Letter pubs.acs.org/Langmuir
Fast Self-Assembly Kinetics of Quantum Dots and a Dendrimeric Peptide Ligand Jianhao Wang,†,‡,# Pengju Jiang,†,# Zuoyan Han,‡ Lin Qiu,† Cheli Wang,† Bo Zheng,‡ and Jiang Xia*,‡ †
School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, China, 213164 Department of Chemistry, The Chinese University of Hong Kong, Shatin, Hong Kong, China
‡
S Supporting Information *
ABSTRACT: Engineered peptide ligands with exceptionally high affinity for metal can self-assemble with nanoparticles in biological fluids. A high-affinity dendrimeric peptide ligand for CdSe-ZnS quantum dots (QDs) exhibited very fast association kinetics with QDs and reached equilibrium within 2 s. Here, we have combined a droplet-based microfluidic device with fluorescence detection based on Förster resonance energy transfer (FRET) to provide subsecond resolution in dissecting this fast self-assembly kinetics in solution. This work represents the first application of microfluidic devices to ligand−particle assembly for the measurement of fast assembly kinetics in solution.
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INTRODUCTION As nanoparticles such as semiconductor nanocrystals (quantum dots, QDs) have been finding increasingly wider-ranging uses in tissue imaging and diagnostics recently, public concerns about their behavior in biological systems and even their presence as a source of biological hazard continue to rise.1−4 When nanoparticles enter a biological fluid, a ligand exchange process usually occurs between the surface of particles and plasma proteins, with eventually the formation of a protein “corona” on the surface of particles that may transmit undesirable biological effects.2,5−10 A self-assembly process driven by metal− hexahistidine peptide interaction has been developed as an efficient and biocompatible method to functionalize the surface of CdSe-ZnS QDs.11−14 Its great success notwithstanding, the binding affinity between oligohistidine and QDs might not be sufficient to shield the surface from ligand exchange. In complex biological fluids or in cytosol, small peptides or histidine-rich endogenous proteins can compete for the binding sites on the surface of QDs.15,16 Engineering peptide and protein ligands with exceptionally high affinity for QDs is thus desirable. Inspired by the discovery that multidentate ligands could achieve much stronger binding interaction with QDs than monodentate ligands,17,18 we designed a 4-arm polyhistidine peptide dendrimer (PHPD) ligand and discovered that PHPD ligand binds to QDs 50 times more tightly than the monomeric hexahistidine peptide.16 More surprisingly, self-assembly between QDs and PHPD reaches equilibrium in less than 2 s after mixing, whereas it took ∼100 s for hexahistidine peptide to assemble with QDs (Figure 1).11,16 Such fast kinetics dwarfs common methods for kinetics measurements of protein− nanoparticle interactions, such as ensemble fluorescence measurement in cuvette,11,19 gel filtration chromatography,9 capillary electrophoresis,15,16,20 and others.21 SPR9 or fluorescence measurement on surface-coated QDs11 might be suitable to resolve the fast kinetics, but the binding kinetics on © 2012 American Chemical Society
Figure 1. PHPD assembles with QDs with fast kinetics. (A) Schematic diagram of the structure of PHPD and FRET resulted from the assembly of PHPD with QDs. (B) Binding kinetics of PHPD (a) and H6 (b) to QDs measured by the increase of emission fluorescent signal at 661 nm in cuvette. (C) Binding kinetic within the first 10 s. a, PHPD; b, H6. [QD] = 20 nM, [peptide] = 80 nM. λex = 420 nm, λem = 661 nm.
surface-immobilized particles might differ from self-assembly in solution. In this report, we designed a droplet-based microfluidic system in conjunction with a fluorescent microscope and an optic spectrometer to measure the ligand−QD assembly in solution with subsecond resolution. By converting reaction/ Received: March 23, 2012 Revised: May 11, 2012 Published: May 14, 2012 7962
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kinetics in solution phase by adopting a microfluidic device in conjunction with fluorescence detection (Figure 2A).
binding time into spatial information, droplet-based microfluidics22 has been proven to be capable of measuring fast kinetics such as rapid enzymatic kinetics,23 RNA−Mg2+ binding,24 and protein−protein interaction.25 Our report showcases for the first time the successful implementation of microfluidic devices to measure the rapid self-assembly kinetics between peptide ligands and QDs in solution, utilizing which we discover that the multidentate ligand PHPD binds to QDs ∼80 times faster than oligohistidine.
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MATERIALS AND METHODS
PDMS microfluidic chip was fabricated by soft lithography method as described previously,23,24 and then was stationed on a fluorescence microscope (IX71, Olympus, Japan). Droplets form when streams of aqueous reagents were injected into the flow of the carrier fluid (silicone fluids, viscosity ∼50 cP, Brookfield, USA) in the microchannel of the chip under the control of syringe pumps (Harvard Apparatus, USA). Fluorescence images of the droplets in the microchannel were taken by EMCCD camera (SPOT Boost BT 1900, Diagnostic Instruments, USA) and analyzed by ImageJ. Alternatively, the emitted fluorescent signal of each droplet was collected by a fiber optic spectrometer (QE65000, Ocean Optics, USA) connected to the microscope.
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RESULTS AND DISCUSSIONS Shown in Figure 1A, polyhistidine peptide dendrimer (PHPD) contains four arms of hexahistidine at the particle-binding end and a fluorescent dye Cy5 at the distal end.16 PHPD was synthesized by solid-phase peptide synthesis and confirmed by mass spectrometry (Figure S1 in Supporting Information). Cy5 forms a FRET pair with CdSe-ZnS core−shell QDs (∼5 nm in diameter) when PHPD associates with the surface of QDs.16 Assembly of PHPD with QDs driven by histidine−metal binding interaction on the surface of particles can thereby be monitored by the increase of fluorescent emission signal at 661 nm (with excitation at 420 nm), concomitant with the decrease of emission signal at 612 nm (Figure S2 in Supporting Information).16 The association kinetics was first monitored in a cuvette using a conventional fluorimeter, by following the change of the FRET signal at 661 nm (with λex = 420 nm) immediately after manually mixing peptides and QDs in buffer solution. Surprisingly, while a monodentate control peptide H6 (sequence Cy5-DDDLVPRGSGP9G2H6) showed an expected association kinetics with an equilibrium time (teq) of about 100 s similar as previously reported,11 PHPD−QD binding resulted in an almost instant increase of FRET signal (Figure 1B). Because it requires at least 2 s to thoroughly mix the two solutions and initiate measurement in this assay, the details of the kinetic changes within the first 2 s cannot be observed (indicated by dashed lines in Figure 1C). Nevertheless, these results suggest that PHPD assembles with QDs rapidly and reaches equilibrium within 2 s. Such fast self-assembly kinetics originates from the multivalency effect of PHPD in ligand− surface binding interaction. Intrigued by the very fast self-assembly kinetics of PHPD, we seek to quantitatively dissect the kinetic details by a technique with subsecond resolution. SPR or fluorescence measurement based on surface coated QDs9,11 might be suitable for monitoring such fast kinetics, but the assembly process on immobilized particles might differ from that in solution. The ligand−particle assembly process in solution more closely resembles the encounter of nanoparticles with proteins in biological fluids. Herein, we resolve the fast self-assembly
Figure 2. Droplet-based microfluidic system for monitoring ligand− particle self-assembly in solution. (A) Schematic diagram of instrumental setup. (B) Design of the Y-junction and winding channel. Circled areas represent positions of microscopic views at which fluorescent signals were collected. (C) Photograph of the microfluidic chip with one Hong Kong dollar. (D) Fluorescent snapshot image of moving droplets carrying the binding solutions of ligand and QDs. The rapid movement of droplets resulted in a seemly continuous flow. See Movie S1 in the Supporting Information for the move of discrete droplets. (E) Conversion of the 9 positions in (B) to reacting/mixing times based on flow rate and the droplet travel distance. Flow rates of the carrier fluid, QD solution, and peptide solution were 3, 1.5, and 1.5 μL•min−1, respectively.
A microfluidic device with size similar to a Hong Kong dollar coin was fabricated.23,24 A simple Y junction design introduces the peptide solution and QD solution into the microchannel to produce a flow of aqueous droplets carried by oil flow (Figure 2B). Each droplet is a miniature of reacting mixtures containing the peptide and QDs at given concentrations defined by the relative flow rates. The miniature size of the device requires only small volumes of ligand and QD solution, as compared to 1 or 2 mL in cuvette (Figure 2C). Notwithstanding that the peptide and QD solutions interface briefly in the stem of the Y junction, premixing of the two solutions before droplet formation at position 1 (time 0) is negligible, as the nanoparticle and macromolecular ligand have very limited diffusion within the traveling time in the Y junction due to their large sizes. See Experimental Section and Figure S3 in Supporting Information for details. The winding channel ensures a rapid and complete mixing process that can be monitored under a fluorescent microscope in real time (Figure 2D).22,26 Rapid mixing of the ligand and QD solution within 0.1 s has been achieved under a microscope.23,24 Droplets flowing through the channel can be monitored by EMCCD under a 40× objective (Movie S1 in Supporting Information). Concomitantly, the fluorescence spectrum of each droplet, when passing through the microscopic view, can be collected by 7963
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Figure 3. Ligand−particle self-assembly in droplets. (A) FRET signals collected when droplets passed through the focused area of the microscopic view at position 9. (B) FRET signals of droplets collected at positions 1 to 9. Positions of droplets have been converted to corresponding binding times based on the flow rate and droplet travel distances. [QDs] = 20 nM, [peptide] = 80 nM. λex = 420 nm, λem = 661 nm.
Figure 4. Comparison of self-assembly kinetics of PHPD (A) and H6 (B) with QDs by droplet-based microfluidic method. Each point represents an average of 11 droplets. [QDs] = 20 nM, [ligand] = 80 nM. λex = 420 nm, λem = 661 nm.
a spectrometer through an optical fiber (Figure 2A). Therefore, our device features a convenient direct observation of the mixing process, as well as quantitative recording of fluorescence spectra of single droplets. Another essential feature of the microfluidic device is that the position of the observatory window set along the trajectory of the droplets (from which the droplet travel distance can be derived) correlates with the reaction/mixing time in the droplet solution. Namely, by measuring the distance between the origin of the inlet and the selected positions of microscopic views, under a constant flow, the reacting/mixing time in the droplets that pass through the view can be calculated (Figure 2E). Choosing microscopic views that are close to the origin of the inlet and adjusting the flow rates thus allows subsecond details of the kinetics to be monitored. PHPD ligand and QD solutions were then flowed into a microfluidic device with carrier to test the detection of FRET signal. Periodic spikes were observed (Figure 3A), with each spike corresponding to the process of a single droplet passing through the microscopic view focused on a specific segment of the microchannel as indicated in Figure 2B. As the reaction solution contained in each droplet passing through the microscopic view has exactly the same composition and reacting/mixing time (due to the same droplet travel distance),
the pattern of the peaks is highly reproducible. For example, Figure 3A shows 11 peaks with almost identical shapes and peak heights of FRET signal (λex = 420 nm, λem = 661 nm) corresponding to 11 droplets that have passed through the microscopic view one by one within the observing time frame. The peak heights represent the FRET signals at the moments when the whole and only one droplet are within the area of microscopic view (Figure 3A). The unsymmetric peak shape is likely due to the shape change of the droplet that is forced by the uneven curvature of the fabricated microchannel or alternatively the unsymmetry of the microscopic view. By focusing on different segments of the microchannel, the ligand−particle self-assembly kinetics can be observed (Figure 3B). With the reproducibility and sensitivity of the microfluidic device verified, the assembly kinetics of peptide ligands and QDs indicated by the increase of FRET signals at different binding times were measured (λex = 420 nm, λem = 661 nm). The assembly of PHPD and QDs reaches equilibrium after about 1.5 s as expected (Figure 4A). The same strategy was implemented to measure the self-assembly kinetics between H6 and QDs (Figure 4B), which shows a similar equilibrium curve as in Figure 1B. This verifies that our droplet-based method is in line with the conventional fluorimeter-based method for 7964
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association kinetics of 100 s scale.11 We defined t1/2(obs) as the time it takes to reach half of the maximal signal for comparison between monovalent H6 and multivalent PHPD ligands. The estimated t1/2(obs) for PHPD is only 0.6 s, 1/80 of that of H6 ligand, 48 s. This suggests that the multidentate PHPD ligand binds to QDs 80 times faster than the hexahistidine peptide. Such a difference accounts for kinetically controlled specificity when both ligands are present in a complex biological fluid.16 The decrease of the emission signal of QDs at 612 nm due to FRET showed a similar trend between the two ligands (Figures S4 and S5 in Supporting Information).
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CONCLUSION Droplet-based microfluidics tailored toward ligand−particle interaction was set up to provide subsecond resolution to the self-assembly kinetics between peptide ligands and QDs in solution. The instrument is feasible for ligand−particle selfassembly kinetics ranging from a few to hundreds of seconds. It provides a facile, sensitive, and disposable device for measurement of ligand−particle interaction. By utilizing this instrument, the detailed kinetics of PHPD, a very fast ligand for QDs was dissected. Our experiments confirm that this multivalent ligand assembles with QDs 80 times faster than the well-studied hexahistidine peptide. Such a marked difference results in kinetically controlled selectivity among different peptide ligands, namely, in a complex biological fluid where many proteins including histidine-rich proteins are present, the rapid kinetics of PHPD will ensures a preferential binding of PHPD ligand to QDs.16 One can thereby take advantage of this multivalency-assisted kinetically controlled assembly to design novel biocompatible ligands for QDs for selective self-assembly in a biological environment.
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ASSOCIATED CONTENT
S Supporting Information *
General materials and instruments, peptide synthesis and purification, preparation of oil-soluble CdSe-ZnS core−shell and GSH stabilized QDs, in-cuvette fluorescence titration, droplet-based microfluidics method, supporting figures S1, S2, S3, S4, and S5 and Movie S1. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions #
These authors contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge financial support from the Direct Grant for Research 2010/11 of the Chinese University of Hong Kong (CUHK 2060385) and the National Natural Science Foundation of China (grant no. 31100530). The authors also acknowledge financial support from “a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions”.
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REFERENCES
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(24) Han, Z.; Chang, Y. Y.; Au, S. W.; Zheng, B. Measuring rapid kinetics by a potentiometric method in droplet-based microfluidic devices. Chem. Commun. 2012, 48, 1601−1603. (25) Srisa-Art, M.; Kang, D. K.; Hong, J.; Park, H.; Leatherbarrow, R. J.; Edel, J. B.; Chang, S. I.; deMello, A. J. Analysis of protein-protein interactions by using droplet-based microfluidics. ChemBioChem 2009, 10, 1605−1611. (26) Song, H.; Tice, J. D.; Ismagilov, R. F. A microfluidic system for controlling reaction networks in time. Angew. Chem., Int. Ed. 2003, 42, 768−772.
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