ZnS Quantum Dot

Article pubs.acs.org/JPCC

Reversible Modification of CdSe−CdS/ZnS Quantum Dot Fluorescence by Surrounding Ca2+ Ions Li Li,† Yun Chen,‡ Guangjun Tian,§ Victor Akpe,† Hao Xu,† Li-Ming Gan,‡,∥ Stanko Skrtic,∥,⊥ Yi Luo,§ Hjalmar Brismar,† and Ying Fu*,† †

Science for Life Laboratory, Department of Applied Physics, Royal Institute of Technology, SE-10691 Stockholm, Sweden Department of Molecular and Clinical Medicine/Clinical Physiology, The Sahlgrenska Academy and University Hospital, University of Gothenburg, SE-41345 Gothenburg, Sweden § Division of Theoretical Chemistry and Biology, School of Biotechnology, Royal Institute of Technology, SE-10691 Stockholm, Sweden ∥ AstraZeneca R&D, S-431 83 Mölndal, Sweden ⊥ Department of Internal Medicine, Sahlgrenska Academy and University Hospital, University of Gothenburg, SE-41345 Gothenburg, Sweden ‡

ABSTRACT: It has been known for a long time that the fluorescence intensity of colloidal quantum dots (QDs) becomes modified when free ions are added to the QD solution. The consequences of removing free ions from the QD solution, however, have not been closely investigated. In this work we studied fluorescence from 3-mercaptopropionic acid (3-MPA) coated CdSe−CdS/ZnS core−multishell QDs when free Ca2+ ions were added to and subsequently removed from the QD solution. It was found that QD fluorescence intensity was reduced when Ca2+ ions were added to the QD solution, while the wavelength of the QD fluorescence peak remained unchanged. QD fluorescence recovered when the concentration of free Ca2+ ions in the QD solution was reduced by adding Ca2+ chelator (ethylene glycol tetraacetic acid, EGTA). It was further observed that the time of single QD fluorescence at on-state and QD fluorescence lifetimes were also reduced after adding Ca2+ and then recovered when EGTA was added. Theoretical study shows that a free Ca2+ ion can attach stably to the system of [QD + surface ligand], attract the photoexcited electron, and repel the photoexcited hole inside the QD core, leading to the reduction of the radiative recombination between the electron and hole, thereafter decreasing the QD fluorescence intensity, on-state time, and fluorescence lifetimes, as observed experimentally. To the best of our knowledge, this is a first study to show that the changes of QD optical properties are reversible under the influence of Ca2+ ions. We further estimated the equilibrium association constant pKa of our QDs with Ca2+, which is much larger than QDs with Mg2+, Na+, and K+, indicating the feasibility of developing a QD-based Ca2+ sensor.

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that the QD fluorescence decreases following the increase of free ion concentration, while the consequences of removing free ions from the QD solution have not been closely investigated since it is much more difficult to remove free ions from the QD solution than to add ions to the QD solution (simply adding highly concentrated stock solutions of ions). Another important aspect of ion sensing is to be able to be ion specific. So far, QD fluorescence intensity always decreases when free ions are added. A quantitative study of the QD fluorescence modification as a function of ion species is thus of great significance. Monitoring Ca2+ in live cells is critical in biological research, especially in neuroscience.17−19 There are several commercial Ca2+ indicators such as Fluo-3 and Fluo-4, but all of them are

INTRODUCTION Colloidal quantum dots (QDs) have been extensively studied and developed as optical contrast elements for bioimaging applications because of their superior optical properties in comparison with traditional organic dyes.1−4 A lot of work has further been devoted to utilize these QDs as biosensors5 to monitor biological parameters such as pH value6,7 and ion concentrations in bioenvironments. The principal sensing mechanism is that QD fluorescence is modified when pH value or ion concentration changes. Many studies show that the fluorescence intensity of QDs was decreased when adding free ions such as Zn2+, Cu2+, Ag+, Fe3+, Hg2+, Na+, K+,8−14 as well as Ca2+15,16 to the QD solution, leading to the potential of developing QD-based ion sensors. A critical criterion for a fully functional QD-based ion sensor is the ability to follow the dynamical change, i.e., both the increase and the decrease, of the ion concentration in the bioenvironment. In laboratory setups, it has been clearly shown © 2014 American Chemical Society

Received: January 24, 2014 Revised: April 24, 2014 Published: April 24, 2014 10424

dx.doi.org/10.1021/jp500853h | J. Phys. Chem. C 2014, 118, 10424−10433

The Journal of Physical Chemistry C

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fluorescence spectral measurement. The pH value of the QD solution was monitored continuously by a pH electrode (InLab, Semi-Micro, Mettler Toledo), showing that the pH value decreased very slightly (less than 0.15) during the experiments and thus the effect of the pH modification on the QD fluorescence was negligible. QD Fluorescence Spectrum Measurement. Fluorescence spectra of the QD solutions were measured by an optical spectrometer (FluoroMax-3, Horiba Jobin Yvon) along with the sequential injections of ions and EGTA. Since the temporal responses of the QD fluorescence to the injection depend on the type and amount of the injected item, we repeatedly measured the fluorescence spectrum after each injection at a time interval of 4 min to reveal the dynamic cross interactions of Ca2+, [QD + surface ligand], and EGTA. Single-QD Fluorescence Imaging. The samples for single-QD fluorescence imaging were prepared by dropping 10 μL of QD solution into circular areas formed by nail polish on microscope slides then covered by glass coverslips. Fluorescence images were monitored by using an AxioObserver.D1 microscope (Carl Zeiss) equipped with a mercury lamp (HBO 100, Carl Zeiss), a filter set (Exciter: FF02-435/40-25, Dichroic: FF510-Di02-25 × 36, Emitter: FF01-500/LP-25, Semrock), an EMCCD camera (Andor), and a 100 × 1.4 NA oil immersion objective (Carl Zeiss). QDs were excited by the spectral line of 415−455 nm centered at 436 nm from the mercury lamp, with an excitation intensity of 10.7 W/cm2. We noticed that QDs needed several minutes to attach to the surfaces of microscope slides to be immobile, and fluorescence images thereafter acquired were stable and used for analysis. Two kinds of single-QD fluorescence images were acquired: (1) Images of bright QD-fluorescence spots in different QD solutions (i.e., Figure 4) were taken with a time interval of 50.91 ms (the exposure time was 50 ms and readout time 0.91 ms), and each image frame contains 512 × 512 pixels (51.2 × 51.2 μm2). (2) For single-QD fluorescence measurements (Figures 5, 6, and 7), one imaging series consisted of 10 000 frames. The size of each frame was 64 × 64 pixels (6.4 × 6.4 μm2), and the time interval was 5.2 ms (4.29 ms exposure time and 0.91 ms readout time). The images were analyzed by using ImageJ software.26 QD Fluorescence Lifetime Measurement. QD fluorescence lifetime was measured by using a time-correlated single-photon counting machine (FluoroMax-3, Horiba Jobin Yvon). QDs were excited by a 495 nm laser. The detector was set at 607 nm (QD fluorescence peak wavelength) with a bandpass of 2 nm. The peak was preset at 10 000 counts. The signal was recorded by DataStation v2.3 software (Horiba Jobin Yvon). The fluorescence spectrum and its temporal development were measured immediately after the injection of ions and EGTA to the QD solution, which shows that it needed more than 20 min for the QD fluorescence spectrum to become stabilized after ion or EGTA injection. Therefore, single-QD fluorescence imaging and QD fluorescence lifetime measurements were performed 0.5−1.0 h after sample preparations to ensure that the cross interactions of [QD + surface ligand], Ca2+, and EGTA were completed before the measurements.

easily bleached.20 Developing a QD-based Ca2+ sensor is thus very attractive, with many exciting new developments such as cell-permeable QDs.21 The effect of adding free Ca2+ to the QD solution was studied before.15,16 We notice that a Ca2+ ion chelator, ethylene glycol tetraacetic acid (EGTA), is able to specifically capture a free Ca2+ ion, usually in a one-to-one relationship.22 Thus, the Ca2+−EGTA interaction provides an excellent tool to study and develop QD-based ion-sensing function. The aim of this work is therefore to study and understand the fluorescence of water-soluble CdSe−CdS/ZnS core− multishell QDs as a function of the dynamical change of free Ca2+ ion concentration in the QD solution by utilizing the Ca2+−EGTA interaction. The fluorescence spectrum of the QDs was carefully examined in terms of the fluorescence intensity, peak wavelength, single-QD blinking, and fluorescence lifetime as functions of the concentrations of Ca2+ and EGTA in the QD solution. Density-functional and solid-state theoretical studies were also performed to understand and quantitatively analyze our experimental data and the interactions among Ca2+, [QD + surface ligand], and EGTA, from which we were able to extract the equilibrium association constant pKa between [QD + surface ligand] and Ca2+, which is shown to be much larger than that for [QD + surface ligand] with Mg2+, Na+, and K+, indicating the feasibility of developing a QD-based Ca2+ sensor. To the best of our knowledge, this is the first time that the changes of QD optical properties are shown to be reversible under the influence of Ca2+ ions.

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MATERIALS AND METHODS QD Sample Preparation. Water-soluble CdSe−CdS/ZnS core−multishell QDs were fabricated in-house by common recipes.23,24 They were coated by 3-mercaptopropionic acid (3MPA) and had a fluorescence peak at 607 nm at room temperature, consisting of a CdSe core, a CdS shell of 2 monolayers, and another shell of 1.5 monolayer ZnS. The total diameter of the QDs was 5.7 nm excluding 3-MPA surface ligands. We emphasize that these QDs together with their surface ligands (3-MPA in this work) are entities that interact with their environment, so when ambiguity may arise, we denote them as [QD + surface ligand]. These QDs were dispersed to a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution containing 50 mM HEPES and 23 mM NaOH with a pH value of 7.2. The QD concentration in the QD HEPES buffer solution was determined by the measurement of the QD absorption spectrum (Pharmacia LKB Biochrom 4060 UV−visible spectrophotometer) and the empirical relationships between the QD concentration and the absorbance,23,25 and the result was 37 nM, which is common for many bioapplications.4 Stock solutions of various ions and EGTA were first prepared by dissolving relevant reagents in HEPES buffer, and the concentrations were 5 M (NaCl), 1 M (MgCl2), 5 M, 1 M, 100 mM, 10 mM, 1 mM, 0.1 mM (CaCl2), and 0.5 M EGTA plus 1 M NaOH, respectively. They were then injected into the QD solution stepwise with very high dilution rates (500−2000 for ions and 100−250 for EGTA so that the volume change of the QD solution was negligibly small after adding various items) to study their effects on the QD fluorescence. After each item injection, we used an Eppendorf pipet to repeatedly (20 times) draw up and then inject back 1 mL of solution from and to the cuvette that contained 2 mL of QD solution so that the injected item was thoroughly mixed in the QD solution prior to

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RESULTS AND DISCUSSIONS Effect of Ca2+ Ions on the QD Fluorescence Spectrum. One series of QD fluorescence spectra after Ca2+ and EGTA injections is shown in Figure 1, where text in each step (1−10) 10425



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Figure 1. Effect of Ca2+ ions and EGTA on QD fluorescence spectrum. Text indicates the final concentration of Ca2+ or EGTA after Ca2+ or EGTA injection.

indicates the final concentration of Ca2+ or EGTA after each injection. More specifically, starting with the QD HEPES solution (step 1), we injected Ca2+ ions into the QD solution to get a final Ca2+ concentration of 0.05 μM at step 2. Ca2+ ions were further added to increase its concentration by another 0.05 μM at step 3 so that the final Ca2+ concentration at step 3 was 0.1 μM. In other words, the final concentrations of injected Ca2+ and EGTA in steps 2−10 were 0.05 μM, 0.1 μM, 1 μM, 10 μM, 0.1 mM, 1 mM, and 2 mM Ca2+, 2 mM Ca2+ + 2 mM EGTA, and 2 mM Ca2+ + 5 mM EGTA, respectively. Figure 1 clearly demonstrates that Ca2+ ions suppress the QD fluorescence, but they do not affect the wavelength of the QD fluorescence peak (inset of Figure 1). Most importantly, such modification is reversible; i.e., the QD fluorescence intensity is totally recovered after EGTA is added to chelatefree Ca2+ ions in the QD solution. The decrease and recovery of QD fluorescence intensity under the influence of Ca2+ ions are more clearly revealed in Figure 2(a) where the QD fluorescence intensity, i.e., the area under the fluorescence peak in the optical range of 550−665 nm, is presented as a function of Ca2+ and EGTA injection. Here the temporal developments of the interactions between QDs and Ca2+ ions as well as between Ca2+ ions and EGTA are revealed (the time interval between successive spectral measurements is 4 min). We notice that the QD fluorescence intensity decreases most drastically immediately after each Ca2+ injection, and it gradually stabilizes (20 min). QD fluorescence intensity increases gradually after adding 2 mM EGTA. However, an extra 3 mM EGTA (step 10) first suppressed the QD fluorescence, which recovered after about 16 min (see also Figure 3 below). These temporal developments reflect the dynamic cross interactions among [QD + surface ligand], Ca2+, and EGTA. Figure 2(b) shows the QD fluorescence intensity as a function of the Ca2+ concentration. Most interestingly, drastic modifications in the QD fluorescence intensity occur in the Ca2+ concentration ranges of 0−1 μM and 0.2−2 mM, which coincide with the intracellular and extracellular Ca2+ variation ranges, respectively. This suggests the possibility of directly using 3-MPA coated QDs to monitor intracellular and extracellular Ca2+ concentrations. Figure 2(c,d) shows data from another measurement, which are very close to Figure 2(a,b). We however observe deviations

Figure 2. (a) Temporal development of QD fluorescence intensity vs Ca2+ and EGTA. Text indicates the final concentration of Ca2+ or EGTA after Ca2+ or EGTA injection. (b) Fluorescence intensity vs final Ca2+ concentration in each step (2−8) in (a). Each data point was obtained from the last measured fluorescence spectrum in the corresponding step. Normal intracellular and extracellular Ca2+ ion concentration ranges are marked for reference. (c,d) Same as (a,b) but from another measurement.

and fluctuations when EGTA was added, i.e., step 9 and 10 in both Figures 2(a) and (c). In general, EGTA is able to capture free Ca2+ in a one-to-one relationship. Our work shows however that 5 mM EGTA was needed to restore the QD fluorescence in the presence of 2 mM Ca2+, and the restoring process was very slow (>100 min). This is most probably due to the cross interactions among [QD + surface ligand], Ca2+, and protonated EGTA. Effect of Ca2+ on the QD Fluorescence Intensity in the Presence of EGTA. To investigate the influence of Ca2+ on 10426



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the QD fluorescence in the presence of EGTA, EGTA was first added to the QD solution, and different amounts of Ca2+ ions were then added stepwise. The fluorescence intensity is presented in Figure 3(a) which demonstrates that EGTA

that the radiative recombination between the electron and hole is reduced, leading to the decrease in the fluorescence intensity. A similar effect should be expected when negative charges are added to the QD solution (Figure 10). We notice in Figure 3 a 30% decrease of the QD fluorescence intensity directly after adding a large amount of EGTA. A similar suppression was also present after adding 3 mM EGTA at step 10 in Figure 2. QD fluorescence intensity recovered about 20 min later. It is known that at pH 7.2 about 98% EGTA in solution exists as H2EGTA2−, 2% as HEGTA3−, and a negligible fraction is in EGTA 4− form,28 indicating that EGTA in solution is protonated and negatively charged. Thus, a large EGTA dose will result in a large statistical probability that protonated EGTA stays close to the QD to reduce the QD fluorescence. However, as will be shown shortly by theoretical studies, [QD + surface ligand] can only stably bind to a positively charged ion, so that the effect of EGTA on the QD fluorescence will gradually diminish. This is most probably what happened in step 10 of Figure 2(a) and in Figure 3(a). Note that in Figure 3(b) there was only a modest decrease in fluorescence intensity when Ca2+ was added in the presence of K+, so that the sensitivity of this Ca2+-induced change is quite low when we intend to develop a QD-based Ca2+ sensor. A simple way to increase the sensitivity is to increase the QD concentration (only 37 nM in the current work) so that there will be more ion-free QDs after binding with K+. Effect of Ca2+ on Single-QD Fluorescence. Figure 4 presents three typical fluorescence images of single QDs in

Figure 3. (a) Effect of Ca2+ on the QD fluorescence in the presence of EGTA. (b) The effect of Ca2+ on the QD fluorescence in a simulated intracellular environment.

totally inhibits the Ca2+-induced quenching effect on the QD fluorescence. Note that QD fluorescence recovered to a higher level than the original solution when 2 mM Ca2+ was added. We notice two factors related to the over-recovery of the QD fluorescence: one was the coexistence of highly concentrated Ca2+ and EGTA, and the other was the long measurement time (>180 min), both of which were expected to cause deviations and fluctuations. However, EGTA inhibition of the Ca2+induced quenching effect was unambiguously demonstrated. Effect of Ca2+ on the QD Fluorescence Intensity in a Simulated Intracellular Environment. To mimic the intracellular environment, 10 mM Na+, 0.5 mM Mg2+, and 140 mM K+ were added to the QD solution stepwise (see Figure 3(b)), showing that all these ions suppress the QD fluorescence. Different amounts of Ca2+ were then injected into the QD solution, and the QD fluorescence intensity decreased even further, as expected. Adding EGTA recovered the QD fluorescence. This confirms that EGTA chelates largely free Ca2+ in the QD solution. We notice however that the QD fluorescence recovered beyond the level detected before adding Ca2+ when Mg2+ ions were present. This is simply due to the fact that EGTA also binds with Mg2+ (see more discussions below).27 We have shown thus far that additions of free ions (Ca2+, Na+, Mg2+, and K+ in the current work) to the QD solution will quench the QD fluorescence, and the removal of free Ca2+ ions from the QD solution will restore the QD fluorescence, indicating clearly a stable binding between QD and free ions, while the binding is relatively weak when compared with the ion chelation. The observed QD fluorescence modification can be understood by the theory we proposed before14 that a positively charged ion in close proximity to the QD attracts the photoexcited electron and repels the photoexcited hole so

Figure 4. Fluorescence images of single QDs in (a) HEPES solution, (b) HEPES solution plus 2 mM Ca2+, and (c) HEPES solution plus 2 mM Ca2+ and 5 mM EGTA.

HEPES solution, HEPES solution plus 2 mM Ca2+, and HEPES solution plus 2 mM Ca2+ and 5 mM EGTA, respectively. In the time domain, a large majority of the bright spots in the three images were all blinking, thus representing light emissions from single QDs. Note that the brightness of these bright QD spots in the images was only relative due to contrast adjustments in the microscope, and the spatial densities of these spots in different images represented spatial densities of QDs deposited on the microscope slides. Therefore, the principal physical parameter to be closely examined is the geometrical size of each bright QD spot, which remains largely the same in the three images of Figure 4. The blinkings and the unchanged geometric sizes of the bright QD spots suggest that QDs did not significantly aggregate after Ca2+ and EGTA additions. Figure 5(a) shows a typical fluorescence trajectory of one single QD in HEPES buffer. Through the analysis of many single QDs, we found that most QDs fluoresced at a level between 800 and 1200 photon counts at on-state, whereas there were a few extremely bright spots fluorescing over 4000 photon counts which were excluded in the following statistical analysis since most probably they were QD clusters. The on− off distribution profile of single QD fluorescence is obtained in 10427



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on- and off-states in the whole fluorescence trajectory, which is denoted as on−off probability density.29 A large probability density means that the QD dwells more frequently at this state. The statistical results of the three groups of QDs are presented in Figure 7. Figure 7(a) indicates that QDs in HEPES were

Figure 5. (a) Fluorescence trajectory of one single QD in HEPES buffer. Inset displays the first 200 frames. (b) On−off distribution of this QD. (c) On−off distribution after excluding rare events. Red dashed line marks the statistical threshold below which the QD is at the off-state and above which the QD is at the on-state.

the following way:24 The fluorescence intensity range, i.e., from 0 to the highest fluorescence intensity, in one fluorescence trajectory, such as Figure 5(a), was divided into 400 subranges. The number of fluorescence occurrences in each subrange was counted, and the result is shown in Figure 5(b), denoted as the on−off distribution. For further statistical analysis we excluded rare events, such as the one marked by an arrow in Figure 5(a) (see more detail in the inset). By setting a cutoff occurrence of 6, the new on−off distribution was obtained and shown in Figure 5(c). We summarized many bright single-QD spots (excluding the extremely bright spots) in the images of 64 × 64 pixels (6.4 × 6.4 μm2) from three different QD solutions, and the average on−off distributions of single-QD fluorescences (22 single QDs in HEPES, 38 in HEPES + 2 mM Ca2+, and 27 in HEPES + 2 mM Ca2+ + 5 mM EGTA, respectively) are presented in Figure 6. It is shown here that after adding 2 mM Ca2+ the on-state

Figure 7. Statistics of on- (red hollow stars) and off-time (black solid stars) durations of single-QD fluorescence in three different QD solutions. Dashed diagonal lines are added to guide the eye.

more likely to dwell at the on-state. Ca2+ induced two effects (see Figure 7(b)). First, the on-time probability density was reduced, which was already reflected in Figure 6. Ca2+ further reduced the probabilities of long time durations, which means that the frequency of switching between the on- and off-states was increased. The latter agrees with our model that Ca2+ at the QD surface facilitates carrier transfers between the QD core and surface region (see more in the Theoretical Studies and Discussions section). EGTA restored the statistical behavior of on/off probability density (see Figure 7(c)). Effect of Ca2+ on QD Fluorescence Lifetime. Fluorescence lifetimes of QDs in different solutions are shown in Figure 8(a). The decay curves of 0.1 and 1 μM would too closely pack in between curve 1 and 2 so are not presented for the sake of clarity. However, their fluorescence lifetime characterizations were analyzed together with other curves shown in Figure 8(a) and are all presented in Figure 8(b−d). Fitting of the fluorescence decay curves by a single exponential decay did not converge, while ⎛ t − t0 ⎞ ⎛ t − t0 ⎞ y = y0 + A1 exp⎜ − ⎟ ⎟ + A 2 exp⎜ − τ1 ⎠ τ2 ⎠ ⎝ ⎝

(1)

describes numerically very well all the fluorescence decay curves. Three exponential decays improved the fitting numerically. However, the obtained fitting parameters varied without any clear trends as functions of the Ca2+ and EGTA concentrations, indicating that the three-exponential fitting was not physically meaningful. The results of eq 1 are presented in Figure 8(b−d) including statistical standard errors. Two processes were clearly distinguished here: one was slow with an initial lifetime τ1 = 14.087 ± 0.067 ns, and the other one was fast with τ2 = 2.292 ± 0.018 ns. Adding Ca2+ ions to the QD solution decreases both τ1 and τ2 (to 7.196 ± 0.060 and 1.576 ± 0.008 ns, respectively, after adding 2 mM Ca2+). The percentage A1 of the slow process was decreased, while the percentage A2 of the fast process was increased by Ca2+. EGTA

Figure 6. Averaged on−off distributions of single-QD fluorescence in three different QD solutions.

ratio of single-QD fluorescence decreased, and the off-state ratio increased. After adding EGTA to chelate free Ca2+ ions, the on−off distribution recovered (though not totally). Knowing the statistical threshold that separated the on- and off-states (see Figure 5(c)), we calculated the on and off time durations, i.e., how long the QD stayed at one state before switching to another state, from which we calculated the ratio between the number of times that the QD stayed at one state for a certain time duration and the number of switches between 10428



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electron and repels the hole, leading to increased interactions between the electron/hole and QD surface states so that nonradiative relaxation will be increased and τ1 decreased. At the same time, the displaced electron and hole can transfer more easily to the QD surface states, i.e., more often to the offstate, thus A1 is reduced and A2 increased. Note that the fast process τ2 is about electron and/or hole transfer to the QD surface states, return to the QD core state, then radiative recombine to fluoresce, where the last step is characterized by τ1 so that τ2 is correlated to τ1. This is exactly so when comparing Figure 8(b) and (c).

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THEORETICAL STUDIES AND DISCUSSIONS To understand the interactions among [QD + surface ligand], Ca2+, and EGTA, we performed a density functional theory (DFT) study. The B3LYP functional was used with a mixed 631+g(d) (for nonmetal atoms) and LanL2DZ (for metal ions) basis set. A Zn6S6−3-MPA cluster was chosen to model our [QD + surface ligand].24 We speculated that the oxygen atom in the carbonyl of the carboxyl group could attract Ca2+ due to its electronegativity. Thus, in the DFT study we placed one Ca2+ ion near the oxygen atom in the carbonyl (see Figure 9(a)). The structure was then fully relaxed for searching its

Figure 8. (a) Fluorescence decays of QDs in different solutions. Black curve: QDs in HEPES. Red curves: 0.1, 1, and 2 mM Ca2+. Blue curve: 2 mM Ca2+ + 5 mM EGTA. (b−d) Fluorescence lifetime parameters τ1, τ2, A1, and A2 as functions of Ca2+ (black triangles in HEPES, red circles/lines with Ca2+) and EGTA (blue stars/dashed lines). Standard errors of τ1, τ2, A1, and A2 are presented.

chelated Ca 2+ and restored QD fluorescence lifetime parameters. Figures 6, 7, and 8 show that the QD fluorescence state and the QD fluorescence lifetime are strongly correlated that the fluorescence lifetime is long when the QD is at the on-state and short when the QD is at the off-state. This agrees with early works that measured the fluorescence lifetime separately at the on- and off-state.30,31 On the basis of the above experimental data, we can firmly conclude that the QD excitation and fluorescence under investigation consist mainly of the following processes: (1) A photon from the excitation laser is absorbed by the QD, resulting in an electron in the high-energy conduction-band state and a hole in the high-energy valence-band state in the QD core. (2) The high-energy electron and hole relax to the ground states in the conduction and valence bands via nonradiative processes, mainly electron−phonon interactions.32,33 The electron at the conduction-band ground state radiatively recombines with the hole at the valence-band ground state, resulting in a photon emission. The typical lifetime of the whole process of (2), i.e., electron and hole relaxation from high-energy states to ground states then radiative recombination, is about τ1 = 14 ns. (3) The highenergy electron and/or hole can also transfer to QD surface states, such as the HOMO and LUMO of [QD + surface ligand],24 resulting in the QD fluorescence blinking, which is characterized by τ2.30,31 (4) Ca2+ at the QD surface attracts the

Figure 9. (a) Initial and (b) optimized structures of the Zn6S6−3-MPA cluster with one Ca2+ ion. (a′) Initial and (b′) optimized structures of 3-MPA and Ca2+.

optimized geometry, which was found and presented in Figure 9(b). Vibrational analysis was performed at the same level, and no imaginary frequency was found, which confirmed that the optimized geometry was located at its energy minima. This shows that the 3-MPA-coated QD is able to attract stably the Ca2+ ion. Similar attachments of Na+, K+, and Mg2+ ions on [QD + surface ligand] were found through DFT studies. We used DFT to study the binding between Ca2+ and free 3MPA and obtained an optimized structure similar to the one of Ca2+ and 3-MPA attached to the Zn6S6 cluster (see Figure 9(a′,b′)). However, the binding energy of 3-MPA−Ca2+ was about half the one of Zn6S6−3-MPA−Ca2+ (2.61 and 4.90 eV, respectively, calculated by DFT in vacuum with thermal corrections to Gibbs free energy at 298 K). The large binding energy between Ca2+ and Zn6S6−3-MPA was due to the extra Coulomb attraction between Ca2+ and the negatively charged S atom in Zn6S6 (see Figure 9(b)). In other words, Ca2+ binds to [QD + surface ligand] much stronger than to the free ligand so that [QD + surface ligand] is the entity that binds tightly with Ca2+. 10429



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shows the radiative recombination rate R as a function of α (xα = 3.7 nm, red dashed line) and xα (α = 1.0, black solid line). As demonstrated by Figure 10, the appearance of an ion at the QD surface quenches the radiative recombination between the electron and the hole in the QD core. Moreover, the distance between the QD and the ion xα is a critical factor in determining the radiative recombination rate; i.e., by increasing xα from 3.7 to 10 nm, R increases from 0.2 to 0.95, which explains well the big drop then the quick recovery of the QD fluorescence when adding EGTA shown in Figure 3. Moreover, an anion (α < 0) affects the radiative recombination rate more strongly than a cation (α > 0) because the effective mass of the hole in the valence band is much heavier than the electron. Figure 2 per se does not show directly that Ca2+ binds to the QD surface. It may indicate collisional quenching since QD fluorescence quenching follows the charge concentrations in the QD solution, which is also reflected in the fluorescence lifetime as a function of the Ca2+ concentration. Since the collisional quenching is temperature dependent, we measured QD fluorescence spectra as functions of temperature (between 22 and 50 °C). It was found that the temperature dependences of these spectra of the QD solutions with different ion concentrations were all the same as the one of the QD HEPES solution. In other words, the temperature dependence of these spectra was dominated by the electric and optical properties of the QD material (see, e.g., refs 40 and 41), so that we were not able to use the temperature dependence data to determine whether or not the collisional quenching was the major cause of the QD fluorescence modification. However, the interaction between the ion and the QD should be more than just collision since the relationship between collisional quenching and quencher concentration is normally linear. Furthermore, Figures 2 and 3 show that it is the charge that affects the QD fluorescence. QDs respond to both positive charges (Na+, K+, Mg2+, Ca2+) and negative ones (protonated EGTA) but in very different ways, while Figure 10 says that both the positive and negative charges should affect the QD fluorescence in a similar way as long as the charges are close to the QD surface. In addition, the physical sizes of Ca2+ and K+ are approximately the same, so are Mg2+ and Na+, for which we should expect two similar collisional quenching effects from Ca2+ and K+ and two similar collisional quenching effects from Mg2+ and Na+, while experiments show otherwise. Together with the DFT study of Figure 9, we therefore conclude that the ion binds to the QD surface, and the binding is ion-species dependent. We now use Figure 10 to analyze the strength of binding between QD and Ca2+ from Figure 2. It can be easily estimated that Coulomb potential energy will be 1.152 eV when two electric charges (each 2e) stay at a distance of 5 nm (such as two Ca2+ ions associate to one QD), which is a very high energy so that the most probable scenario is that a single Ca2+ ion associates and dissociates to a single QD dynamically. We write the equilibrium status of the association and dissociation process as in standard textbook format42

Quite different from the quenching effect due to the neighboring fluorescent particles,34 the appearance of an ion at the QD surface modifies the QD fluorescence through modifying the spatial distributions of the electron and hole photoexcited in the QD core by the Coulomb potential of the ion. The situation is rather similar to the work of Jha and Guyot-Sionnest where an external electric field was applied to modify the wave functions of the electron and hole.35 There are other ways to modify the electron and hole wave functions such as core/shell structure25 and surface ligands.36,37 The radiative recombination rate R between the electronoccupying conduction-band ground state ψc0(r) and the hole at the valence-band ground state ψv0(r) is proportional to38 R=|

∫ ψc0*(r)ψv0(r)dr|2

(2)

We adopted the solid-state theory38 to calculate the wave functions of the electron and hole in the QD core under the influence of an electric charge (αe, where e is the charge unit) then calculated R. The electron and hole in the QD were described by the commonly used solid-state model.39 For our CdSe−CdS/ZnS core−multishell QD (CdSe core radius 2 nm, 2 monolayer CdS, 1.5 monolayer ZnS, total QD diameter 5.7 nm; see subsection QD Sample Preparation), the calculated transition energy between ψc0(r) and ψv0(r) is 2.039 eV (608 nm), in agreement with the experimental fluorescence peak wavelength (607 nm). The electric charge was positioned at (xα,0,0) (see inset of Figure 10(c)). Figure 10(a) and (b) is contour plots of ψc0(r) and ψv0(r) at z = 0 when α = 0.5 and xα = 3.7 nm, demonstrating clearly that the electron is attracted to the positive charge while the hole is pushed away. Figure 10(c)

QD−Ca 2 + ⇌ QD + Ca 2 +

Figure 10. (a, b) Wave functions of the electron ψc0(r) and hole ψv0(r) at z = 0 when α = 0.5 and xα = 3.7 nm. (c) Radiative recombination rate R as a function of |α| (xα = 3.7 nm, red dashed line: α > 0, red dashed line with solid red stars: α < 0) and xα (black line: α = 1.0, black line with hollow black stars: α = −1.0). Inset shows the schematic geometrical structure of a QD with an electric charge αe at (xα,0,0).

(3)

its equilibrium dissociation constant is Kd = 10430

[QD]·[Ca 2 +] [QD−Ca 2 +]

(4)



Article

Table 1. Equilibrium Association Constants pKa of QDs with Ca2+, Mg2+, Na+, and K+a QD−Ca2+ pKa a

QD−Mg2+

3.20 27

pKa = 3.20

QD−Na+

2.61 2+

between EGTA and Mg

and pKa = 6.91

1.23 28

1.10

between EGTA and Ca

■

where [QD], [Ca2+], and [QD−Ca2+] denote concentrations of unbound QD, unbound Ca 2+ , and bound QD−Ca 2+ , respectively. The equilibrium association constant is given by

pK a = −log10 Kd

QD−K+ 2+

EGTA−Mg2+ 3.20

27

EGTA−Ca2+ 6.9128

at pH = 7.4 are also listed for comparison.

CONCLUSION We observed that the increase of Ca2+ concentration in the QD solution decreased QD fluorescence intensity, fluorescence lifetime, and on-state time of single QDs. After adding EGTA to chelate-free Ca2+ from the QD solution, QD optical properties largely recovered. Theoretical analysis confirmed that a positively charged Ca2+ ion attaches stably to the [QD + surface ligand], separates the photoexcited electron and hole in the QD core, and helps the photoexcited carriers to transfer to the trap states on the QD surface, reducing QD fluorescence intensity, QD radiative lifetime, and on-state ratio of single QDs. Our work demonstrates that effects of the surrounding ions on the QD fluorescence properties in the QD solution are multiple folds including fluorescence intensity and radiative lifetime modifications, and most importantly, these effects are reversible. Moreover, the qualitative modifications of QD optical properties as functions of the ion concentrations are in general similar for the four ion species under investigation (Ca2+, Mg2+, Na+, and K+), but the binding between QDs and Ca2+ is much tighter than others. Thus, water-soluble 3-MPAcoated QDs are promising to be developed as Ca2+ sensors to monitor dynamic change in the Ca2+ ion concentration.

(5)

Since Figure 10 shows that the existence of a positive charge at the QD surface quenches the fluorescence of the QD, we calculate pKa by the following way: [QD] is proportional to the remaining fluorescence intensity after adding Ca2+, and [QD− Ca2+]/[QD] equals the ratio between the reduced fluorescence intensity (i.e., the original fluorescence intensity before adding Ca2+ minus the remaining fluorescence intensity after adding Ca2+) and the remaining fluorescence intensity. The estimation of unbound Ca2+ is difficult, which however can be approximated as the total added Ca2+ concentration when it is much higher than the QD concentration. As stated in the subsection QD Sample Preparation, the QD concentration was only 37 nM. Using the fluorescence intensities of steps 7 and 8 in Figure 2, we obtained pKa = 3.28 and 3.20, respectively, which were close to each other so that the estimation scheme was valid. We performed similar equilibrium association constant calculations for Mg2+, Na+, and K+ by using Figure 3, and the results are summarized in Table 1. Here we observe that the binding between QDs and Ca2+ (also Mg2+, Na+, and K+ under investigation) is much weaker than the one between EGTA and Ca2+. This agrees with our experimental observation that EGTA easily chelated Ca2+ from the QD solution and restored the QD fluorescence. Note that the binding between EGTA and Mg2+ with pKa = 3.2027 is tighter than QD−Mg2+ (pKa = 2.61) so that adding EGTA to the QD solution with Mg2+ also restored partially the QD fluorescence, as shown in Figure 3. Furthermore, the binding between QDs and ions is though not clearly ion-species specific (i.e., see Figure 3), Table 1 indicates that the binding between QDs and Ca2+ is much tighter so that it is possible to develop a QD-based Ca2+ sensor through quantitative spectral analyses. With this, the theory we proposed before14 explains well the effects of the ions on the QD fluorescence that a positively charged ion in close proximity to the QD attracts the photoexcited electron and repels the photoexcited hole so that the radiative recombination between the electron and hole is reduced, leading to the decrease in the fluorescence intensity (see Figures 1, 2, and 3), accompanied by the decrease of the QD fluorescence lifetime (Figure 8). Such a physical mechanism also explains well the experimental data of single QD fluorescence characterization in Figures 6 and 7 and the fluorescence lifetime in Figure 8. The blinking of single QD fluorescence has been attributed to the transfer of the electron or hole from the QD core to trap states on the QD surface.24,43−46 An extra ion on the QD surface pushes the electron and hole toward the QD surface, thus facilitating these carrier transfers, resulting in a decreased on-state ratio and a reduced QD fluorescence lifetime. The removal of the surface Ca2+ ion by adding EGTA restores the radiative recombination between the electron and hole in the QD core, precisely as demonstrated experimentally in this work.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The work was supported by Swedish Research Council (6212011-4381), Swedish Foundation for Strategic Research (Strategisk Mobilitet 2010), Swedish Vinnova (Project number P35914-1), and the PDC Centre for High Performance Computing (PDC-HPC) at the Royal Institute of Technology for computation resources. We sincerely thank Professor Jerker Widengren from Department of Applied Physics, Royal Institute of Technology, for technical support and scientific discussions.

■

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