TEM images of dye loaded silica nanoparticles. Inset: Particle size distribution from

TEM images of 200 silica nanoparticles.

Shape and size of nanoparticles Surface functionalization of silica nanoparticle with D and A in different ratios has been achieved in three steps: preparation of the nanoparticle, surface modification with silanecoupling agent (APTS) and surface functionalization with fluorophores, as discussed in the experimental section. The nanoparticles are found to be spherical, with diameters of ca. 29 ± 4 nm, as constructed from samples of 200 particles (Figure 1). These dye-silica nanoconjugates have been referred to as S-dye-SNP in the subsequent sections. The covalent binding of dye in silica has been characterized by the absence of isothiocyanate peak (2150-1990 cm-1) of free dye in FT-IR spectra of silica bound fluorescein (Figure S2) and rhodamine B (Figure S3). Siloxane groups like Si-O-Si and Si-O-C have characteristic bands in the range of 1095 – 1075/1055 – 1020 cm-1 and 1110 – 1080 cm-1, respectively. These bands become predominant and mask the


8

Page 9 of 23

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


fingerprint region of the dyes to a very great extent. In an attempt to optimize the D:A ratio, the amount of dye ratio has been varied at fixed amounts of surface modified nanoparticles (Table 1, Figure S4).

Nanoconjugates with low level of dye loading Before proceeding with the spectroscopic study, we realized that the analytical D: A ratio maintained during the synthesis cannot be used in the subsequent discussion, as D and A do not have equal propensities to bind to the nanoparticles. The surface charge of the nanoparticles is positive, as indicated by their zeta potentials.31,32 The absorption spectrum of FITC-APTS indicates that it exists in the solution as an anion. RITC, on the other hand, has an inherent positive charge. So, the binding of fluorescein (D) is assisted by the electrostatic factor while the binding of rhodamine B (A) is hindered. The ratios used in the present work are estimated by determining the amounts of D and A actually bound to the nanoparticles. This is done by etching the silica matrix of a known amount of dye-silica (∼ 1-2 mg) nanoconjugate by overnight dispersion in 1 mL of 0.5 M NaOH, resulting in the release of the dyes in the solution. Their concentration is then estimated using Beer-Lambert law and hence, the number of dye molecules is calculated. The number of nanoparticles is estimated considering them to be silica spheres of diameter = 29 nm with density = 1.96 g cm–3.32 Hence, the number of dye molecules per nanoparticle is calculated to be 75 ± 16 in the first set of experiments (i.e. low concentration) and 850 ± 150 in the second set (high concentration). For spectroscopic measurements, the S-dye-SNP samples have been re-dispersed in ethanol. The absorption spectra of S-dye-SNP indicate effective binding of FRET pair in definite


9


ratios (Figure 2a). Since the sample is a dispersion, the absorption spectra are contaminated with scattered light. Upon excitation 440 nm, which is not absorbed by the A to a large extent, D is

0.4

D A 0.6:1 1:1 1.7:1 2.2:1 4.6:1 7.6:1

(a)

0.3 0.2 0.1 0.0 60

F.I.

Absorbance

0.5

(b)

D/4

λex = 440 nm

40 20

F.I. (Normalized)

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 10 of 23

0

(c)

400

450

500

550

600

650

700

λ /nm Figure 2. (a) Absorption spectra (b) Emission spectra (c) Normalized emission spectra at D emission of S-dye-SNP with different D:A ratio at low fluorophore concentration (

FRET on Surface of Silica Nanoparticle: Effect of Chromophore

Recommend Documents