Quenching Fluorescence of Quantum Dots by Using Gold Nanocrystals in Quantum and Classical Size Regime

: This paper investigates the factors which can influence the quenching effect from the perspective of energy transfer. The quenched fluorescence of the CdSe semiconductor quantum dots (SQDs) by using plasmonic Au nanocrystals (p-AuNCs) and molecule-like Au nanocrystals (m-AuNCs) in aqueous suspensions and spin-coated films is comparatively investigated. In the aqueous suspensions, the p-AuNCs have larger quenching effect than the m-AuNCs. In the spin-coated films, the p-AuNCs and m-AuNCs have comparable quenching factor. Furthermore, the experiments show that the p-AuNCs simultaneously enhance the radiative and nonradiative rates. But the m-AuNCs only enhance the nonradiative rate of the SQDs, which reveals the difference of quenching process between the p-AuNCs and m-AuNCs. This result of the research has guiding significance for the detection technique based on the fluorescence quenching.


Introduction
In recent years, noble metal nanocrystals have attracted greatly-increasing interests from researchers because of their unique properties. In materials science, the structure of material determines the material properties, and the various preparation methods of noble metal nanocrystals provide possibilities for materials that can adapt to different functional requirements [1][2][3] . Therefore, the researchers are committed to research the noble metal nanocrystals which have tunable optical response by adjusting their surface factors such as size, shape, and component [4][5][6][7][8][9][10] , which leads to diverse applications ranging from surface enhanced Raman scattering, bio-imaging, photothermal cancer treatment, photocatalysis, to ultrafast optical information processing [11][12][13][14] . Especially, the unique optical responses of the Au and Ag nanocrystals (AuNCs and AgNCs) in the quantum size regime have been made many practical achievements [15][16][17][18] . For instance, the plasmon resonance wavelength of the individual ligand-free AgNCs blue-shifts 500 meV as the nanocrystals' diameter decreases from 20 nm to less than 2 nm, which is revealed to be caused by the prominently increased quantum confinement effect in the quantumsized nanocrystals [19][20][21][22] .
Noble metal nanocrystals with strong plasmon resonance are widely used to tune optical emission behaviors of the nearby nanoemitters. Fluorescence detection technology is an effective tool in the communities of biomedicine and geological exploration, the luminescence modulation process of which has always been an important subject. For the metal surface plasma has significant extinction and field enhancement effects, the fluorescence could be largely enhanced or significantly quenched by the metal nanocrystals depending on the competition of the enhanced radiative and nonradiative processes, which can be controlled by adjusting the size of metal nanocrystals or the separation distance between metal nanocrystals and nanoemitters [23][24][25][26] . When the size of the metal nanocrystals decreases to very small, the plasmon resonance disappears and the molecule-like behavior of the atomic clusters exhibits. The molecule-like metal nanocrystals could not be used to enhance emission of the nearby optical emitters but could be used to quench fluorescence and have also prospective applications in bio-sensor [27][28][29] .
In our previous studies, we observed the nonmonotonic shift in the spectrum of AuNCs ranging from the classical to the quantum size, due to the competition between quantum and classical effect [30] . Because of the different performance of AuNCs in quantum and classical size, we design the fluorescence quenching experiment of AuNCs in these two size regions, respectively. In this paper, we investigate fluorescence of CdSe SQDs quenched by plasmonic Au nanocrystals (p-AuNCs) and molecule-like Au nanocrystals (m-AuNCs) in aqueous suspensions and solid films. We find that both p-AuNCs and m-AuNCs exhibits strong quenching effect, but there are essential differences in quenching modes between p-AuNCs and m-AuNCs. After analyzing the experimental data, our results have shown that the p-AuNCs enhance both radiative and nonradiative rates but the m-AuNCs only enhance nonradiative rate of the SQDs.

Materials and Methods
CdSe SQDs with the central emission wavelengths of ~655 nm are purchased from Invitrogen Corporation. The m-AuNCs and p-AuNCs were prepared by thermal etching the same concentration of Au nanorod solution according to a previously reported work by our team [30] . We found that the size of AuNCs is solely depended on the reaction temperature. The m-AuNCs and p-AuNCs are prepared by adjusting the etching temperature to 260 ℃ and 205 ℃, respectively. The CdSe SQDs and AuNCs are mixed to form suspensions. The concentration of m-AuNCs in the mixed suspensions is adjusted by the volume (V Au ) of aqueous liquid of m-AuNCs. The complex film consisting of the CdSe SQDs and the p-AuNCs or m-AuNCs are prepared by spin-coating mixed suspensions.
The transmission electron microscopy (TEM) images and high-resolution TEM images are performed by a JEOL 2010 HT and JEOL 2010 field effect transistor TEM at an accelerating voltage of 200 kV, respectively. The extinction spectrum is recorded by a TU-1810 UV-Vis-NIR spectrophotometer. The fluorescence spectrum excited by a Xe lamp is recorded by a Hitachi F-4500 fluorescence spectrophotometer. The fluorescence signal excited by a laser is collected in reflection geometry, filtered with two filters and recorded by a spectrometer (Spectrapro 2500i, Acton) with a liquid-nitrogen-cooled charge-coupled device (SPEC-10, Princeton). The time-resolved fluorescence emission decay traces are recorded by using a time-correlated single-photon counting system (PicoQuant GmbH), and the pulsed laser is provided by a mode-locked Ti: Sapphire laser (Mira 900, Coherent) equipped with an optical frequency doubling system.

Results
The TEM images of the p-AuNCs and m-AuNCs are shown in Fig. 1(a) and 1(b), respectively. The p-AuNCs have an average size of ~34 nm and strong resonance absorption at the wavelength of 528 nm (Fig. 1(c)). On the contrary, the quantum-sized m-AuNCs have a very small diameter of ~2 nm, which have an absorption band edge around 500 nm and the plasmon resonance absorption around 530 nm is efficiently suppressed (see Fig. 1(c)). The excitonic absorption band edge of the CdSe SQDs around 630 nm is also shown in Fig. 1(c).
In our experiment, the relationship between the quenching effect and the concentration of AuNCs was investigated (Fig. 2). The fluorescence of CdSe SQDs was almost completely quenched by a small amount of p-AuNCs. Therefore, only the experimental data of m-AuNCs are shown below. Figure 2(a) clearly demonstrates that the fluorescence of the suspended CdSe SQDs is quenched by the m-AuNCs. It is shown that the quenching effect has an analogous logarithmic relationship with the concentration of metal solution. The quenching efficiency is defined as q = 1-I FL /I FL,0 , where I FL and I FL,0 are the SQDs' fluorescence peak intensity with and without AuNCs, respectively. The volume of m-AuNCs (V Au ) added to the SQDs are 0, 5, 10, 20, 30, 40, 50, 70, 100, 150, 200, 300, and 400 L. The q values dramatically increase from 0 to 0.58 as V Au increases from 0 to 40 L, and then slowly increases to 0.81 as V Au further increases to 400 L (see the inset in Fig. 2(a)). As the number of AuNCs increases, there are more absorption cross sections in the system. The quenching effect in-  Quenched fluorescence spectra (b) and normalized TRFL (c) of SQDs with p-AuNCs or m-AuNCs creased with the increase of concentration can be attributed to the increase of whole absorption cross section. As comparison, the p-AuNCs exhibits stronger quenching effect than the m-AuNCs and the corresponding q value reaches as high as 0.95 (see Fig. 2(b)).
To investigate the physical mechanism of the fluorescence quenched by the p-AuNCs and m-AuNCs, the time-resolved fluorescence (TRFL) of the CdSe SQDs with and without AuNCs is recorded at the central emission wavelength of the SQDs at 655 nm. As shown in Fig.  2(c), the bare CdSe SQDs have a single exponential decay, with decay rate Γ 0 ≈0.030 6 ns -1 . The SQDs with m-AuNCs also exhibit a single exponential decay, but the decay rate Γ increases to 0.044 6 ns -1 . This indicates that all SQDs are coupled to the small-sized m-AuNCs. The fluorescent group of quantum dots is regarded as a pair of dipoles, and the electromagnetic field generated by them interacts with the m-AuNCs, which is accompanied by the nanosurface energy transfer (NSET). The energy transfer rate is approximately estimated by the relation, Γ ET = Γ -Γ 0 = 0.014 ns -1 .
On the contrary, the SQDs with p-AuNCs have two-exponential decay processes, which can be fitted by where A f (A s ) and τ f (τ s ) represent weight factor and the lifetime of the fast (slow) decay processes, respectively. From Fig. 2(c), we obtained τ f = 1.4 ns, τ s = 38.1 ns, A f /(A f + A s ) = 0.31, and A s /(A f + A s ) = 0.69. The lifetime of the slow process τ s is very close to that of the bare SQDs, which indicates this slow decay process is attributed to the SQDs uncoupled to p-AuNCs. Except for the slow decay of quantum dots, the curve of fluorescence life shows a fast decay process which is attributed to the SQDs strongly coupled to p-AuNCs. This leads to a very strong coupling energy transfer. The energy transfer rate is approximately estimated by Γ ET = 1/τ f -Γ 0 ≈ 0.683 ns -1 . The fluorescence quenching effect is strongly dependent on the complex nanostructure of the metal nanocrystals and the optical nanoemitters. Then, we further investigate the fluorescence behaviours of the complex films consisting of CdSe SQDs and p-AuNCs or m-AuNCs. The film samples are prepared by spin-coating of the complex suspensions onto the fused quartz substrate. Figure 3(a) and 3(b) presents quenched fluorescence spectra and TRFL of the bare CdSe SQDs and those with p-AuNCs or m-AuNCs.
In the complex film, the p-AuNCs have weaker quenching effect (q = 0.79) than the ones in the suspensions,

Fig. 3 Quenching fluorescence spectra (a) and normalized TRFL (b) of CdSe SQDs by p-AuNCs and m-AuNCs in complex film
the fast decay rate slightly decreases to 0.370 3 ns -1 , the slow decay rate increases to 0.041 2 ns -1 , and the corresponding weight factor A f /(A f + A s ) increases to 0.5 (more detailed data are presented in Table 1). Since the total decay rate  is the sum of the radiative and nonradiative rate ( =  rad +  nonrad ), and the fluorescence intensity is proportional to the radiative rate  rad , therefore, the relatively smaller quenching factor with comparable decay rates in the complex films can be explained by the plasmon-enhanced radiative rate [31] . Part of the energy transferred to the gold nanoparticles drives the plasma state. The electrons emit photons in the transition process of the plasmon state to the ground state. The radiation of photon can reduce the quenching effect.
On the other hand, there is no plasmon enhancement on the radiative rate of the SQDs' fluorescence when the m-AuNCs are used, which result in a prominent quenching effect with one-component exponential decay fluorescence of the CdSe SQDs with m-AuNCs in both aqueous suspensions and complex films.

Conclusion
In summary, we comparatively investigate the quenched fluorescence of the CdSe SQDs by p-AuNCs and m-AuNCs in the suspensions and complex films. In both suspensions and films, the m-AuNCs have comparable quenching effect (q values are about 0.69 and 0.62 respectively), and the quenched fluorescence of the SQDs follows a single exponential decay process. On the other hand, the p-AuNCs demonstrate stronger quenching effect than the m-AuNCs. The fluorescence quenching factor of the SQDs with p-AuNCs reaches as high as 0.97 in the suspensions. The prominent two-decay processes induced by plasmon-enhanced radiative rate are observed in the complex films. These observations could provide an alternative route to the prospective applications in bio-sensing and bio-labeling.