Structure-Correlated Dual Fluorescent Bands in BSA-Protected Au25

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Structure-Correlated Dual Fluorescent Bands in BSA-Protected Au25 Nanoclusters Xiaoming Wen,* Pyng Yu, Yon-Rui Toh, and Jau Tang* Research Center for Applied Sciences, Academia Sinica, Taipei, Taiwan ABSTRACT: In this work, we investigated the temperature-dependent luminescence of bovine serum albumin-protected Au25 nanoclusters and the correlation with their structure. Our experiments reveal that the red luminescence consists of two bands, namely, band I at 710 nm and band II at 640 nm. The temperature dependence of band I exhibits similarity to semiconductors, such as a red shift of emission and bandwidth broadening upon increasing temperatures due to electron−phonon and electron−defect/surface scattering. In contrast, band II exhibits different temperature dependence. It is concluded that band I exclusively originates from the icosahedral core of 13 Au(0) atoms and band II dominantly arises from the [−S−Au(I)−S−Au(I)−S−] staples. Moreover, with increasing temperatures, the intensity of band I and band II decreases. A similar activation energy was extracted, which is attributed to thermally activated defect/surface trapping. In addition, the relaxation from band II to band I was found to be inactive from 300 K down to 77 K.

1. INTRODUCTION Gold nanoclusters (NCs) comprising few to tens of atoms have been shown to possess distinct optical, magnetic, and catalytic properties.1,2 With the size comparable to the Fermi wavelength of electrons, these NCs exhibit discrete energy levels and molecule-like properties in the absorption and fluorescence features.2−4 The luminescence of gold NCs has drawn considerable research interest for both fundamental understanding and applications in bioimaging and photonics.2,5,6 The crystal and electronic structures of Au25 NCs have been extensively studied.7−10 It has been shown that Au25 NCs have a core−shell structure in which 13 Au(0) atoms form an icosahedral core surrounded by six Au2(SR)3 staples,3,5 and Au25 NCs are embedded in the thiol group of cysteine in bovine serum albumin (BSA).11 The optical properties of Au25 NCs have been studied by various techniques,8,12 such as femtosecond laser pump−probe techniques,13,14 ultrafast upconversion luminescence,15,16 and temperature dependence.17,18 Zhu and co-workers have correlated the structure with the optical transitions.8 Link et al. observed two luminescence bands at 1.5 and 1.15 eV in glutathione-protected Au28 NCs and assigned to fluorescence and phosphorescence, respectively.6 The strong luminescence in the red has been observed by many groups, and different origins were proposed. To date, it is debating that the red luminescence originates from the intraband transition of the lowest unoccupied molecular orbital (LUMO) to the highest occupied molecular orbital (HOMO)6 or arisen from the interband transition between the 6sp conduction band and the filled 5d band.3,15,19 Despite many theoretical and experimental works on the optical properties of Au25 NCs, fundamental understanding of the fluorescence is far from complete. Several significant questions, the mechanism and the origin of the luminescence, © 2012 American Chemical Society

the electron−phonon interactions, the role of the core, and the semiring and ligands in NCs, still remain unclear.3,5,20 It has been shown that temperature dependence of optical could provide physical insight of the nature for fluorescent gold NCs. Temperature-dependent optical properties in gold NCs have been reported rarely.17,18,20,21 The detailed fundamental understanding, in particular for the luminescence of Au25 NCs, is still not available. The significant difficulties include the broad luminescence band without structure in Au25 NCs and variation with capping ligands. To acquire a deep insight for the fundamental understanding, in this work, we focus on the temperature-dependent luminescence of Au25 NCs. Experiments reveal that the red luminescence consists of two bands. The band I behaves similarly to semiconductors because of correlating exclusively to the icosahedral core, and the band II exhibits very different temperature dependence due to dominant contribution originating from six −S−Au(I)−S−Au(I)−S− staples. The roles of electron−phonon scattering, Au(I)−Au(I) interaction, and exterior ligands will be addressed.

2. EXPERIMENTAL METHODS 2.1. Synthesis of BSA/Au25 NCs. The Au25 NCs used in this study were synthesized using a biomineralized approach developed by Ying et al.22 Typically, 5 mL of 10 mM HAuCl4 was mixed with 5 mL of 50 mg/mL BSA and kept at 37 °C overnight in the incubator. The clusters composed of 25 gold atoms were formed and stabilized by the thiol group of cysteine in BSA. Received: April 12, 2012 Published: May 7, 2012 11830

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2.2. Spectroscopy Measurements. In fluorescence measurements, a 406 nm CW laser was used as an excitation source. Fluorescence was collected into a MicroHR spectrometer (HORIBA Jobin Yvon) and recorded by a cooled CCD (SynapseTM CCD). For temperature-dependent experiments, the sample was installed in a cryostat (ST500). The lifetimes were measured by time correlated single photon counting (TCSPC) technique on a Microtime-200 system (Picoquant) with an excitation of 467 nm laser. Au25 film samples were fabricated by conventional drop casting.

to the red band, we observed a fast component with a lifetime of 3.2 ns followed by a significantly slow component with a lifetime of 1.5 μs at room temperature, similar to other observations.3,19 At 500 nm, corresponding to the weak blue band centered at 470 nm, the fluorescence exhibits a lifetime of 2.5 ns. This is different from that reported by Devadas et al. in which they found as fast as 100 fs decay in Au25(C6S)18 and Au25(GS)18 in the blue band.15 This likely is due to the influence of different ligand.3 The luminescence of Au25 NCs was measured as a function of temperature, as shown in Figure 3. With increasing temperatures,

3. RESULTS AND DISCUSSION As shown in Figure 1, the absorption peak appears at around 520 nm, and the strong red fluorescence locates around 690 nm,

Figure 3. Luminescence of Au25 NCs as a function of the temperature. The dashed lines represent two Gaussian components of the luminescence at 300 K.

Figure 1. Absorption and luminescence of pure BSA and Au25 NCs. The luminescence band (blue band) of BSA significantly decreases when Au25 NCs forms.

the intensity of luminescence monotonously decreases. At each temperature, the red band can be nicely fitted by two Gaussian functions, with the fitted parameters summarized in Table 1. These findings suggest that the red band originates from two transitions.

22

consistent with the observation by Xie et al. It should be noted that the absorption spectrum is different from those of glutathione15 and −SRSC6H13 or SC12H253 protected in which an absorption peak appears around 650 nm. In BSAprotected Au25 NCs, an absorption peak appears between 470 and 550 nm, and no structure was observed after 600 nm. 22 A weak blue fluorescence was observed around 470 nm, which could be attributed to BSA emission because the pure BSA exhibits similar fluorescence in this region, and a decreased blue emission was observed when forming BSA-protected Au25.23,24 Figure 2 shows time evolution of fluorescence in BSAprotected Au25 NCs measured by TCSPC. At 600 nm, corresponding

Table 1. Fitted Parameters of the Red Band at 300 K Using Two Gaussian Functions

band I band II

central wavelength (nm)

bandwidth (meV)

704.1 ± 0.29 (1.76 eV) 639.1 ± 0.27 (1.94 eV)

255.1 ± 1.0 (253.5 meV) 164.9 ± 2.4 (164.8 meV)

intensity ratio AI/AII 9.47

We extracted the fitted parameters of the band I and band II as a function of temperature from 77 K to 300 K. With increasing temperature, the energy gaps of band I exhibit a red shift of 11 meV, as shown in Figure 4. In contrast, band II displays a blue shift of 10 meV. The temperature-dependent energy gap has been extensively studied in semiconductor bulk and quantum dots.25,26 The dominant mechanism is renormalization of band energies by electron−phonon interactions, while thermal lattice expansion typically has a negligible effect. A red shift of tens of meV in the energy gap was usually observed in semiconductor quantum dots for temperature changes from 77 K to room temperature.25−27 The temperature dependence of the band gap is often explained using empirical Varshni relation or an expression proposed by O'Donnell and Chen for core/shell quantum dots based on an analysis of the electron−phonon coupling mechanism.28,29 Eg (T ) = Eg (0) − 2SEphonon /[exp(Ephonon /KBT ) − 1]

Figure 2. Time evolution of luminescence in Au25 NCs at 500 and 600 nm, corresponding to the blue band and the red band, respectively.

(1) 11831

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energy band gap decrease and a red shift. At present, the detailed lattice expansion data and the correlation with the red shift in Au25 NCs are not available. Therefore, it is difficult to estimate the red shift that arises from the thermal expansion. The evolution of emission energy could be nicely fitted by eq 1, which suggests that the dominant mechanism is electron−phonon scattering. Similar to semiconductor quantum dots, the red shift that arises from decreased quantum confinement due to thermal expansion is negligible.27 Consequently, band I exhibits a red shift upon increasing temperature. In contrast, band II exhibits a blue shift. We suggest that band II dominantly originates from the −S−Au(I)−S−Au(I)− S− staples. Fackler and co-workers have investigated the luminescence properties of various Au complexes.35,36 It has been shown that Au(I) has a d10 electron structure, there exists intramolecular or intermolecular Au(I)−Au(I) interaction for many Au(I) complexes, and the Au(I)−Au(I) interaction results in aurophilicity and plays an important role in the determination of the emission wavelength.35−39 In particular, Au(I) thiolate complexes were synthesized and characterized, and it was concluded that the emission can be affected by both the presence of Au(I)−Au(I) interactions and the substituents on the thiolate ligands.36 The emission band of Au(I) compound exhibits a blue shift with increasing temperatures due to an increasing Au(I)−Au(I) separation.36,40 The relationship between the emission energy and the separation between Au(I)−Au(I) has been used to explain the photophysical properties of several Au(I) complexes in solid states.35 In Au25 NCs, it is expected that the six [−S−Au(I)−S−Au(I)−S−] staples can act as Au(I) complexes. Upon increasing temperature, the separation of Au(I)−Au(I) increases due to thermal expansion,35 and thus, the emission energy exhibits a blue shift. It should be emphasized that BSA also exhibits thermal expansion with increasing temperatures. It was shown that the occupied volume of a protein increases by 3% from 80 to 300 K.41 With further increasing temperatures, the blue shift of emission tends to saturate near room temperature, most likely due to the spatially restraint of BSA. With increasing temperature, the volume of BSA increases, which can spatially restrain the expansion of staples at a higher temperature. Therefore, above 250 K, the separation of Au(I)−Au(I) cannot be increased further. The emission energy of band II will not increase further with further increasing temperatures. The bandwidth also exhibits temperature-dependent variation, as shown in Figure 5. Basically speaking, the bandwidth of both band I and band II increases with increasing temperatures. It has been shown that acoustic phonon scattering and surface/ defect/ionized impurity scattering are the major mechanisms for bandwidth broadening in core/shell semiconductor nanocrystals.42 Spectral bandwidth essentially is determined by the electron relaxation time,43,44 Γ = (1/2πc) × (1/T2) = (1/2πc)[(1/2·T1) + (1/T2*)], where T1 is the population time (radiative and nonradiative processes), T2 represents the total dephasing times, and T2* is the pure dephasing time, which may originate from collision with electrons. For metal materials, the electron relaxation time is determined by electron−electron, electron−phonon, and electron−surface/defect scattering processes. In particular, electron−surface/defect scattering has an increased effect in NCs due to a significant increase in the surfaceto-volume ratio. It has been shown that the electron−phonon interaction is the dominant factor for gold bulk, and the collision of conduction electrons and surface would result in a significant

Figure 4. Energy gap of the band I and band II as a function of temperature. The solid line is the fitted curve based on eq 1.

where S is the Huang−Rhys factor and Ephonon is the average phonon energy. The energy gap of band I could be nicely fitted using eq 1 with the extracted parameters as: Eg(0) = 1.769 ± 0.005 eV, S = (5.91 ± 1.2) × 10−4, and ELO = 52.0 ± 3.7 meV. The relative small S represents the weaker electron−phonon coupling, which is consistent to the results in other studies.30,31 S = 1.95 and S = 1.57 were found in InP/ZnS quantum dots and CdSe/CdS dots-in-rods, respectively.25,32 The energy of phonons is close to the result obtained from the temperaturedependent absorption (43 ± 6 and 54 ± 7 meV for Au25 and Au38, respectively) by Devadas et al.20 However, the total shift of 11 meV is much smaller than the shift of 90 meV extracted from the absorption.20 Crystal and electronic structures of thoilate-protected Au25 NCs have been studied.7,8 The Au25 NC consists of an icosahedral core in which 12 Au(0) atoms form the vertices of the icosahedron and enfold one central Au(0) atom. Then, six dimeric −S−Au−S−Au−S− staples form a semiring surrounding the core.7,33 It is noteworthy that the BSA-protected Au25 NCs comprising the icosahedral core and the Au(I)−S semiring have been demonstrated recently.11 Pradeep et al. studied the evolution of Au25 NCs formation in BSA and confirmed that Au25 NCs form through a protein-bound Au(I) intermediate and subsequent emergence of free protein and Au(I)−S staple.34 These staples are directly anchored to the surface via Au−S bonding, while the staples interact with the icosahedral core via Au−Au bonds. In this structure, each Au25 NC consists of 13 Au(0) in the core and 12 Au(I) in the staples, as well as various Au−Au and Au−S bonds. They are closely correlated to the optical transitions.8,10 Time-dependent density functional theory (TDDFT) calculations of thiol-protected Au25 NCs have been performed by Zhu et al.8 It has been shown that some states, such as HOMO, LUMO, and LUMO + 1, are composed mostly of atomic orbital contribution from the core. The other states originate from the staples. A different contribution source could result in very different properties. On the basis of the experimental observation and the correlation between the electronic structure and the optical transitions, we propose that the band I originates predominantly from the icosahedral core. With increasing temperatures, enhanced electron−phonon interactions can result in a red shift of emission energy. At the same time, the lattice length within the icosahedral core of Au25 NCs and the diameter of the core increase due to thermal expansion, which results in an 11832

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structure is quite complicated. It is expected that the vibration of Au(I) in the semirings will be affected by the ligands. Au(I)− phonon scattering and Au(I)−defect/surface scattering are dominant in the low-temperature regime, and thus, the bandwidth increases with increasing temperatures. With further temperature increases, enhanced phonon scattering and thermal expansion are expected, not only for Au25 NCs but also for BSA ligands.41 It should be noted that each Au25 NC is embedded in a thiol group of cysteine in BSA.11 With increasing temperatures, Au(I) gradually touches the thiol group of cysteine more tightly. This structure will interfere with the intrinsic phonon mode of Au(I) and restrain the electron− phonon scattering in the semirings. Similar to the observation in other compounds, large branches of oligomer can dramatically influence their electronic properties and photophysical properties.46 The origin of the red band still remains unclear. The similar red emission around 640−750 nm was observed in Au25 NCs with various ligands, which suggests that the red band closely correlated to the cluster itself and roughly obeys the N1/3 law based on quantum confinement.3,4,15,19 On the other hand, it has been shown that ligands can significantly affect the emission, both wavelength and spectrum shape.2,3,15,17,22 Luminescence of Au25 NCs was observed by many groups, and different fluorescent wavelengths were found for Au25 NCs protected by different ligands between 640 and 750 nm, such as BSA at 640 nm22 and 674 nm,47 phenyl ethane thiol at 750 nm,3 glutathione at 700 nm,15 MSA at 700 nm,17 pepsin at 670 nm,48 and DHLA at 684 nm.49 According to Zhu's calculations, thiol-protected Au25 NCs have a HOMO−LUMO gap at 1.37 eV.8 Therefore, the red band cannot originate from the transition of LUMO to HOMO. On the basis of the electronic structure of Au25 NCs and luminescence experiments,7−9 one possibility is that band I and band II correspond to the transition of LUMO + 1 to HOMO and LUMO + 2 to HOMO, respectively. The band I exhibits a relatively larger bandwidth, 253.5 meV, because LUMO + 1 consists of three degenerated states, while LUMO + 2 is a single one, and thus a small bandwidth of 164.9 meV. The separation between band I and band II, 180 meV, is reasonably close to the gap of LUMO + 1and LUMO + 2 of about 200 meV.8 Moreover, it has been shown that LUMO + 1 is composed mostly of atomic orbital contribution from the icosahedral core8 and LUMO + 2 dominantly originates from the Au(I)−S staples; it is consistent with the observed temperature-dependent behavior in band I and band II. The calculated absorption gap of 2.314 eV (535 nm) is consistent to the absorption and luminescence excitation spectra with a peak around 520 nm. However, to gain a strong luminescence from the transitions of LUMO + 1 to HOMO and LUMO + 2 to HOMO, the relaxation of LUMO + 1 and LUMO + 2 must be very slow. Otherwise, the red band should be very weak. The time evolution of the red luminescence exhibits a μs lifetime; this suggests that the relaxation should be extremely slow. It is still an open question why the relaxation from LUMO + 1 and LUMO + 2 is not active. To study the nonradiative processes in the relaxation, we analyzed the temperature dependence of luminescence intensity. The integrated intensity of each band decreases with increasing temperatures. At 406 nm excitation, electrons are excited into a higher excited state. According to absorption spectra (Figure 1), both BSA and Au25 NCs can absorb the photons for 406 nm excitation. In other words, electrons are

Figure 5. Luminescence bandwidth of the band I and band II as a function of the temperature. The solid line is the fitted curve according to eq 2.

decrease in the mean free path.30,45 Therefore, gold bulk exhibits an extremely fast dephasing time in the range of a few fs.45 For gold NCs, the interaction of electron−electron is temperature-independent and results in the homogeneous broadening term of Γe−e. A very broad luminescence spectrum has been observed even at low temperatures for few NCs. On the other hand, the electron−phonon interaction and electron− surface/defect scatterings are closely relevant to thermal activation, and they lead to bandwidth broadening with increasing temperatures. Following the above discussion, we could classify bandwidth broadening into a temperature-independent Γe−e component and a temperature-dependent component that includes the contributions from electron−phonon scattering and surface/ defect/ionized impurity scattering. As expressed: ⎛ E ⎞ Γ(T ) = Γe − e + σT + α exp⎜ − S ⎟ ⎝ KBT ⎠

(2)

where σ is the electron-acoustic phonon coupling coefficient, ES is the activation energy for surface/defect/impurity states, and α is the bandwidth due to fully activated surface/defect/ impurity states. It is found that band I can be nicely fitted by eq 2 with parameters Γe−e = 243.3 ± 3.6 meV, σ = 11.3 ± 1.6 μeV/K, and ionized energy ES = 26.6 ± 5.2 meV. This finding confirms that electron−phonon scattering and electron−defect/surface scattering are the mechanisms for bandwidth broadening. In comparison to the parameters extracted from Au10 NCs protected with histidine,21 Γe−e = 127.7 ± 0.6 meV, σ = 5.9 ± 0.6 μeV/K, and ES = 65.5 ± 5.9 meV, the bandwidth of Au25 NCs is significantly larger. The broadened bandwidth is most likely attributed to the stronger electron−electron interaction for the larger NCs. Au25 NCs exhibit a larger σ, suggesting an increased contribution of acoustic phonon to bandwidth broadening than Au10 NCs. In addition, the ionization energy of Au25 NCs is evidently smaller, likely influenced by both the structure and the ligands. On the other hand, the bandwidth of band II does not follow eq 2. With increasing temperatures, the bandwidth increases at low temperatures and then saturates at high temperatures, above 250 K. This result suggests that there exists another mechanism to affect the bandwidth of band II. In an Au25 NC, the semiring locates between the icosahedral core and the outer ligands. Each Au25 NC is surrounded by ligands that the spatial 11833

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extracted, respectively. This similarity suggests two transitions experience the similar nonradiative trapping. It is interesting to note the intensity ratio between band I and band II is roughly temperature-independent between 77 and 300 K, as shown in Figure 7. In general, upon increasing temperature, phonon scattering is enhanced, and the induced relaxation also increases. Taking into account the band I and band II dominantly originating from the icosahedral core and Au−S semiring, the temperature-independent ratio of band I to band II confirms that the relaxation from the semirings into the core is ineffective, and there may be a relatively large energy barrier between the semiring and the core.

initially generated at both BSA and Au25 NCs. Following the excitation, most of the excited electrons will be trapped by defect/surface states because of the relatively lower quantum efficiency (