Doped Semiconductor Nanocrystals and Organic Dyes: An Efficient

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Doped Semiconductor Nanocrystals and Organic Dyes: An Efficient and Greener FRET System Suresh Sarkar,†,‡ Riya Bose,†,‡ Santanu Jana,†,‡ Nikhil R Jana,‡ and Narayan Pradhan*,†,‡ †

Department of Materials Science and ‡Centre for Advanced Materials, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India

ABSTRACT We report here the F€ orster resonance energy transfer (FRET) from transition metal ion (Mn and Cu)-doped semiconductor nanocrystal (d-dots) donors to different organic dyes acceptors. Longer excited-state lifetime, high quantum yield in water, smaller size, and higher spectral overlap integral with acceptors make these doped nanocrystals efficient FRET donors with efficiency (E) up to 90%. Moreover, these doped nanocrystals whose emission covers almost the entire region of the visible window with variable excitations can couple with a large variety of organic dyes for energy transfer. Additionally, these are free from toxic cadmium element, which allows the exploration of their applications as greener and safer donors for FRET in different biological events. SECTION Kinetics, Spectroscopy

€rster resonance energy transfer (FRET) is a photoo physical process through which an electronically excited fluorescent “donor” molecule transfers its excitation energy to an “acceptor” molecule nonradiatively through a long-range dipole-dipole interaction.1,2 It mostly depends on the spectral overlap integral, center-to-center distance (typically 1-10 nm), relative orientations of donors and acceptors, as well as on the excited-state lifetime and quantum yield of donors.1,3-5 FRET in different organic dyes,6 proteins7-9 and quantum dot (q-dot)-dye10 pairs has been widely studied and proved to be a powerful spectroscopic technique to probe very small changes in the interseparation distance between donor and acceptor fluorophores.6 FRET using q-dots as a donor has generated much more attention over that using dyes because of their higher photostability, sharp and tunable emission spectra, and a wide range of excitation wavelengths.5,10-15 Moreover, these q-dots have also additional configurational advantage where a single donor can interact with several acceptors at a time.10,13 Hence, this is successfully used in biology to detect molecular binding events, protein and DNA conformation analysis, receptors/ligand interactions, and so forth.9,13,16-21 To pursue FRET in biology, CdSe/ZnS q-dots have been proved to be efficient donors but contain the toxic cadmium element. We explore here the recently developed high-quality transition metal ion (Mn2þ, Cu2þ)-doped semiconductor nanocrystals22,23 as efficient FRET donors for their long excited state lifetime,24,25 high emission quantum yield,22 wide spectral overlap, and smaller size. Unlike CdSe, these nanocrystals do not need a shell with higher bandgap materials to retain their emission intensities during surface functionalizations.23 Using both Cu-doped (d-dots-Cu) and Mn-doped (d-dots-Mn) semiconductor nanocrystals (ZnS and/or ZnSe), whose emissions cover almost the entire window of the visible

F

r 2010 American Chemical Society

spectrum, a wide variety of organic dyes can be chosen as acceptors to study the energy transfer. Because of broad emission spectrum for both Cu and Mn dopant emissions, d-dot-dye conjugate systems also show higher spectral overlap (Figure 1) compared to q-dot-dye conjugates.10 The concept of energy transfer from dopant emission to dye has been reported recently26 using as-synthesized Mn-doped ZnSe nanocrystals, but we report here a series of alternative doped nanocrystals having pure dopant emissions with various surface functionalizations to make a versatile system that can couple with a wide variety of organic dyes both in aqueous and nonaqueous medium. d-Dots-Mn shows a narrow range of tunable pure dopant emission within 575 to 600 nm with full width at half maxima (fwhm) of ∼50 nm in aqueous as well as nonaqueous solution27 and have excited state lifetime in milliseconds (ms).24,28 Similarly, d-dots-Cu emission tunes in the range of 480 to 540 nm in the visible spectrum22 with fwhm ∼ 65 nm with ∼90 ns excited state lifetime. These d-dots are ligand exchanged with short-chain ligands cysteamine hydrochloride (CYST), mercapto propionic acid (MPA), and dimercapto succinic acid (DSA) to make them water-soluble. d-Dots-Mn in either of the above ligand exchange shows ∼40% quantum yield (QY) in aqueous solution. For Cu-doped ZnSe, which is stabilized with a thin shell of ZnS, there is ∼30% QY in water after thiol functionalizations. Surface ligands for d-dots are chosen according to the dye used for possible oppositely charged electrostatic interaction to bring the donor and acceptor in close proximity (Experimental section in the Received Date: December 15, 2009 Accepted Date: January 8, 2010 Published on Web Date: January 15, 2010

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DOI: 10.1021/jz9004015 |J. Phys. Chem. Lett. 2010, 1, 636–640

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Figure 1. Spectral overlap of donor d-dots emission and acceptor organic dye absorption. Dotted lines represent emission spectra of donors and solid line for absorption spectra of respective acceptors. d-Dots-Mn and d-dots-Cu represent Mn:ZnS and Cu:ZnSe/S, respectively. Excitation wavelength for d-dots-Mn is 320 nm, and that of d-dots-Cu is 350 nm, respectively.

Figure 2. Successive titration emission spectra of d-dots with different organic dyes. Each panel from a to c is labeled for the d-dots and dye conjugates. The excitation wavelength for d-dots-Mn is 320 nm, and that of d-dots-Cu is 350 nm, respectively.

Supporting Information). Here, we have designed three FRET pairs using different surface charged d-dots having tunable dopant emissions and suitable organic dyes. Cresyl Violet and Texas Red C5 Cadavarine (Texas Red) dye acceptors conjugated with d-dots-Mn having MPA- and CYST-capped ligands, respectively, and Rhodamine B with MPA-capped d-dots Cu are used to study the energy transfer. As d-dots show large Stoke shift, the chosen excitation in each FRET pair exclusively excites these donor dots, minimizing the cross talk with dye acceptors. Figure 1 shows the spectral overlap of these FRET pairs with cartoons showing possible electrostatic interactions between d-dots and dyes. Figure 2 shows the successive titrated emission spectra of chosen d-dots-dye conjugates. Decrease of d-dots emissions and enhancement of acceptor dye emissions indicate FRET in their conjugates. For the d-dots-Mn-Cresyl Violet FRET pair, the emission of donor d-dots (Mn:ZnS-585 nm, 41% QY in water) quenches above 90% at a ratio of 1:4 of dots to dyes (Figure 2a, and Figure S1a). However, for q-dots-dye conjugates more than 1:10 ratio of dots to dye has been reported for similar FRET efficiency.10 FRET efficiencies are calculated against the decrease of integrated emission intensity of donor d-dots, and maximum FRET efficiency for d-dots-Mn (Mn: ZnS) and Cresyl Violet has been found to be 90% (Figure S2). For this case, the pH of the mixture has been maintained at 6.5 using MES buffer (10 mM) where the dye shows the


emission at 623 nm with absorption maxima at 585 nm and d-dots-Mn has 585 emission with 320 nm absorption (Figure 1a and 2a). Similarly, for positively surface charged d-dots-Mn-Texas Red dye and negatively surface charged d-dots-Cu-Rhodamine B dye FRET pairs, the experimental details are provided in the Supporting Information, and their FRET parameters are shown in Table 1. The numbers of d-dots are calculated according to the reported method29 by identifying the mass of Zn and Se though inductive coupled plasma (ICP) measurements and from the size measured from transmission electron micrograph (TEM). A representative TEM picture for d-dots-Mn showing nearly monodisperse d-dots (average diameter ∼3.8 nm) has been provided in the Supporting Information (Figure S3). The number of dyes is calculated from their absorption maxima and reported extinction coefficients (Supporting Information, page S4). The FRET efficiency calculation has been provided in the Supporting Information, page S3 and Figure S2. Mn:ZnSe d-dots also show similar energy transfer like Mn:ZnS d-dots with different organic dyes. We also studied the FRET with CYST-capped d-dots-Cu having positively charged surface with Lissamine Rhodamine B Ethylenediamine dye in aqueous medium and hexadecylamine amine-capped d-dots-Mn (Mn:ZnS) with Nile Blue Dye in hydrophobic dispersion (Supporting Information, Figure S4), and found efficient energy transfer from d-dots to dyes. This

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Table 1. Overlap Integrals, Quantum Yield of Donors, FRET Efficiency and F€ orster Radii of Different d-dots-Dye Conjugatesa donor

acceptor

J(λ) (M-1 cm3)

QYD (in water)

E for (A/D = 1) (%)

E* for (A/D = 1) (%)

EMax (%)

F€ orster radii (Ro) Å

Mn:ZnS

Cresyl Violet

8.8 10-13

0.41

69

63

90

66

Mn:ZnS

Texas Red

9.8 10-13

0.41

67

65

86

65

Cu:ZnSe

Rhodamine B

4.2 10-13

0.28

58

54

76

69

a

J(λ) = spectral overlap integral; QYD= quantum yield of donor; E = FRET efficiency calculated from d-dots quenching data; EMax = maximum FRET efficiency; A/D = acceptor/donor; E* = efficiency calculated from lifetime decay measurement.

Figure 3. Excited-state lifetime decay plots of d-dots-Mn (Mn:ZnS) with Cresyl Violet (a),Texas Red (b) and d-dots-Cu with Rhodamine B (c). The sequential decrease of lifetime with increasing number of acceptor dyes indicates the energy transfer from d-dots to respective dyes. Black dots indicate the lifetime of d-dots in the absence of acceptors and colored dots for the presence of successive amounts of added acceptors.

Supporting Information, Figure S5) to 1.61 ms (Figure 3b, blue curve) with increase of dyes-to-d-dots ratio from 0 to 1, showing 65% FRET efficiency. Similar results are also obtained for other d-dots-dye conjugates including d-dots-Cu. Calculated FRET efficiency from these decrease of lifetime values for other FRET pairs are summarized in Table 1. The FRET efficiency from time resolved spectroscopy measurement shows close agreement with those values measured from the emission intensity decay. However, the small discrepancy might be due to the parasitic excitation of acceptors, which is not exactly evaluated here. However, this needs further and detailed study for a quantitative estimation. The F€ orster distance for each set of FRET pairs has been calculated (Table 1), and the values are found to be a little higher compared to reported q-dots-dye pairs.10 The higher value is expected here due to the dynamic interactions of donor-acceptor pairs in solution. The advantage of these ddots are the long excited-state decay lifetime, which makes the FRET efficiency higher compared to q-dots, in spite of having a wide separation distance and higher F€ orster radii. For the case of d-dots (Mn:ZnS), the excited electron relaxes through the additional dopant energy states and show higher excited state lifetimes.25For the case of Mn dopant, the exciton relaxes through the forbidden Mn-d-states (6A1-4T1), which results in the 106-fold enhancement of excited-state lifetime compared to undoped q-dots. In the presence of acceptors, the radiated photons transfer to the acceptors nonradiatively, which reduces the emission intensity of donors and enhances the emission of acceptors. A schematic presentation of the possible mechanism is presented in Figure 4. In conclusion, we report here the efficient fluorescence resonance energy transfer from d-dots (Cu and Mn) donors to different organic dye acceptors. This leads to a greener FRET

Figure 4. Schematic presentation of energy transfer from donor Mn-doped nanocrystals to acceptor organic dyes. (1) Donor excitation, (2) vibrational relaxation, (3) nonradiative donor dopant state energy transfer, (4) nonradiative acceptor excitation, (5) acceptor emission.

supports the versatile combination of d-dots with various dyes in different dispersion medium to study the energy transfer. The FRETand its efficiency have also been studied by time resolved spectroscopy. During titration, we have measured the lifetime of donors with sequential introduction of acceptor dyes to a fixed number of d-dots. The decrease of excited-state lifetime of d-dots with increase of dye-to-d-dots ratios indicates the energy transfer from donor d-dots to acceptor dyes. Figure 3 shows the decrease of the lifetime of donor d-dots with increase of the numbers of dye molecules. For the case of Texas Red dye acceptor, we observed the decrease of lifetime of d-dots-Mn from 4.59 ms (Figure 3b, black curve and


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system opening an alternative door to CdSe/ZnS-based nanocrystals in almost entire visible spectrum to couple with a wide variety of acceptor dyes irrespective of their charge and dispersion medium. All factors for effective FRET;(1) small size, (2) high QY in water, (3) higher excited state lifetime, and (4) wide spectral overlap;favor doped semiconductor nanocrystals as efficient donors for organic dyes. Wide separated absorption and emission bands of these nanocrystals also minimize the crosstalk with coupled acceptor dyes. Additionally, these are free from heavy metals, which would help their applications in different biological and other related processes.

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SUPPORTING INFORMATION AVAILABLE Experimental methods, supporting figures, and a F€ orster distance calculation. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION Corresponding Author: *To whom correspondence should be addressed. E-mail: camnp@ iacs.res.in.

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ACKNOWLEDGMENT DST of India is acknowledged for funding.

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N.P. acknowledges LNJ Bhilwara Research Fellowships. S.S. and S.J. acknowledge CSIR for scholarships. (19)

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Doped Semiconductor Nanocrystals and Organic Dyes: An Efficient

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