Unique Energy Transfer in Fluorescein-Conjugated Au22

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Surfaces, Interfaces, and Catalysis; Physical Properties of Nanomaterials and Materials 22

Unique Energy Transfer in Fluorescein-Conjugated Au Nanoclusters Leading to 160-Fold pH-Contrasting Photoluminescence Kyunglim Pyo, Nguyen Hoang Ly, Sang Myeong Han, Mohammad Bin Hatshan, Abubkr Abuhagr, Gary P. Wiederrecht, Sang-Woo Joo, Guda Ramakrishna, and Dongil Lee J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b02130 • Publication Date (Web): 30 Aug 2018 Downloaded from http://pubs.acs.org on September 4, 2018

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The Journal of Physical Chemistry Letters

Unique Energy Transfer in Fluorescein-Conjugated Au22 Nanoclusters Leading to 160-Fold pH-Contrasting Photoluminescence Kyunglim Pyo,1 Nguyen Hoang Ly,2 Sang Myeong Han,1 Mohammad bin Hatshan,3 Abubkr Abuhagr,3 Gary Wiederrecht,4 Sang-Woo Joo,*2,5 Guda Ramakrishna,*3 Dongil Lee*1 1

Department of Chemistry, Yonsei University, Seoul 03722, Korea

2

Department of Chemistry, Soongsil University, Seoul 06978, Korea

3

Department of Chemistry, Western Michigan University, Kalamazoo, MI 49008, USA

4

Center for Nanoscale Materials, Argonne National Laboratory, Chicago, IL 60439, USA

5

Department of Information Communication, Materials Engineering, Chemistry Convergence Technology, Soongsil University, Seoul 06978, Korea

* Address correspondence to : [email protected], [email protected], or [email protected]

ACS Paragon Plus Environment

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Abstract

Accurate measurements of intracellular pH are of crucial importance in understanding the cellular activities and in the development of the intracellular drug delivery systems. Here we report a highly sensitive pH probe based on a fluorescein-conjugated Au22 nanocluster. Steadystate photoluminescence (PL) measurements have shown that, when conjugated to Au22, fluorescein exhibits more than 160-fold pH-contrasting PL in the pH range of 4.3-7.8. Transient absorption measurements show that there are two competing ultrafast processes in the fluorescein-conjugated Au22 nanocluster: the intracore-state relaxation and the energy transfer from the non-thermalized states of Au22 to fluorescein. The latter becomes predominant at a higher pH, leading to dramatic PL enhancement of fluorescein. In addition to the intrinsically low toxicity, fluorescein-conjugated Au22 nanoclusters exhibit high pH-sensitivity, wide dynamic range, and excellent photostability, providing a powerful tool for the study of intracellular processes.

TOC GRAPHICS

KEYWORDS: ultrafast energy transfer, intracellular pH sensing, gold nanoclusters, fluorescein, transient absorption spectroscopy


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Intracellular pH plays a critical role in many cellular processes, including cellular metabolism, signaling, proliferation, apoptosis and vesicle trafficking,1-3 and is tightly regulated in organelles. For a typical mammalian cell, the intracellular pH value varies from 4.7 in lysosome to 8.0 in mitochondria.4 Abnormal intracellular pH may reflect inappropriate cell functions and is observed in some common disease types such as cancer and Alzheimer’s.5-6 Since intracellular pH is strictly regulated7, small pH changes could dramatically alter the intracellular behaviour. For example, compared with the normal cells (pH 7.2) the pH of the transformed cells is only higher by 0.3-0.5 pH unit and apoptosis occurs at 0.3-0.4 decreased value8-9. Therefore, it is of crucial importance to develop a highly sensitive method that can reliably detect the small pH changes in a wide pH range. Methods that can measure the intracellular pH include H+ ion selective microelectrodes, nuclear magnetic resonance (NMR), analysis of metabolites or pH indicators inserted in the cells, absorption and fluorescence spectroscopy using pH-sensitive probes.10-13 Among these, fluorescence spectroscopy offers a special advantage in monitoring of the intracellular pH of intact cells and subcellular compartments with excellent spatial and temporal resolution. Organic dyes have been extensively developed as pH probes for intracellular pH sensing.14 These pHsensitive fluorophores, however, typically exhibit poor photobleaching resistance, limiting their use in imaging experiments for an extended period of time.15 Moreover, they tend to aggregate in the cell or undergo rapid cell leakage, which also limits their use in long-term monitoring of the intracellular pH.14 Alternatively, fluorescent probes based on semiconductor quantum dots (QDs) that exhibit bright luminescence and excellent photostability have been developed.16-17 In particular, QDs have been found to be useful in a Förster resonance energy transfer (FRET) pH


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sensor that comprises QD donor and pH-sensitive fluorophore acceptor.18-20 However, their intrinsic toxicity keeps them from their wide use in biological applications. 21-23. Thiolate-protected gold nanoclusters have received much attention recently because of promises offered by their unique optical, electrochemical and catalytic properties.24-29 These ultrasmall nanoclusters have special stability at certain compositions, and, therefore, atomically monodisperse nanoclusters can be obtained from various size-selective syntheses. It has been reported that these ultrasmall gold nanoclusters display discrete electronic transitions, indicating their

molecule-like

properties.24-27

In

particular,

these

nanoclusters

exhibit

notable

photoluminescence (PL) that has attracted significant research interest in the development of optical probes for biological sensing and imaging.30-32 In a previous study, we reported design strategies to prepare highly luminescent Au22 nanoclusters via rational surface engineering. By conjugating pyrene to Au22, the PL of Au22 shows a dramatic increase by the rigidifying effect and the resonance energy transfer sensitization by pyrene.33 In this work, we present a novel strategy to enhance the pH sensitivity by conjugating pH-responsive dye, aminofluorescein (AF), to gold nanoclusters, such as Au22. As opposed to the expected resonance energy transfer from AF to Au22, ultrafast energy transfer from the nonthermalized states of Au22 to AF was observed from combined steady-state PL and transient absorption measurements. The ultrafast energy transfer sensitization in AF-conjugated Au22 led to remarkable pH-contrasting luminescence in the pH range of 4.3-7.8. In addition, the AFconjugated Au22 displayed high pH-sensitivity, excellent photostability, and low toxicity for intracellular pH measurements.


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Figure 1. (a) Synthetic scheme of Au22-AF. (b) Positive mode MALDI mass spectra of Au22SG18 (black), Au22-CBz (blue) and Au22-AF (red). Arrows indicate the m/z value corresponding to Au22SG18 (bottom), Au22 protected with 18 CBz (middle), and Au22 protected with 18 CBz and 2 AF (top). (c) Absorption spectra of Au22SG18, Au22-AF and SG-AF in pH 7.8 PBS buffer solution. (d) PL spectra (λEX = 490 nm) of SG-AF and Au22-AF in pH 7.8 PBS buffer solution. The concentrations of SG-AF and Au22-AF solutions were adjusted to exhibit the same AF absorbance (0.013).

Figure 1a shows the synthetic scheme for the preparation of fluorescein-conjugated Au22SG18 (Au22-AF), where SG is glutathione. The starting Au22SG18 nanoclusters were synthesized following a procedure reported elsewhere32. The Au22 nanoclusters are highly water soluble and display broad emission around 665 nm with a quantum yield of 6.5 %. To prevent potential interparticle coupling, the primary amine group of glutathione was first protected with benzyl chloroformate (CBz-Cl).33-34 AF was then conjugated to the CBz-protected Au22 nanocluster

through

an

amide

coupling

reaction

by

using

(1-ethyl-3-(3-


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dimethylaminopropyl)carbodiimide hydrochloride (EDC) under carefully controlled pH conditions. (see Supporting Information for detailed synthetic procedure) The resulting AFconjugated Au22 will be abbreviated as Au22-AF. AF-conjugated glutathione (SG-AF) was synthesized similarly. The synthesized Au22-AF was confirmed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry and 1H NMR spectrometry. As can be seen in Figure 1b (black line), the mass spectrum of Au22SG18 shows an intact ion at m/z 9,865 Da along with the fragment ions in the m/z range of 8,000–9,500 Da, corresponding to Au18SG14, Au19SG15, Au20SG16, and Au21SG17 cations. The mass spectrum of CBz-protected Au22 (Au22-CBz, blue line) exhibits a broad peak in the m/z range of 10,400-12,300 Da, which is composed of fragment ions generated from Au22-CBz. The arrow indicates the m/z value of 12,261 Da corresponding to Au22 cation fully protected with 18 CBz groups. The mass spectrum (red line) of Au22-AF also shows a broad peak in the m/z range of 12,000-13,700 Da, which consists of the intact and fragments ions of Au22-AF. Specifically, the arrow indicates the m/z value (12,936 Da) corresponding to Au22-AF ion conjugated with 18 CBz and 2 AF. 1H-NMR analysis shown in Figure S1 (Supporting Information) also shows similar result that there are on average 16 CBz and 2 AF conjugated to Au22 nanocluster. These results unequivocally suggest that the integrity of Au22 nanocluster was well preserved during the surface functionalization processes.


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Figure 2. (a) Absorption spectra and (b) PL spectra (λEX = 490 nm) of Au22-AF in various PBS buffer solutions (pH 4.3-7.8). (c) Point-to-point line graph of the pH-dependent PL intensity for Au22-AF and SG-AF. (d) Excitation spectra of SG-AF and Au22-AF in pH 7.8 PBS buffer solution (λEM = 517 nm). The excitation spectra were normalized at 490 nm to show the PL difference in 300-460 nm clearly. Inset shows the excitation spectrum (λEM = 665 nm) of Au22SG18 in pH 7.8 PBS buffer solution. The absorption spectrum of Au22-AF in Figure 1c clearly shows the characteristic absorption of AF at 490 nm. Comparing the absorbance of Au22-AF at 490 nm after correcting for the Au22 absorption with that of SG-AF, it can be confirmed that there are on average 2 AF conjugated to the Au22-CBz. This is in good agreement with mass spectrometry and NMR analysis. In Figure 2d, however, it can be found that the PL intensity of Au22-AF is much higher than that of SG-AF. Note that the concentrations of both Au22-AF and SG-AF in phosphatebuffered saline (PBS) solution (pH 7.8) were adjusted to exhibit the same absorbance at 490 nm.


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The dramatically enhanced AF luminescence suggests that there is certain contribution from Au22 in the PL process of AF in Au22-AF. It is well known that fluorescein derivatives, such as AF, carboxyfluorescein and fluorescein isothiocyanate (FITC), display pH dependent emission.35 The pH dependence is associated with the acid dissociation and spirocycle ring-opening processes when pH is higher than the pKa of the fluorescein derivative as illustrated in Figure 1a. The dianionic form of AF (pKa = 6.3)36-37 is responsible for the higher fluorescence observed at elevated pHs. To better understand the role of Au22 in the PL enhancement of AF in Au22-AF, we monitored absorption and PL spectra of Au22-AF at various PBS buffer solutions with pH from 4.3 to 7.8 and compared with those of SG-AF obtained at the same AF concentration. In Figure S2, the absorbance of SG-AF at 490 nm increased by 8-fold when the solution pH was raised from 4.3 to 7.8. Similarly, the PL intensity at 515 nm shows 10-fold increase from 4.3 to 7.8 (Figure S2). Comparing the pH-dependent increases of absorbance and PL, one can conclude that the PL increase mainly arises from the pH-dependent absorption of SG-AF. In Figure 2a, the absorption of AF in Au22-AF at 490 nm also increased by 8-fold when the solution pH increased from 4.3 to 7.8. However, the PL intensity of Au22-AF at 517 nm dramatically increased by 165-fold when pH was raised from 4.3 to 7.8. The PL increases with increasing pH for Au22-AF and SG-AF are compared in Figure 2c. This is surprising and may indicate an important role of Au22 in the PL increase of AF in Au22-AF. The excitation spectrum of Au22-AF in Figure 2d evidently corroborates the role of Au22 in the PL of AF. As can be seen in the figure, in addition to the spectral features of AF with peaks at 285, 320 and 490 nm there is a broad excitation found in 300-460 nm region. Interestingly, the broad excitation profile matches with that of Au22SG18 (Figure 2d inset),


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suggesting the contribution of Au22 in the PL enhancement of AF in Au22-AF. The broad excitation profile increases along with the AF peaks as pH increases from 4.3 to 7.8 as shown in Figure S3. It is well documented that fluorescence quenching occurs in the vicinity of gold nanoparticles38-41 via the Förster resonance energy transfer process.42-43 In a previous study of pyrene-conjugated Au22 system33, we have shown that resonance energy transfer from pyrene to Au22 efficiently occurs when the electron transfer deactivation pathway is blocked, leading to dramatic PL enhancement of Au22. Considering the emission energies of AF (517 nm) and Au22 (665 nm) and the spectral overlap between the emission and absorption, one would expect efficient resonance energy transfer from AF to Au22. Surprisingly, the excitation spectra of Au22AF in Figure 2d and Figure S3 suggest that the reverse energy transfer, i.e., from Au22 to AF, increases with increasing pH.

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Figure 3. Transient absorption spectra at short time delays for (a) SG-AF at pH 7.5, (b) Au22SG18, (c) Au22-AF at pH 4.5 and (d) Au22-AF at pH 7.5. To probe the role of Au22 in the PL enhancement of AF, pH-dependent transient absorption measurements were carried out. Transient absorption measurements have been very powerful in investigating the excited-state interactions in gold nanoclusters28, fluorophore-conjugated gold nanoclusters.33,

51-53

44-50

as well as

Figure 3a shows the transient absorption

spectra at different time delays for SG-AF in a pH 7.5 buffer after excitation at 370 nm. Immediately after excitation, a negative absorption with a maximum around 495 nm was observed that can be attributed to a combination of ground state bleach and stimulated emission. As can be seen from Figure 3a, the negative absorption recovered quickly and more than 99 % was recovered within 100 ps. The bleach recovery was fitted to a bi-exponential function (∆ = / + / ; where a1, a2 are the amplitudes and , are lifetimes) with time constants of 1.8 ps and 15 ps. (Figure S4a) Average lifetime ( = ∑ / ∑ of 11.5 ps was determined from the obtained lifetimes. In Figure 3b, the transient absorption analysis of Au22SG18 shows decay at a shorter wavelength region ( 600 nm), indicating the intracore-state relaxation.33 In Figure S4b, the species-associated spectra obtained after global fit analysis show 220 fs intracore-state relaxation, followed by long-lived excited state. Significantly different transient features are observed for Au22-AF in two buffer solutions of pH 4.5 and 7.5; one is lower and the other is higher than the pKa of AF. Figure 3c shows the transient absorption spectra from 100 fs to 1.5 ps for Au22-AF at pH 4.5 after excitation at 370 nm. From Figure 1c, one can see that the 370 nm excitation preferentially excites the Au22 as very little is absorbed by AF at this wavelength. The initial transient absorption spectral feature of Au22-AF at pH 4.5 is rather similar to that of Au22SG18 with prominent decay at a shorter


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wavelength region and growth at a longer wavelength region, suggesting the intracore-state relaxation. In Figure 3d, the transient spectral features at different time delays from 100 fs to 650 fs at pH 7.5 show significantly different spectral features. The transient absorption spectrum immediately after excitation at 100 fs shows small positive absorption below 500 nm that decays immediately to give rise to a negative absorption with a maximum of 505 nm. Again, the 370 nm excitation mostly excites the Au22 rather than AF. This result strongly suggests that there is energy transfer from Au22 to AF as the negative absorption at 505 nm indicates the contribution from AF. In Figure S4c, the species-associated spectra obtained from global fit analysis for Au22AF at pH 7.5 show three main components (200 fs, 22 ps and long). The 200 fs spectrum shows a positive absorption at 505 nm, the reverse of the absorption of SG-AF shown in Figure 3a, implying the growth of AF with this time constant. The 22 ps component matches well with the SG-AF transient spectrum as compared in Figure S4d. The long (> 1 ns) component reasonably matches with the > 1 ns transient of Au22SG18 (Figure S4b, red line). These results unequivocally suggest that ultrafast energy transfer from Au22 to AF occurs at pH 7.5 when Au22-AF is excited at 370 nm. To understand the origin of the pH dependence, we carried out transient absorption measurements of Au22-AF at five different pHs starting from 4.5 to 7.5. The spectra at 4.5 and 7.5 show significantly different features and the transient spectra in the intermediate pH regions show a combination of both. The species associated spectra obtained from global fit analysis for Au22-AF at pHs 4.5-7.0 (Figure S5a-d) show the similar ultrafast relaxation, followed by a picoseconds growth component that matches with the stimulated emission of AF and the longlived transients matching with Au22 nanocluster. As discussed above, there is an ultrafast (220 fs)


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component for Au22 nanoclusters that is associated with the intracore-state relaxation. Interestingly, the ultrafast component of Au22-AF shows very distinct pH dependence as can be seen in Figure S5. That is, this decay component is longer than 300 fs at shorter wavelength regions at pH 4.5 and 5.5 and it becomes significantly shorter (200~230 fs) when pH is higher than 6.5. This ultrafast component is comparable or even faster than the intracore-state relaxation at higher pH regions above the pKa. (a)

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250 200 150 100

4.5 5.0 5.5 6.0 6.5 7.0 7.5 pH

Figure 4. Kinetic decay traces at different pHs for Au22-AF at (a) 485 nm and (b) 700 nm. (c) Plot of lifetimes at 485 nm and 700 nm as a function of pH.


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To understand this phenomenon more clearly, kinetic decay traces of Au22-AF at different pHs are compared at two different wavelengths, 485 nm and 700 nm. The 485 nm wavelength is chosen as it is close to the absorption maximum of AF while the 700 nm represents the region where the contribution is only from Au22 as little transient signal is observed for SG-AF at 700 nm. The kinetic decay comparison at 485 nm shown in Figure 4a clearly shows that the decay is slower; 280 fs and 310 fs at pH 4.5 and 5.5, respectively, but becomes faster (190-210 fs) when pH is higher than 6.5. On the other hand, the kinetic decay comparison at 700 nm (Figure 4b) shows that the intracore-state relaxation is faster (220 fs) for Au22-AF at pH 4.5 and becomes gradually slower with increasing pH. The fitted faster decay components at 485 nm and growth component at 700 nm are plotted as a function of pH in Figure 4c. The decay at 485 nm reflects the energy transfer from Au22 to AF while the growth at 700 nm represents the intracore-state relaxation. As seen in Figure 4c, the energy transfer becomes significantly faster above pH 6.5 (black circles) and at the same time, the intracore-state relaxation is gradually slowed down with increasing pH (red circles). The reason for the slower intracore-state relaxation at higher pH is unclear at this juncture, but it has been reported that the intracore-state relaxation can be affected by the ligand shell surrounding the Au13 core in Au25 and other gold nanoclusters.50 The dianionic form of AF formed at higher pH may induce some changes in the ligand shell, which in turn influence the intracore-state relaxation as was observed for Au25 nanoclusters.50 The comparison shown in Figure 4c shows the competing nature of two ultrafast excitedstate relaxation processes; the intracore-state relaxation and energy transfer at various pHs. The results indicate that, at lower pHs, the intracore-state relaxation is dominant while the energy transfer is dominant above the pKa of AF. There have been a number of studies showing the


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competing nature of the ultrafast relaxation processes.54-58 In the study of metalloporphyrin and [Ru(bpy)3]2+ dyads, Harriman et al. have shown the competition between the internal conversion and the ultrafast energy transfer from the non-relaxed states.55 Recent studies have shown that generalized resonance energy transfer theories can be applicable to the excitation energy transfer from non-thermalized excited states that also relies on the spectral overlap between the donor emission and acceptor absorption.59-60 Figure 2a shows that the absorption integral of AF increases with increasing pH. Thus, it is expected that the higher spectral overlap is expected at higher pHs, leading to faster energy transfer rate and greater energy transfer efficiency. That is, as the spectral overlap integral increases with increasing pH, the rate of energy transfer increases and becomes dominant over the intracore-state relaxation. The competing ultrafast energy transfer and the intracore-state relaxation are illustrated in Scheme 1.

Scheme 1. Schematic diagram depicting the competing pathways of the intracore-state relaxation and the non-thermalized energy transfer from Au22 to AF in Au22-AF.

As the energy transfer is found to be faster than the intracore-state relaxation at elevated pHs, it probably involves the non-thermalized excited states of Au22. To the best of our knowledge, this is the first example showing the non-thermalized energy transfer in gold


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nanoclusters. The non-thermalized energy transfer has been observed only in particular molecular systems such as porphyrins and carotenoids where the higher excited states are fairly long-lived.58,60,63 It has also been found that sub-picosecond excitation energy transfer can be utilized in photosynthetic light harvesting systems.61 The present results of Au22-AF have clearly demonstrated that it is indeed possible to harness the hot excited states of gold nanoclusters and to realize energy transfer from such states. Unlike the gold nanoparticles that exhibit ultrafast relaxation with lifetimes shorter than the instrument response limit,50 the molecule-like discrete energy level structure of Au22 with retarded intracore-state relaxation allows for efficient energy transfer from the non-thermalized states, as depicted in Scheme 1. We note that the nonthermalized Au22–to-AF energy transfer is much faster than the time constants typically observed for the Förster resonance energy transfer in fluorophore-conjugated gold nanoclusters.33 The pHdependent kinetics monitoring the intracore-state relaxation has shown slower dynamics at higher pHs. In contrast, the kinetics monitoring excitation energy transfer has shown faster rate at higher pHs. The increase in the rate of energy transfer can be ascribed to the enhanced spectral overlap at higher pHs. The pH-dependent transient absorption results presented here unequivocally show the evidence for ultrafast energy transfer from Au22 to AF at higher pHs, leading to remarkable luminescence contrast. Accurate measurements of intracellular pH are of crucial importance in understanding the cellular activities and in the development of the intracellular drug delivery systems.62-63 The remarkably pH-dependent PL of Au22-AF has prompted us to explore the possibility of using it as an intracellular pH indicator. To monitor the intracellular pH, the fluorescence images of HeLa cells co-incubated with Au22-AF were taken using an inverted fluorescence microscopy with a single excitation wavelength of around 490 nm. For comparison, images of HeLa cells co-


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incubated with SG-AF were also obtained. The concentrations of Au22-AF and SG-AF coincubated with the HeLa cells were adjusted to have the same amount of AF. The cells were thoroughly washed to remove the adsorbed fluorophores on the cell membrane so that Figure 5 represents the fluorescence images of Au22-AF and SG-AF located in the cells.64-65 The pH was then adjusted by using Dulbecco's phosphate-buffered saline (DPBS) in the presence of nigericin that homogenizes the intracellular pH and extracellular pH.66

Figure 5. (a) Fluorescence images of HeLa cells co-incubated with Au22-AF and SG-AF, after adjusting the pH using DPBS in the presence of nigericin. (b) Bar graph showing pH dependent PL intensities of Au22-AF and SG-AF in HeLa cells shown in (a).


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As can be seen in Figure 5a (and Figure S6), the PL of the Au22-AF in HeLa cells increases dramatically with increasing pH. The pH-dependent fluorescence intensity in Figure 5b shows more than 30-fold increase as pH is raised from 4.5 to 7.5, whereas the fluorescence increase found for HeLa cells incubated with SG-AF is only 1.2-fold. Apparently, these are less than those found in Figure 2, 165-fold and 10-fold for Au22-AF and SG-AF, respectively, suggesting that there are some interferences present in the inner cell environment. Nonetheless, the pH-contrasting fluorescence found for Au22-AF in HeLa cells is remarkable. In addition, it can be observed in Figure 5b that the pH-dependent fluorescence is fairly linear in the pH range between 5.0 and 7.5. It is often found that the pH range is limited by the pKa of the pH-dependent fluorophore, which typically exhibits a sigmoidal calibration curve in a pH range of the pKa±1.67 In Au22-AF, there are two AF moieties conjugated to Au22 that is protected with 18 glutathione, a polyprotic acid. It is thus expected that the dissociation of AF can be considerably affected by the presence of polyprotic acids in the ligand shell, leading to a fairly linear calibration curve in the wide pH range as shown in Figure 5b. It has been found that the rapid photobleaching of pH-sensitive organic dyes poses a major disadvantage in the study of intracellular environment that often requires time-course fluorescence imaging for an extended period of time to track cellular processes.68-69 For example, it was reported that a common pH indicator, FITC, is photobleached by more than 80 % within 5 min70-71 Also, cyanine dyes (Cy3, Cy5, Cy7) are known to be photobleached almost completely within 20min.72-73 Au22 nanoclusters have been found to exhibit excellent photostability.34 It is thus expected that the energy transfer sensitization from Au22 to AF may enhance the photostability of AF in Au22-AF. To examine the photostability of Au22-AF, live cell images of Au22-AF co-incubated with HeLa cells were taken under the irradiation of a mercury lamp for 45


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min. using an excitation filter (band pass 482/35 nm) as shown in Figure S7. Live cell images of SG-AF were also obtained for comparison. As can be seen in the figure, the fluorescence decreases observed for Au22-AF and SG-AF were found to be 10 % and 34 %, respectively, during 45 min., indicating improved photostability for Au22-AF. This can be attributed to the effect of the energy transfer sensitization by the photostable Au22. This effect becomes more significant when Au22-AF is excited at

Unique Energy Transfer in Fluorescein-Conjugated Au22

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