Assessing the Blinking State of Fluorescent Quantum Dots in Free

Oct 7, 2014 - ... nearly one order of magnitude less than the ones measured with the ..... M. Random telegraph signal in the photoluminescence intensi...
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Assessing the Blinking State of Fluorescent Quantum Dots in Free Solution by Combining Fluorescence Correlation Spectroscopy with Ensemble Spectroscopic Methods Chaoqing Dong,* Heng Liu, and Jicun Ren* School of Chemistry & Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China S Supporting Information *

ABSTRACT: The current method for investigating the blinking behavior is to immobilize quantum dots (QDs) in the matrix and then apply a fluorescent technique to monitor the fluorescent trajectories of individual QDs. So far, no method can be used to directly assess the blinking state of ensemble QDs in free solution. In this study, a new method was described to characterize the blinking state of the QDs in free solution by combining single molecule fluorescence correlation spectroscopy (FCS) with ensemble spectroscopic methods. Its principle is based on the observation that the apparent concentration of bright QDs obtained by FCS is less than its actual concentration measured by ensemble spectroscopic method due to the QDs blinking. We proposed a blinking index (Kblink) for characterizing the blinking state of QDs, and Kblink is defined as the ratio of the actual concentration (Cb,actual) measured by the ensemble spectroscopic method to the apparent concentration (Cb,app) of QDs obtained by FCS. The effects of certain factors such as laser intensity, growth process, and ligands on blinking of QDs were investigated. The Kblink data of QDs obtained were successfully used to characterize the blinking state of QDs and explain certain experimental results. confocal laser scanning microscopy (CLSM).19 And then blinking behaviors of “one-by-one” dots are analyzed based on the fluorescence trajectory of individual dots. On one hand, the observed blinking phenomena from QDs immobilized in the matrix cannot represent the “real” blinking state in free solution because of the influence of microenvironment in the matrix on their blinking behaviors.20−22 It was observed that single CdSe/ ZnS quantum dots in agarose gel exhibited suppressed blinking behaviors.22 On the other hand, if QDs are not immobilized in the matrix, it is difficult for these methods to investigate the blinking without the accurate photoluminescent (PL) intensity trajectories due to their fast Brownian movement in free solution. Fluorescence correlation spectroscopy (FCS) is an ultrasensitive and noninvasive single molecule/particle detection technique using statistical analysis of the fluorescence fluctuations emitted from a small, optically well-defined open volume, which is measured in the solution.23−32 Recently, FCS has been successfully used to characterize some parameters of QDs including their hydrodynamic diameters, surface charges, dark fraction, and so forth.33−39 For example, since the presence of dark QDs in the samples was observed by Ebenstein et al.,40 FCS methods were developed by Doose et al.,33 Yao et al.,38 and Murthy et al.39 to determine the dark fraction induced by long-lived nonirradiant or blinking QDs.

1. INTRODUCTION Semiconductor quantum dots (QDs) are very important optical nanomaterials with a wide range of potential applications1,2 due to their excellent physical and chemical properties such as about 10 times higher brightness, enhanced stability against photobleaching, and size-tunable optical properties compared with organic dyes.3,4 However, QDs show severe fluorescence intermittency (or blinking) on a wide time scale from milliseconds to minutes, switching randomly between bright (“on”) and dark (“off”) states, which have been considered as an intrinsic drawback to limit certain applications requiring continuous emission such as single nanoparticle tracking and detection in biology, and light-emitting diodes.5−7 It is initially explained that the blinking of QDs is probably attributed to Auger ionization-charging mechanism. “On” and “off” durations correspond to the neutral and charged states, respectively.6,8,9 Recently sufficient experimental findings10−16 have shown that the “off” state of QDs might not be explained by a sole Auger recombination mechanism, and multiple physical mechanisms seemed to be related with the blinking of QDs, such as the carrier trapping model.17,18 So far, few effective methods can be used to characterize the blinking state of QDs in free solution. In the previous methods, investigation on blinking is carried out on the QDs highly dispersed and immobilized in the matrix. Typically, the fluorescence image videos of immobilized QDs are first obtained with fluorescence imaging techniques such as total internal refection fluorescence microscopy (TIRFM) and © 2014 American Chemical Society

Received: July 31, 2014 Revised: September 28, 2014 Published: October 7, 2014 12969

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were then adjusted to pH 8.0 with 1 M NaOH. The solution was deaerated with N2 for 30 min. Under vigorous stirring, the oxygen-free NaHTe solution newly prepared was injected. The concentration of the precursor solutions was 2.5 mM with reference to the Cd2+. The mixture solution was heated in a water bath (∼90 °C), and the size of the prepared QDs could be tuned by changing reaction temperature and reaction time. The QDs with the different emission wavelengths were drawn from the reaction stock in the different reaction times. The numeric character (“2.5”) in the “2.5MPA-QD593” represents the molar ratio of thiol-ligand to Cd in the precursor and “593” indicates the PL emission peak of QDs at 593 nm. NAC, MSA, or TP-capped CdTe QDs were also prepared through the similar method for MPA-capped QDs except that different thiols were used as surface ligands instead of MPA and pH values of their precursor were adjusted accordingly. For NAC, MSA, and TP-capped QDs, the optimal pH values of their precursor were 9.5, 7.0, and 11.0, respectively. Characterization. UV−vis absorption spectra of QDs were measured using a UV−vis spectrophotometer (Tianjin Gangdong Instruments Co., China). Samples were prepared by diluting CdTe QDs solutions with ultrapure water. Photoluminescence spectra were recorded on a fluorescence spectrophotometer (Tianjin Gangdong Instruments Co., China). All optical measurements were carried out at room temperature. Rhodamine 6G (ethanol as solvent) was chosen as a reference standard (QY = 95%). TIRFM Imaging of Individual QDs and Blinking Analysis Procedure. PL time trajectories from individual QDs were acquired on TIRFM imaging system constructed on an Olympus IX 71 inverted fluorescence microscope. Samples were excited with 488 nm argon ion laser (ILT Ion Laser Technology, Shanghai, China), and the laser power monitored in front of the microscopy objective (NA1.45/60×, Olympus) was measured to be 0.22 mW after it is attenuated. Fluorescence from the sample was collected by the same objective, separated from the excitation light by a dichroic mirror (505DRLP, Omega Optical, Brattleboro, VT) and emission filters (595AF60 or 590 long-pass), and then focused into a cooled-CCD camera (Cascade 650, Photometrics). Image acquisition and processing were performed using the MetaMorph software (Universal Imaging). All measurements were performed at room temperature. The coverslips for TIRFM imaging were thoroughly cleaned by in hot chromic acid mixture, 0.1 M NaOH solution, ethanol, and ultrapure water (Millipore) (each for 15 min) and dried in a jet of N2. Before measurements, all QDs samples were centrifuged with 12 000 rpm to remove the potential agglomeration. Then samples were prepared by spin-casting freshly diluted newly-prepared aqueous QDs and commercial QDs in 1% PEO (w/w) (MW 1 000 000, Sigma) and left for 60 s to allow complete drying of PEO. To clearly distinguish between the on and off events, we set the threshold separating “on”-duration time from “off”-duration time at 6 times the standard deviation (6σ) of the average background noise level. A PL time trajectory from a random pixel array (6 × 6) but in that no QDs are included was chosen as the background. A software program was compiled with MATLAB to acquire both on- and offduration times by analyzing the PL intensity trajectories of single QD gained from ImageJ software. The probability distributions of different on- and off-duration time, ratio of overall off-duration time to whole sampling time (off%), and percentage distributions of QDs samples in different on-duration time were calculated based on PL intensity trajectories of about 120 ± 10 bright dots. FCS Measurement on QDs. FCS measurements were performed with a home-built FCS system.34 In brief, the 488 nm laser line from argon ion laser (Ion Laser Technology, Shanghai, China) was attenuated to ∼20 μW by using a circular neutral density filter, and then expanded by using a telescope to just fill the back aperture of the objective lens. The expanded laser beam was focused by a water immersion objective (NA1.2/60×, UplanApo, Olympus, Japan) into a small volume within the diluted sample. The excitation intensity of laser beam was measured by an optical power meter (HIOKI 3664, Shanghai, China) before the laser was led into the objective. The excited fluorescence signal was collected by the same objective and

And effort was also done to investigate QDs blinking based on their FCS curves with Monte Carlo methods.41 Although no functional form can directly quantify the blinking degree of QDs from the shape of correlation curves, the influence of QDs blinking on the amplitudes of correlation curves may be used to assess the blinking state of QDs in free solution. In this work, we report a new method to directly characterize the blinking state of QDs in free solution by combining FCS with some ensemble spectroscopic methods. The principle is based on the fact that the apparent concentration of bright QDs obtained by FCS is far less than its actual concentration measured by ensemble spectroscopic method due to the severe blinking effect of QDs. Herein we proposed a blinking index (Kblink) for characterizing the blinking state of QDs in solution, and Kblink is defined as the ratio of the actual concentration (Cb,actual) to apparent average concentration (Cb,app) of QDs. The measurement strategy of our proposed approach is illustrated in Figure 1. The apparent concentration (Cb,app)

Figure 1. Scheme of direct assessment on ensemble QDs blinking in free solution by combining FCS with ensemble spectroscopic methodologies. The apparent concentration (Cb,app) of bright QDs was determined with our developed FCS method (1), and the actual concentration (Cb,actual) of bright QDs was measured with UV−vis spectroscopy method (2). f is the PLQYs of QDs samples. A is the absorbance values of QDs measured at the first exciton absorption peak. ε is their molar extinction coefficients, and L is the optical path length.

and actual concentration (Cb,actual) of bright QDs were determined with FCS and other spectroscopy methods, respectively. Using the method, the blinking indexes of different QDs samples are measured including aqueous CdTe QDs prepared with different thiols in our lab and commercial watersoluble QDs, and the results are compared with TIRFM-based blinking analysis method. Certain effect factors such as laser intensity, types of ligands, and growth process of QDs, on blinking behaviors of QDs, are systematically investigated.

2. EXPERIMENTAL SECTION Materials. Mercaptopropinic acid (MPA), mercaptosuccinic acid (MSA), and N-acetyl-L-cysteine (NAC) were acquired from SigmaAldrich Co. (Shanghai, China). Tiopronin (TP) was obtained from TCI Co. (Shanghai, China). CdCl2·2.5H2O (99%) and NaBH4 (96%) were obtained from Shanghai Reagent Co. (Shanghai, China). Qdot 655 ITK Carboxyl Quantum Dots (QD655-COOH) and Qdot 655 ITK Amino (PEG) Quantum Dots (QD655-NH2) were from Life Technologies (Shanghai, China). All solutions were prepared with ultrapure water purified on a Millipore Simplicity apparatus (Millipore, Billerica, MA). Preparation of Aqueous QDs. Different thiol-capped CdTe QDs were prepared according to our previous method.42 The typical molar ratio of Cd2+/NaHTe/thiol of as-prepared QDs was 1:0.2:1.2 and 1:0.2:2.5. As an example, the synthesis procedure of MPA-capped CdTe QDs was shown in the following. Cd precursor solutions were prepared by mixing a solution of CdCl2 in the presence of MPA, and 12970

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passed through a dichroic mirror (505DRLP, Omega Optical, Brattleboro, VT). Finally, the fluorescence was coupled into a 35 μm pinhole at the image plane in the front of single-photon counting module (SPCM-AQR16, PerkinElmer EG&G, Canada). The fluorescence fluctuations were correlated with a digital correlator card (correlator.com, Shenzhen, China). The obtained raw FCS curve data were fitted with eq 1 based on the Levenberg−Marquardt algorithm assuming that fluorophors were diffusing in a threedimensional Gaussian volume element and nonlinearly fitted.43 G(τ ) =

1 1 N 1+

the ones measured with the UV−vis spectroscopy method (Figure 2).

1 τ τD

1+

2

( ) ω0 z0

τ τD

(1)

where τD is the characteristic diffusion time of fluorophors, which is related to the diffusion coefficient, D:

τD =

ω0 2 4D

(2)

According to the FCS principle, the amplitude of FCS curves at zero correlation time (G(0)) is equal to the inverse average number (N) of fluorophors in the detection volume (V); therefore, the concentration (C) can be measured by FCS (eq 3) as shown in Figure 1.23,43 C b,app =

N NAV

Figure 2. FCS curves of aqueous NAC-capped CdTe QDs extracted from the raw reaction solution at different reaction time (a). Their fitting residual curves can be found in Figure S-1. All raw prepared QDs solutions were diluted with ultrapure water to ensure the concentration for FCS measurements (about 10 nM concentration). The laser intensity was about 50 μW. The measuring time per sample was 60 s. The concentration change of bright QDs with reaction time was measured by FCS (b). UV−vis absorption spectra of QDs obtained at different reaction times (c). Total concentration change of QDs with reaction time was measured by UV−vis spectroscopy (d).

(3)

where NA is the Avogadro constant. V is referred to as the detection volume, which is by a factor of 23/2 larger than the confocal volume (eq 4),44 and it can be calculated after the lateral radius (ω0) and axial radius (z0) of the volume are measured by fitting the FCS curves of Rhodamine green (RG) with eq 1. V = 23/2π 3/2ω0 2z 0

(4)

The FCS method was applied to measure the concentration of bright QDs similar to organic dyes.23,43 Before FCS measurements, all QDs solutions were centrifuged with 12 000 rpm to remove the potential agglomeration and the supernatants of QDs were diluted with ultrapure water to about 10 nM concentration.

Meanwhile, the concentration evolution law of QDs with reaction time measured by FCS was completely opposite to the results by the UV−vis spectroscopy method. As shown in Figure 2a, the G(0) values of correlation curves decreased with reaction time. It suggests that the concentration of QDs gradually increases with reaction time as eqs 1 and 3 (Figure 2b). On the contrary, the results with UV−vis spectroscopy method show that the concentration of QDs gradually decreases with reaction time (Figure 2c and d). Figure 2c shows that the first exciton absorption peaks of different raw QDs solutions shift from 552 to 629 nm with reaction time and the peaks broaden with reaction time due to the polydispersion of QDs. But the decrease of absorbance values proves the gradual decrease of concentration with size growth, where effect of its size distribution has been considered (Figure 2d). The similar decrease law has been widely observed in the synthesis of other nanoparticles, which is attributed to the classical Ostwald ripening process in the growth of nanoparticles.46 The above results demonstrate that there is a remarkable difference in the concentrations of QDs obtained by FCS and UV−vis spectroscopy method. One obvious contribution for this difference is due to the presence of long-lived nonirradiant QDs or dark QDs due to surface defects. The presence of dark QDs in the samples was indeed observed by Ebenstein et al.40 and confirmed by Doose et al. in FCS measurements.33 In the UV−vis measurement, its molar concentration of QDs is determined on the basis of its absorbance (Figure 1).45 Hence, both nonirradiant and irradiant QDs are included in the concentration values, which are not related to the blinking behaviors of QDs. However, in FCS measurements, only irradiant QDs in the detection volume regardless of whether they are long-lived irradiant QDs

3. RESULTS AND DISCUSSION The Difference in QDs Concentrations Obtained by FCS and UV−Vis Spectroscopy and Principle of This Method. Figure 1 shows the principles for concentration measurements of QDs by FCS and UV−vis spectroscopy method, respectively. The feasibility of FCS to measure the concentration of fluorescent molecules has been confirmed.23 As shown in Table S-1 in the Supporting Information, FCS was used to measure the different concentrations of RG samples and compared with UV−vis spectroscopy method. Only a minor difference was found in the concentration range (from 2 to 10 nM) measured by FCS to the expected concentration measured by UV−vis spectroscopy method. So in the following FCS experiments, all QDs were diluted to the certain concentration range before measurements. And the concentrations of QDs were also determined with UV−vis spectroscopy method on the basis of their absorbance at the first exciton peak and their corresponding molar extinction coefficients according to the Beer−Lambert law (Figure 1).45 In the method, the absorbance values have been calibrated with the full width at half-maximum (fwhm) of the PL spectrum of QDs samples for which the effect of their size distribution was taken into account. When FCS and UV−vis spectroscopy methods were applied to measure the concentration of aqueous CdTe QDs and investigate their growth process in the reaction solution, the concentrations of QDs at different reaction times measured by FCS were nearly one order of magnitude less than 12971

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Figure 3. Temporal evolution of representative fluorescence trajectories of individual QDs from TIRFM imaging experiments. Samples were excited by a continuous 488 nm argon laser with an intensity of 0.22 mW in front of the microscopy objective. The temporal resolution is 200 ms for labfabricated samples (NAC-QD596 (a), MPA-QD589 (b), MPA-QD593 (c), MPA-QD601 (d)) due to low brightness and 20 ms for commercial samples (QD655-COOH (e), QD655-NH2 (f)) due to high brightness. The dashed blue line is employed as the threshold to define fluorescent “on” and “off” intervals that were calculated based on the background (black solid line). Off% is ratio of off-duration time to whole sampling time.

Table 1. Kblink Values and Percentage Distributions of QDs Samples in Different On-Time Fractions percentage of QDs in different on-time fractions (%)

a

samples

0%−10%

10%−30%

30%−50%

50%−70%

70%−90%

90%−100%

PLQY

CFCS (μM)

NAC-QD596 MPA-QD589 MPA-QD593 MPA-QD601 QD655-COOH QD655-NH2

98.4 71.6 4.6 0.0 0.0 0.0

1.6 21.0 8.3 0.0 0.0 0.0

0.0 4.9 6.1 5.8 0.0 3.5

0.0 2.5 12.2 8.2 0.0 0.0

0.0 0.0 22.1 13.5 25.0 15.6

0.0 0.0 53.3 72.5 75.0 80.9

0.498 0.591 0.627 0.365 0.84a 0.81a

0.32 0.49 0.82 0.62 6.31 7.49

± ± ± ± ± ±

0.03 0.02 0.03 0.04 0.52 0.65

CUV−vis (μM) 8.66 9.12 8.76 5.78 8.3a 8.6a

± ± ± ±

0.12 0.21 0.16 0.23

Kblink 13.5 11.0 6.7 3.4 1.1 0.9

± ± ± ± ± ±

1.3 0.7 0.3 0.2 0.1 0.1

The PLQY and concentration values were provided by the manufacturer.

behaviors, and two typical TIRFM imaging movies can be found in the Supporting Information. Obviously, in Figure 3, QDs (a) take on “on−off−on” slow blinking events with longer toff and bigger off% compared with QDs (f). In FCS, if fast fluorescence fluctuation happens similar to the triplet state of organic dyes, a fast decay peak appears in the correlation curves in the beginning several microseconds.47 As shown in Figures 2a and S-2, all FCS curves of QDs are very well fitted with eq 1 and look like data from purely diffusing molecules when they were measured at very low excitation intensity. So the absence of a fast decay peak in FCS curves merely implies the absence of fast blinking behaviors in the low excitation intensity. It is consistent with the reported fact that the “on−off−on” fast blinking events within the diffusion time scale were quite rare in the low excitation power.38,48 Herein the influence of blinking effect on FCS measurements is more attributed to the temporary “off” state in the “on−off−on” slow blinking events as shown in Figure 3a−d. As described in Figure 4, when one blinking QD with toff > τD transverses through the detection volume at “off” state, the bright QD can wrongly be regarded as dark one due to no fluorescence contribution to FCS measurements. The “on−off−on” slow blinking events cannot be reflected in the shape variation of FCS curves but the induced false-negative recognition results in the amplitude increase of FCS curves. It leads to that the apparent concentration of bright QDs (Cb,app) obtained by FCS is less than the actual concentration of bright QDs (Cb,actual). And the

or temporarily irradiant QDs can be included in the concentration but nonirradiant QDs in the volume are excluded (Figure 1). So the concentration of aqueous QDs by FCS is less than that determined by UV−vis spectroscopy method. It is consistent with the reported results.33 Meanwhile, it has been reported that the ratio of bright QDs ( f) in the whole QDs (f = Cb,actual/Ctotoal) was directly determined by PL quantum yields (PLQYs) of samples and it was very close to PLQYs of QDs in the FCS experiments.25,44,52 So now the contribution of longlived nonirradiant QDs to total concentration can be evaluated.21,38,40 However, the different finding to the above results is that the concentration value of aqueous CdTe QDs determined by FCS was still far less than the actual concentration of QDs obtained by UV−vis spectroscopy method after the dark fraction was excluded from the samples and the concentration decrease of bright QDs due to small aggregates was taken into account.33 Hence another more remarkable contribution for this difference was assumed to be from the temporarily “off” state of QDs due to the blinking (switching from “on” to “off” state in the detection volume) as shown in Figure 3a−d. The influence of temporary “off” state from blinking on FCS measurements is highly dependent on blinking behaviors such as “off”-duration time (toff, interval between two adjacent “on” states), the off-percentage distribution and ratio of off-duration time to whole sampling time (off%). As shown in Figure 3 and Table 1, different QDs show remarkably different blinking 12972

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Meanwhile, potential agglomeration of QDs can result in the number decrease of bright QDs detected by FCS and the change of its blinking state such as the nonblinking of aggregated QDs. Attempts were performed to eliminate the effect of potential agglomeration such as high-speed centrifugation on QDs samples before measurements and abandonment on FCS measurement data with high fluorescence bursts induced by agglomeration.38,41 The above discussion revealed that another main reason for the concentrations of QDs by FCS being far less than those determined by UV−vis spectroscopy method was the presence of temporary “off” state due to QDs blinking besides the presence of long-lived nonirradiant QDs. Based on the above fact and discussion, the principle for characterizing the blinking state of QDs in free solution was proposed by combining FCS with UV−vis spectroscopy method, as shown in Figure 1. Kblink is defined as the blinking index to describe the blinking state of QDs in free solution, which is the ratio of the actual concentration (Cb,actual) to apparent concentration (Cb,app) of QDs as eq 5.

Figure 4. Scheme of blinking effect on concentration measurement of bright QDs with FCS. The red dot indicates that the blinking dot is at “on” state, and the green one indicates that the blinking dot is at “off” state in the detection volume. When QDs diffuse through the volume at “off” state (1) due to blinking effect, the amplitude of correlation curves (G(0)) increases compared with those at “on” state (2).

experimental results confirmed it. As shown in Figure 3 and Table 1, for QDs (from d to a) with longer off-duration time and higher off%, the determined apparent concentration (Cb,app) is far less than the actual concentration of bright QDs (Cb,actual). On comparison, the concentration of these commercial QDs (e, f) determined by FCS was actually very close to these by UV−vis spectroscopy method if the dark fraction was deducted. The result is consistent with that reported by Doose et al.33 The main reason is that their off% is minor and probability of low counts (less than the threshold) is low. So the dark fraction of these commercial QDs (e, f) is mainly contributed by the existence of long-lived nonradiant QDs. The possible influences from other factors were also discussed, but they were found to be negligible including brightness variations of QDs with their sizes and distribution36 and low relative concentrations of very bright species due to potential agglomeration.49 In FCS, the possible effect of brightness variations with sizes on the amplitude is dependent on whether the brightness is within fluorescence saturation intensity in the detection volume. As shown in Figure S-3, although the brightness of RG increases 10-fold with the laser intensity, the effect of brightness variation on the amplitudes is minor within a certain excitation power range because brightness distribution in the detection volume is not saturated. In the experiments of aqueous QDs, the brightness change with their sizes is less than 10-fold under the excitation of low laser intensity due to the decrease of PLQY.36 And the effect of brightness variation due to size distribution is also minor because the size polydispersity is usually less than 5%. Herein it is assumed that the effect of brightness variation of QDs on the amplitudes is negligible.

Kblink = C b,actual /C b,app

(5)

Therefore, the larger Kblink value implies that the greater apparent concentration is deviated from the actual concentration, which indicates that QDs are in the more serious blinking state. Here the blinking index of six QDs samples in free solution was determined based on the method as shown in Figure 1, and compared with the results by blinking analysis procedure based on TIRFM imaging and MATLAB software. Table 1 shows that the determined Kblink values of NAC-QD596 are about 13.5, which demonstrate that it is in a severe blinking state according to the definition in eq 5. And the fluorescence trajectory of NAC-QD596 (Figure 3a) shows that its “on” duration time was very short and the calculated off% was 96.2%, suggesting that most of the duration time is waiting at the “off” state. And blinking analysis results in Table 1 demonstrate that the percentage of NAC-QD596 with the on-duration time < 10% QDs was 98.4%. Herein it suggests that Kblink values can truly reflect the blinking state of QDs. Meanwhile, the Kblink values of these six QDs samples are in the range of 13.5−0.9. And it was observed (Figure 3) that off% values gradually decreases with the decrease of Kblink. The percentage of QDs with the on-time < 10% gradually decreases, but the percentage of QDs with the on-time > 90% gradually increases. For QD655-COOH in the minor blinking state, the percentage of QDs with the on-time > 90% is larger than 75%. Accordingly, its Kblink value is close to 1. The above discussion suggests that the result from Kblink value is consistent with that

Figure 5. FCS curves of NAC-capped QDs excited with the different laser intensities (a). Calculated detection volume at the different laser intensities using RG as a reference dye with known diffusion coefficient (b). Dependence of blinking behaviors of QDs on laser intensity (c). 12973

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Influences of Ligands on Blinking. The effect of capping thiol-ligands on the blinking behaviors of QDs was compared with Kblink data. In the experiment, different QDs were prepared with different ratios of thiol-to-Cd (1.2 and 2.5) but other synthesis conditions were fixed. The compared QDs samples have similar PL emission peak positions. Figure 7 shows the

observed from TIRFM-based blinking analysis. Herein the Kblink value can be a useful parameter to directly scale the degree of blinking state of QDs. More importantly, this approach allows direct characterization on blinking of QDs in free solution. As the apparent concentration of nanoparticles can be measured by FCS, Kblink can also facilely be extended to characterize the blinking behaviors of other fluorescent nanoparticles including CdSe,45,50 PbSe,51 PbS,52 CuInS2, and ZnCuInS3 QDs53 and nanowires.54 Blinking Index at Various Laser Intensities. Using blinking index, the effect of laser intensity on blinking behaviors of QDs was investigated. Figure 5a displays FCS curves of NAC-capped QDs excited with different laser intensities of a 488 nm argon ion laser. It was observed that the amplitude of correlation curves (G(0)) greatly increased with the increase of laser intensity. Meanwhile, Figure 5b shows that the expansion of the detection volume is negligible within the low laser intensities range that was calibrated using RG as a reference.48 Thus, the increase of G(0) implies that the measured concentration of bright QDs (Cb,app) deceases with the increased laser intensity. Figure 5c reflects the dependence of Kblink on the excitation intensity. The Kblink increases with laser intensity, which suggests that the increased laser intensity leads to the more seriously blinking behaviors of QDs. The dependence of blinking on excitation intensity was consistent with the previous reports.55,56 According to the model in Figure 4, it should be attributed to a near-exponential fall off of ontime probability distributions at long times due to the photoinduced process of laser irradiation.39 Suppressed Blinking with Size Growth. Figure 6 shows the change in Kblink values of NAC-capped QDs samples

Figure 7. Blinking comparison of QDs prepared with different thiolligands. 1.2TP-QD597 (a), 2.5TP-QD604 (b), 1.2MSA-QD589 (c), 2.5MSA-QD593 (d), 1.2NAC-QD606 (e), 2.5NAC-QD602 (f), 1.2MPA-QD593 (g), and 2.5MPA-QD597 (h).

Kblink comparisons of QDs samples capped with different ligands (TP, MPA, MSA, and NAC). For different thiol-capped QDs, it was observed that their Kblink decreased with the increased ratio of thiol-to-Cd. This result demonstrated that blinking behaviors of these QDs was suppressed by increasing the ratio of thiols-to-Cd in the synthesis. The mechanism for blinking suppression induced by thiols was probably attributed to the elimination of QDs surface traps due to strong coordination of thiol ligands in these sites.59 Meanwhile, it was reported that, the decomposition of thiol compounds in the aqueous synthesis of QDs could slowly release active “S” and then “S” bound to surface trap sites of QDs.60 However, different ligands show different blinking suppression efficiency. In contrast to strong suppression of MPA, TP and MSA on the blinking of QDs, Kblink values show that QDs capped with NAC appear to have weak blinking suppression efficiency with the increase in the ratio of NAC-to-Cd. This is possibly related to the different quenching capacity of ligands on surface traps. The results extracted from the measured Kblink values are consistent with the as-reported results based on the power-law blinking statistics on QDs. In the previous findings, the off-time probability distributions for QDs shows that the power-law off exponents (moff) remarkably increase as the ratio of thiol-to-Cd increases or the laser intensity decreases.61 It indicates that the probability of blinking with long “off”-duration time (toff) decreases but the probability of blinking with short toff increases. As described in Figure 4, the decrease of the probability with long toff blinking results in that more QDs can be detected by FCS system, which can lead to the decrease of Kblink values.

Figure 6. Dependence of blinking behaviors of QDs on reaction time.

prepared with different reaction times. The samples are excited with the same intensity of laser in the FCS measurements. On one hand, the blinking index value of QDs prepared within the initial several hours is observed to be larger than 10.0. It suggests that these small sizes QDs locate a seriously blinking state. This result is mainly attributed to lots of trap sites formed in the surface of small size QDs.57 On the other hand, it was found that the Kblink decreased with the increased reaction time. It implies that the growth of QDs is a blinking suppression process. The possible reason is that, with the epitaxial growth of CdTe QDs, their surface is gradually smoothed and more surface traps are gradually modified. The size-dependent blinking suppression is also observed in the other experiments.58 Ito et al. believed that the blinking suppression induced by size growth could be attributed to the electron tunneling effect between a QD and the matrix.58

4. CONCLUSION In this work, a new method was described for direct characterization of QDs blinking states in free solution by combining FCS and ensemble spectroscopic methods. The principle is based on the fact that the apparent concentration of aqueous QDs measured by FCS is far less than the actual concentration measured by ensemble spectroscopic method due to QDs blinking. A blinking index with the ratio of actual concentration to apparent concentration was proposed to assess the blinking of different QDs samples. This method can 12974

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directly exhibit the “real” blinking state of QDs in free solution, avoiding interference from the local microenvironment on blinking when QDs are immobilized in the matrix. Furthermore, this method was successfully used to investigate the effect of laser intensity, growth process, and surface ligands of QDs on their blinking. In future applications, it is significant to characterize the blinking suppression process in the synthesis of nonblinking QDs using the blinking index.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information about TIRFM imaging movies of typical QDs, concentration measurements on RG with UV−vis spectroscopy and FCS methods, FCS curves of different QDs and their fitting residual curves, and the influence of brightness variations on FCS. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-21-54746001. Fax: +86-21-54741297. E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by NFSC (21075081, 21135004, 21327004, and 21475087), Innovation Program of Shanghai Municipal Education Commission (14ZZ024), and SMC-Chenxin Young Scholar project sponsored by Shanghai Jiao Tong University.

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