Rapid and Quantitative Measurement of Single Quantum Dots in a

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Rapid and Quantitative Measurement of Single Quantum Dots in a Sheath Flow Cuvette Shuo Wang, Lihong Li, Shenghao Jin, Weifeng Li, Wei Hang, and Xiaomei Yan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01885 • Publication Date (Web): 18 Aug 2017 Downloaded from http://pubs.acs.org on August 19, 2017

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Analytical Chemistry

Rapid and Quantitative Measurement of Single Quantum Dots in a Sheath Flow Cuvette

Shuo Wang, Lihong Li, Shenghao Jin, Weifeng Li, Wei Hang, Xiaomei Yan*

Collaborative Innovation Center of Chemistry for Energy Material, MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (P. R. China)

*To whom correspondence should be addressed. E-mail: [email protected]

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ABSTRACT Semiconducting quantum dots (QDs) are finding a wide range of biomedical applications due to their intense fluorescence brightness and long-term photostability. Here, we report precise quantification of the fluorescence intensity of single QDs on a laboratory-built high-sensitivity flow cytometer (HSFCM). The nearly uniform illumination of the particles at the intense portions of the radiation field resulted in narrowly distributed signals with high signal-to-noise ratios. By analyzing thousands of QDs individually in as little time as 1 min, intrinsic polydispersity was quickly revealed in a statistically robust manner. Applications of this technique in QD quality assessment, study of metal ion influence, and evaluation of aggregation upon biomolecule coupling are presented. Moreover, an accurate measurement of the QD particle concentration was achieved via single-particle enumeration. HSFCM is believed to provide a powerful characterization tool for QD synthesis and application development.

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INTRODUCTION Semiconducting quantum dots (QDs) are highly fluorescent nanocrystals that exhibit intense brightness, broad excitation and narrow emission spectra, long-term stability against photobleaching, and size-tunable optical properties.1-3 Among the types of QDs, water-soluble QDs have been exploited in an ever-increasing array of biomedical applications, including single molecule tracking, cell and tissue imaging, flow cytometric analysis of single cells, bioanalysis, and others.4-11 However, despite all of the benefits that QDs offer, considerable heterogeneity in the fabrication process has been reported, which leads to intrinsic polydispersity in their photophysical properties.3,12-14 For example, in QDs with a CdSe/ZnS core/shell structure, variations in the size of both the CdSe core and the ZnS shell as well as incomplete shell growth all give rise to differences between particles in a given preparation. Therefore, quantitative measurement of QDs at the single-dot level is important for understanding the photophysics of semiconductor nanocrystals as well as for further development as reliable bright fluorophores.15 Despite the use of a variety of single molecule/particle detection techniques such as total internal refection fluorescence microscopy, confocal fluorescence microscopy, and fluorescence correlation spectroscopy for the analysis of single QDs,14-19 a high-throughput and quantitative method to examine the heterogeneity of QDs in free solution (not subjected to surface-induced artifacts) remains undeveloped. Adopting strategies for single-molecule fluorescence detection in a sheathed flow, we recently developed a high-sensitivity flow cytometry (HSFCM) method.20-23 With this technique, real-time light-scattering detection of single 7-nm gold nanoparticles, 24-nm silica nanoparticles, and 27-nm viruses has been achieved. The method uses a sheath flow 3



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cuvette in which the sample fluid is hydrodynamically focused to a very narrow stream (e.g. 1-2 µm) so that analyte particles pass individually through the center of the laser beam (e.g. 10 µm) with the same trajectory and speed. The nearly uniform illumination of the particles at the intense portions of the radiation field results in even and strong signals, which builds the foundation for quantitative analysis of single nanoparticles. Therefore, different signal intensities can be used to infer different physicochemical properties of the nanoparticles. For example, by directly measuring the scattered light intensity of single nanoparticles, comparable size resolution to that of electron microscopy has been achieved.22,23 In the present study, we report quantitative fluorescence measurement of single QDs. In order to achieve sufficient detection sensitivity, the microscope objective and the flow chamber were redesigned to increase light collection efficiency. Applications of HSFCM in the assessment of QD quality, metal ion-induced QD fluorescence quenching, QD aggregation upon biomolecule coupling, and QD particle concentration are presented.

EXPERIMENTAL SECTION Reagents and Chemicals. Qdot655, Qdot655-Sav, and 200-nm yellow-green fluorescent polystyrene beads were purchased from Molecular Probes/Invitrogen (Carlsbad, CA, USA). Biotin-conjugated Donkey Anti-Mouse IgG was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Na2HPO4 (≥99.5%) and K2HPO4 (≥99.5%) were purchased from Sigma (St. Louis, MO, USA). Tween 20 was purchased from BioBasic (Markham, Ontario, Canada). C10H14N2Na2O8·2H2O (EDTA), CuCl2·2H2O, FeCl3, and PbCl2 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, 4


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China). All the materials were used as received. Distilled, deionized water supplied by a Milli-Q RG unit was filtered through a 0.22 µm filter and used for the buffer preparation and served as the sheath fluid of the HSFCM. Laboratory-built

High-sensitivity

Flow

Cytometer.

The

laboratory-built

instrument described previously22,23 was used in the present study with three modifications. To match with the excitation wavelength of QDs, the original 532-nm laser was replaced with a 200-mW 488-nm laser. To collect more light emitted by single QDs passing through the detection volume, the original achromatic objective lens with a numerical aperture (N.A.) of 0.55 was replaced with a custom-designed, long-working distance, gel-coupled objective with N.A. of 1.2. To accommodate the larger light collection angle endorsed by the new objective, a custom-designed flow chamber was fabricated. Based on the theoretical calculation, the increase from N.A. 0.55 to N.A. 1.2 of the objective can enhance the light collection efficiency 4.7-fold. The experimental setup is schematically illustrated in Figure 1. Briefly, the sample fluid was injected from a tapered capillary (40 µm i.d., 240 µm o.d.) into the center of a custom-made cuvette (Jingke Optical Instrument, Yixing, Jiangsu, China) with 250 × 600 µm rectangular quartz flow channel where it was hydrodynamically focused by the sheath fluid to a very fine stream (~1.4 µm in diameter). A 200-mW 488-nm laser was used as the excitation source. A half-wave plate and a polarizing beam splitter provided polarization control and continuously variable attenuation of the laser light. The laser beam was focused to a ~10 µm diameter spot by an achromatic-doublet lens onto the hydrodynamically focused sample stream. The light emitted by QDs was collected perpendicularly to both the laser beam and the sample stream using a custom-designed, long-working distance objective 5



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with N.A. of 1.2 (AZURE Photonics, Fuzhou, Fujian, China). After being spectrally filtered by an edge filter (BLP01-532R-25, Semrock) and a bandpass filter (FF01-660/52, Semrock), the fluorescent light was focused by a lens (AZURE Photonics) onto a single-photon counting avalanche photodiode detector (APD, Excelitas Technologies model SPCM-AQRH-12, dark count 1000 c/s) for detection. The detection volume, defined as the overlap of the focused laser spot and the sample stream, was calculated to be ~15 fL. At a QD concentration of 3 × 109/mL, the probability that two QD particles would pass through the 15-fL probed volume simultaneously is 0.1%, which is negligible. A custom program written in LabVIEW 2012 (National Instruments) was used for data acquisition and processing. In brief, the bin width was set to 100 µs. For each sample, 1 min of data acquisition was performed.

Figure 1. Schematics of the laboratory-built high-sensitivity flow cytometer (HSFCM). (a) Instrument configuration: P, half-wave plate; S, polarizing beam splitter; M, mirror; L1 and L2, focal lens; C, cuvette; EF, edge filter; BP, bandpass filter; APD, single-photon counting avalanche photodiode detector. (b) An enlarged view of laser illumination upon hydrodynamic focusing in a sheath flow cuvette.

Coupling QDs with Biomolecules. Qdot655-Sav stock solution (1 µM) was diluted 100-fold with phosphate buffer (pH 7.4) containing 0.05% Tween 20. Biotin-conjugated 6


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Donkey Anti-Mouse IgG was then added with a Qdot655-Sav : biotinlylated-Ab molar ratio of 1:10, 1:20, or 1:50. The mixture was incubated at room temperature for 3 hours before detection. QD Concentration Measurement Using the ICP-MS & TEM Method. The TEM sample was prepared by briefly dipping a TEM grid in a suspension of QDs and letting it dry under ambient conditions. TEM images of Qdot655 were acquired using a JEOL JEM-2100 (HR) transmission electron microscope. ICP-MS samples were prepared by digesting 10 µL of a Qdot655 suspension with 10 µL of aqua regia at 100 °C for 10 min and then diluting the mixture solution with ultrapure water. ICP-MS measurement was performed on an Agilent 4500 ICP-MS. The molar masses are 191.36 g/mol for CdSe (MCdSe) and 97.44 g/mol for ZnS (MZnS), respectively. The densities of CdSe and ZnS were assumed to be the same as those of the bulk material, with ρCdSe of 5.81 g/cm3 and ρZnS of 3.98 g/cm3. The concentration of Qdot655 was calculated using the following equations: CCd × MCdSe + CZn × MZnS = CQD × (ρCdSe × VCdSe + ρZnS × VZnS),

(1)

VCdSe + VZnS = VQD,

(2)

(CCd × MCdSe) / (CZn × MZnS) = (ρCdSe × VCdSe) / (ρZnS × VZnS).

(3)

Of which, CCd and CZn are the molar concentrations of Cd and Zn, respectively; VCdSe, VZnS, and VQD are the volumes of CdSe, ZnS, and QD, respectively; and CQD is the particle concentration of QD in the unit of particles/mL. The final molar concentration of QDs can be obtained by dividing CQD by Avogadro’s number (6.02×1023) and then multiplying by 1000.

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RESULTS AND DISCUSSION High Sensitivity Fluorescence Detection of QDs via HSFCM. Figure 2a depicts the representative fluorescence burst trace from 655 emitting CdSe/ZnS core-shell carboxyl quantum dots (Qdot655, Invitrogen). The peak height of single QDs was detected to be 879 ± 209 counts/bin, which is well above the background. The signal-to-noise ratio (SNR) reached 88:1. With an analysis rate up to 10000 particles per minute, a statistically representative distribution histogram of fluorescence burst height was obtained in minutes. As demonstrated in Figure 2b1, the distribution profile of Qdot655 was quite narrow with a coefficient of variation (CV) of 23.8%. To the best of our knowledge, this is the smallest CV for QD measurements reported to date. Therefore, HSFCM provides a more accurate measurement of the intrinsic heterogeneity of QDs’ brightness by facilitating uniform trajectory and illumination of each individual QD. Taking advantage of the high-throughput analysis of single QDs, HSFCM can be used to assess the quality of QD preparations, such as batch-to-batch under the same conditions or via different routes. Figure 2b2 shows the fluorescence burst height distribution histogram of Qdot660 purchased from another company, which has a very close fluorescence emission spectrum to that of Qdot655 (Figure S1). Compared to Qdot655, a weaker fluorescence intensity (616 ± 371 counts/bin) and a broader distribution (CV of 60.2%) was identified.

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Figure 2. Quantitative fluorescence measurement of single QDs. (a) Representative fluorescence burst trace of Qdot655. (b1, b2) Fluorescence burst-height distribution histograms of Qdot655 (b1) and Qdot660 (b2) derived from data collected over 1 min. Laser excitation power: 200 mW; emission bandpass: 660/52 nm; bin width: 100 µs; QD concentration: 5 pM (3 × 109/mL) in ultrapure water.

Effect of Laser Excitation Intensity on the Fluorescence of QDs. Single QDs have demonstrated a number of interesting optical phenomena, including fluorescence intermittency (blinking), photoluminescence activation (PLA), and photodarkening (bleaching).24-26 For the present HSFCM setup, the irradiation time, i.e., the time it takes for a single QD to traverse the interrogation volume, was approximately 0.8 ms. Thus, the on-off blinking normally observed under continuous illumination on a time scale from tens of milliseconds to hundreds of seconds can be ignored,27 as can photobleaching. The PLA of QDs was studied by varying the laser excitation power in the regime of 20 to 200 mW, which corresponds to excitation intensity from 25.5 to 255 kW/cm2. Yellow-green fluorescent (505/515 nm) polystyrene (PS) beads with a diameter of 200 nm were used as a reference. The fluorescence burst trace and histogram data given in Figures 3a and 3b 9



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indicate that the brightness of QDs excited at 200 mW was more intense and uniform than those excited at 20 mW. In contrast to the symmetric intensity distribution at 200 mW, a tailing towards low intensity was identified at 20 mW, which indicated that a portion of the QDs could not be efficiently excited at a low energy density. With a continuous increase in the laser excitation power from 20 mW to 200 mW, the mean fluorescence intensity of QDs steadily increased and the CV decreased, indicating an increasingly uniform and brighter fluorescence signal of the QDs (Figure 3c). Though a similar trend in fluorescence brightness with laser power was observed for the control sample of 200 nm fluorescent PS beads (the same edge and bandpass filters were used, and an ND filter of 1.5 was placed before the detector to bring about comparable fluorescence intensity to that of the Qdot655 at 200 mW), the CV remained relatively constant (Figure 3, d-f). Because the photoactivation rate of QDs increases with laser power, we attributed the increased uniformity of QD fluorescence at a higher laser irradiation intensity to the rapid photoactivation with more “dark” or “dim” QDs turned into “bright” QDs.26,28 To ensure a more uniform and intense QD signal, 200 mW laser excitation was used for the following experiments despite a linear increase in the background signal with increasing laser power (Figure S2).

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Figure 3. Laser excitation power dependence of fluorescent QDs and 200-nm PS beads. (a) Representative burst traces of Qdot655 at 20 mW and 200 mW laser excitation powers. (b) Fluorescence burst height distribution histograms for Qdot655 at 20 mW (black line) and 200 mW (red line). (c) Line chart of mean value and CV% of fluorescence burst height distribution histogram obtained at different laser excitation powers. (d, e, and f) Data for 200 nm fluorescent PS beads. QD concentration: 5 pM (3 × 109/mL) in ultrapure water.

Influence of Metal Ions on the Fluorescence of QDs. For the numerous biomedical applications of QDs, the environments QDs may encounter are quite different, e.g. buffers of different metals and concentrations, cytoplasma or other intracellular conditions, and all kinds of matrix of real samples. For example, many reports have demonstrated that the fluorescence of QDs can be quenched by a variety of metal ions, and this feature has been utilized to construct QD-based metal ion sensors.29,30 However, fluorescence quenching can be detrimental to the utility of QDs as reliable bright 11



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fluorescence probes. Although the fluorescence quenching of QDs induced by metal ions can be easily measured by bulk methods such as spectrofluorimetry, rapid single-QD analysis may provide a more straightforward and effective approach because the heterogeneity in QD response can be revealed by the statistical distribution. Meanwhile, the amount of sample required for accurate measurement can be can be significantly reduced. We tested three metal ions commonly found in biological systems. For example, the total copper concentration in blood plasma is approximately 1.2 mg/L, which corresponds to 19 µM.31 The representative burst traces and fluorescence histogram data shown in Figure 4 indicate that, compared to phosphate buffer at pH 7.4 (PB), the presence of 10 µM Pb2+ or Fe3+ caused partial reduction of Qdot655 fluorescence. Copper ions are strong quenchers, and the presence of 10 µM Cu2+ resulted in complete quenching of the QD fluorescence (Figure 4, a and e). The major quenching pathway for Cu2+ could be ascribed to the formation of small CuSe particles on the CdSe surface owning to the potential penetration of copper ions into the capping layer and diffusion into the CdSe core.30,32,33 However, the presence of 1 mM EDTA in phosphate buffer can efficiently chelate Cu2+ and preserve the fluorescence of QDs. These data suggest that care must be taken when QDs are to be employed as fluorescent probes under complicated environments, and HSFCM can aid in the development of effective staining protocols.

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Figure 4. (a) Representative fluorescence burst traces for Qdot655 in phosphate buffer (PB) in the presence of 10 µM Pb2+, Fe3+, or Cu2+, and 1 mM of EDTA and 10 µM of Cu2+. (b-e) Fluorescence burst height distribution histograms of Qdot655 in these solutions.

Aggregation Analysis of QDs upon Biomolecule Conjugation. By virtue of their unique optical attributes, QDs are well suited for ultrasensitive multiplexed biological detection and molecular profiling when conjugated with biomolecules.5,10,34 Coupling biomolecules to QDs can be achieved primarily via direct covalent crosslinking, noncovalent interactions of biotin-avidin and protein A/G-antibody, and nickel-based histidine tagging.35,36 A growing set of high-quality “ready-to-use” QDs with functional groups, especially when covalently attached with streptavidin (Sav), can be easily purchased and conjugated to nearly all of the biotinylated biomolecules of interest. However, the multivalence of both the QD and biological molecules (e.g., antibody and 13



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streptavidin) often results in aggregation upon conjugation, which can jeopardize the utility of QDs as fluorescent labels. Because the fluorescence intensity of the aggregates is an integer multiple of the monomer, the degree of QD aggregation could be directly revealed in the fluorescence distribution histogram if the instrument is sensitive enough to discriminate monomer, dimer, trimer, and larger aggregates at the single-particle level. Figure 5a displays the schematics of coupling biotinylated Ab to Qdot-Sav at low or high molar ratios. Multi-peak fitting was applied to distinguish monomers and aggregates in the fluorescence distribution histogram obtained from HSFCM and estimate their proportions. Figure 5b illustrates that the fluorescence of Qdot655-Sav (Invitrogen) was quite uniform with negligible levels of aggregation. When the molar concentration of biotinylated-Ab was ten times the amount of the Qdot655-Sav (Figure 5c), 55% of the QDs aggregated upon 3 h of incubation (the total number of quantum dots was estimated by weighted sum of the monomers and oligomers), and the proportion of monomers was 65%. With a further increase in the biotinylated-Ab concentration, the population of monomers increased to 75% and 89% at biotinylated-Ab/Qdot655-Sav molar ratios of 20:1 and 50:1, respectively. This can be explained by that when the concentration of Biotin-Ab is much higher than that of Qdot-Sav, Qdot-Sav will have greater probability to be bound with free Biotin-Ab rather than with the Biotin-Ab already conjugated to the other Qdot-Sav. These data demonstrate that HSFCM provides a powerful method for quantitatively measuring the aggregation of bioconjugated QD probes, which has been a

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challenging task despite its great importance in ensuring the successful bioapplications of QDs.

Figure 5. Quanification of QD aggregation upon bioconjugation. (a) Schematics of Qdot-Sav conjugation with biotinylated-Ab at low or high molar ratios. (b-e) Fluorescence burst height distribution histograms obtained for conjugates at the biotinylated-Ab/Qdot655-Sav molar ratios of 0:1 (b, control), 10:1 (c), 20:1 (d), and 50:1 (e). Superimposed are the multi peak fits (red line) of the histograms. The bar graph insets indicate the proportions of monomer, dimer, trimer, and tetramer.

Quantification of QD Concentration via Single Particle Enumeration. For every synthesis, functionalization, and application of QDs, precise control of their particle concentration is critically important.37 With prior knowledge of absorption coefficients and quantum yields, QD concentrations can be estimated by UV-Vis spectrophotometry and spectrofluorimetry.38 However, such information is rarely available for a new preparation. The widely acceptable approach is the so-called inductively coupled plasma 15



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mass spectrometry (ICP-MS) & transmission electron microscopy (TEM) method, which is labor-intensive and time-consuming. Conversely, for single-particle techniques, once the fluorescence signals emitted from single QDs can be unambiguously distinguished from the background noise, single-particle counting can be utilized to quantify the on-fraction particle concentration of QDs with a standard of known particle concentration.15,39 Owing to the approximately 100% detection efficiency of HSFCM, absolute quantification of fluorescence polystyrene beads and gold nanoparticles has been demonstrated with excellent accuracy.20,40 Here, the on-fraction QD concentration quantification was exploited with Qdot655 by using 200-nm fluorescent PS beads of known particle concentration as an external standard. The calibration curve between the rate of events and the particle concentration of PS beads exhibited a linear correlation with an R2 value of 0.9994 (Figure 6a). The Qdot655 stock solution was then diluted by different folds and analyzed under the same instrument conditions. The on-fraction concentration of the Qdot655 stock solution was measured to be 7.03 µM. The molar concentration of the same Qdot655 sample was also measured by the ICP-MS & TEM method. Figure 6b shows the representative TEM micrograph of the Qdot655, and the shape of the CdSe/ZnS particle resembles a circular truncated cone. To simplify the size measurement and calculation, the circular truncated cone was treated as a cylinder with a radius equivalent to the radius of the middle cross-section of the circular truncated cone, and the height equivalent to that of the circular truncated cone. The average volume of Qdot655 was measured to be 336 ± 83 nm3 upon examining ~200 individual QDs from the TEM micrographs. The measured stock solution of Qdot655 was 10.6 µM by the ICP-MS & TEM method. Note that the QD particle concentration

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provided by the manufacturer (measured via absorption method) is 8.4 µM. The deviation between HSFCM method and the other two methods can be mainly attributed to the fact that a dark fraction of QDs that is undetectable by fluorescence measurement always exits.16,41 Figure 6c reveals that in the QD concentration range of ~100 fM to 10 pM, a linear relationship (R2=0.9999) was obtained between the results by HSFCM analysis and the theoretical values based on ICP-MS & TEM measurement. The slope of 0.67 indicates that the on-state proportion of Qdot655 was 67%. This number becomes 84% when divide the QD concentration measured by HSFCM by that provided by the manufacture. Of course, the difficulty in volume calculation owing to the irregular shape and varied size of QDs also contributed to the deviation between the ICP-MS & TEM determination and the true value. Nevertheless, our data agrees with the findings that the bright fraction of QDs measured by single-particle techniques is proportional to the ensemble quantum yield (80% here, provided by the Manufacture), of which QDs of different quantum yields have been tested.12,16

Figure 6. Particle concentration measurement of QDs. (a) Standard calibration curve between the events detected in a minute and the particle concentration of fluorescent PS beads. (b) Representative TEM image of Qdot655. (c) Correlation between QD concentrations 17



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measured by HSFCM enumeration and the theoretical values based on ICP-MS & TEM measurement.

CONCLUSIONS In conclusion, by employing hydrodynamic focusing in a sheath flow cuvette, a large N.A. objective lens, and the single-photon-counting APD, we have developed a rapid and highly sensitive method for the precise quantification of the fluorescence intensity of single quantum dots. The as-developed method can be used to assess the brightness and monodispersity of QD preparations for good quality control, to examine the influence of matrix conditions on the photoluminescence of QDs to avoid fluorescence quenching, and to measure the particle concentration of QDs. Theoretically, by changing the sheath fluid to organic solvent, HSFCM could be applied to the quantitative measurement of oil-soluble QDs, which are largely used in light-emitting diodes and solar cells. HSFCM is believed to provide a powerful characterization tool for QD synthesis and application development.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge via the ACS Publications website at http://pubs.acs.org.

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Figures S1 and S2 (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel: 86-592-2184519. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We thank the National Key Basic Research Program of China (2013CB933703), and the National Natural Science Foundation of China (21225523, 21475112, 21027010, 21627811, and 21521004) for the financial support.

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Table of Content

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Rapid and Quantitative Measurement of Single Quantum Dots in a

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