Functional-Group-Dependent Formation of ... - ACS Publications

Article pubs.acs.org/JPCC

Functional-Group-Dependent Formation of Bioactive FluorescentPlasmonic Nanohybrids Jan-Philip Merkl,*,†,‡,§ Christian Schmidtke,† Fadi Aldeek,‡,¶ Malak Safi,‡,# Artur Feld,† Hauke Kloust,† Hedi Mattoussi,*,‡ Holger Lange,†,§ and Horst Weller*,†,§,∥,⊥ †

Institute of Physical Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany Department of Chemistry and Biochemistry, Florida State University, 95 Chieftan Way, Tallahassee, Florida 32306, United States § The Hamburg Center for Ultrafast Imaging, University of Hamburg, Luruper Chaussee 149, 22761 Hamburg, Germany ∥ Center for Applied Nanotechnology (CAN) GmbH, Grindelallee 117, 20146 Hamburg, Germany ⊥ Department of Chemistry, Faculty of Science, King Abdulaziz University, P. O. Box 80203, Jeddah 21589, Saudi Arabia ‡

S Supporting Information *

ABSTRACT: We detail the assembly, driven by metal-affinity coordination, of fluorescentplasmonic hybrid constructs that are also biologically active. The hybrid constructs are prepared by first assembling polymer-encapsulated luminescent quantum dots that present amine-, carboxy-, and lipoic acid-terminated groups (QD-FG) and plasmonic gold nanoparticles capped with rather low density of lipoic acid-appended zwitterion ligands (AuNP-LA-ZW). The dual QD-AuNP constructs were then coupled to polyhistidineappended maltose binding proteins, yielding the final trifunctional assemblies. The coordination of amine-, carboxy-, and lipoic acid-terminated QDs with AuNP-LA-ZW was characterized using steady-state and time-resolved fluorescence quenching measurements. We measured rather different coordination affinities between the functional groups on the QDs and the AuNP surfaces. This assembly mode still allowed the partially exposed AuNPs in the inorganic/polymer hybrid to bind to polyhistidine-appended proteins. This protein assembly was confirmed using amylose affinity chromatography, which also confirmed the structural integrity of the hybrid and biological activity of the bound protein. Owing to the high colloidal stability of the surfacemodified QDs and AuNP-LA-ZW, combined with flexible functionalization, we anticipate that this strategy could facilitate the integration of hybrid inorganic/polymer constructs with specific photophysical properties into biological systems.

■

INTRODUCTION Hybrid nanostructures prepared by assembling single particles into “superstructured” composites with distinct properties are an attractive new class of materials. They combine the unique properties exhibited by each individual constituent/nanoparticle within a nanoscale superstructure and can potentially be used for designing multimodal probes/platforms for imaging, sensing, and theranostic applications.1−5 The preservation or enhancement of the individual properties of each component within the hybrid structure is an important goal but also represents a formidable challenge.6−8 Various hybrid nanostructures have been developed including core/shell iron oxide/Au nanoparticles, self-assembled AuNP-iron oxide NP composites, vesicles containing iron oxide NPs and luminescent quantum dots (QDs), and iron oxide NP-QD silica microspheres.9−11 AuNP-QD hybrid nanostructures can exhibit strong exciton−plasmon interactions,6,12,13 and several reports have shown that AuNPs act as highly effective energy quenchers of dye and QD photoemission when the two are brought in close proximity.14−20 In this study we apply photoluminescence (PL) quenching measurements as a convenient, straightforward tool to test the self-assembly between surface-functionalized, polymer-encapsu© 2016 American Chemical Society

lated QDs and AuNPs in solution phase. More precisely, we introduce a simple route to conjugate QDs, surface-functionalized with amine, carboxy-, and lipoic acid-terminated polymer capsules (QD-FG; QD-NH2, QD-COOH, QD-LA) with AuNPs that are capped with lipoic acid-modified zwitterionic (LA-ZW) ligands. The AuNPs, prepared using one-phase growth route as described in ref 26, are capped with low density of LA-ZW ligands (NPs with sparse surface coverage). The exposed nanoscale surface patches on the AuNPs are targeted for metal coordination with the functional groups on the QD (QD-FG), yielding hybrid superstructures. The zwitterionic character of the ligand coating imparts high colloidal stability to the AuNPs and eliminates aggregation during the hybrid assembly.21 Additionally, further targeting of these same exposed surface patches on the AuNPs (within the hybrid material) has allowed us to apply metal-affinity-driven conjugation between polyhistidine-appended maltose binding protein (MBP-His7) and the hybrid superstructures. This strategy relies on the direct coordination between C- or NReceived: May 23, 2016 Revised: August 11, 2016 Published: September 21, 2016 25732

DOI: 10.1021/acs.jpcc.6b05204 J. Phys. Chem. C 2016, 120, 25732−25741

Article

The Journal of Physical Chemistry C

mol) was synthesized using CDI coupling of PI to diethylenetriamine (DETA).36,37 The lipoic acid appended with a zwitterion group, LA-ZW, was synthesized using methanesulfonyl chloride activation of LA followed by reaction with N,N′-dimethyl-1,3-propanediamine and 1,3-propane sultone as detailed in ref 23. The LA-ZW ligand was used for the Au nanoparticle growth (see below). Growth of the Quantum Dots and Phase Transfer Strategy. CdSe/CdS/ZnS core−shell−shell nanocrystals were grown following the procedure originally described by Talapin et al.,38 but we substituted the bis(trimethylsilyl) sulfide and diethyl zinc with hydrogen sulfide and zinc(II) acetate as precursors for shell growth. Tri-n-octylphosphine (TOP), tri-noctylphosphine oxide (TOPO), and hexadecylamine (HDA) were used as high temperature boiling solvents. Briefly, the CdSe core was grown first using hot injection of cadmium(II) acetate and TOP-Se precursors in TOP. Growth of the CdS and the ZnS shells on the CdSe cores was subsequently carried out using cadmium(II) acetate and zinc(II) acetate dissolved in TOP and hydrogen sulfide as a sulfur source. Additional details of the original growth method along with subsequent modifications can be found in refs 39 and 38. Encapsulation of the above-mentioned QDs within the PI-bPEO diblock copolymer was carried out stepwise as described in refs 37, 36, and 39. Briefly, the hydrophobic QDs were precipitated twice with ethanol and then dispersed in a solution of PI-DETA and incubated in n-hexane for at least 3 h (molar ratio PI-DETA:QD 300:1). This facilitates a removal of the native cap (TOP, TOPO and HDA). The nanocrystals were then precipitated with ethanol and redispersed in THF. A molar excess of 300 PI-b-PEO-FG and 2,2′-azobis(2-methylpropionitrile) (1/3 equiv per PI-double bound) were added. Afterward, the solution was injected into 18 mL of water using an automated flow system.36,39 The mixture was maintained at room temperature for ∼15 min and then heated to 80 °C for 4 h to initiate the cross-linking of PI moieties.40 The reaction mixture was purified using a hydrophilic syringe filter (0.45 μm) and washed three times with water using a membrane filtration device (Amicon Ultra-15; 100 kDa). The set of encapsulated QD exhibits a narrow PL spectrum centered at 575 nm and a fluorescence quantum yield of ∼42% (Figure 1). The concentration of the QD was calculated using the method reported by Mulvaney and co-workers with ε(540 nm) = 1.8 × 106 M−1 cm−1.41 Growth of LA-Zwitterion-Capped AuNPs. LA-ZWcapped gold nanoparticles were grown in aqueous solution using chemical reduction of tetrachlorauric acid with sodium borohydride in the presence of LA-ZW, as described in ref 42. The nanoparticles were purified using a membrane filtration device and further characterized using transmission electron microcopy; an average diameter of 8.5 ± 1.5 nm was measured. This protocol yields partially capped nanoparticles (NPs with incomplete ligand coverage).26 Further passivation of the NPs is achieved after the reaction is complete (i.e., growth is arrested) by adding small amounts of LA-ZW to the dispersions; here ligands were added to reach a final Au:LAZW molar ratio of 50:1 (partially passivated) or 1:1 (fully passivated) nanoparticles.26 The concentration of the AuNPs was determined using the absorption data combined with the extinction coefficient of the nanoparticles, ε(at 516 nm) = 5.1 × 107 M−1 cm−1.43 Fluorescence Quenching Measurements and Analysis. The fluorescence quenching data tracking the self-

terminal polyhistidine sequence (His7-tag) appended on proteins or peptides and metal-rich nanoparticle surfaces. Though the strategy was originally developed for conjugating various proteins and peptides onto fluorescent QDs with Znrich surfaces, it has been recently extended to AuNPs.22−26 We have shown that only AuNPs with partially exposed surfaces (i.e., partially passivated NPs), or capped with weakly bound ligand, can self-assemble with polyhistidine-tagged peptides and proteins via metal−histidine coordination.26 The protein binding was tested using an amylose affinity chromatography assay, which also confirmed the structural integrity of the entire hybrid system. Although the superstructure formation does not rely on covalent chemical coupling,15,27−35 relatively stable constructs have been formed, where the effects of varying the nature of the coordinating group and reagent concentrations have been tested, and distinct binding behaviors have been measured depending on the functional group used.

■

EXPERIMENTAL SECTION Chemicals. Air and/or water sensitive chemicals were handled using standard Schlenk technique (argon or nitrogen atmosphere). Tetrachlorauric(III) acid trihydrate (HAuCl4* 3 H2O, 99.9%), α-lipoic acid (LA), sodium borohydride, organic solvents, triethylamine, D-(+)-maltose, s-buthylithium, 1,1′carbonyldiimidazole (CDI), 2,2′-azobis(2-methylpropionitrile) (AIBN), trioctylphosphine (TOP), ethylene oxide, selenium, and isoprene were purchased from Sigma-Aldrich (St. Louis, MO). Trioctylphosphine oxide (TOPO) and hexadecylamine (HDA) were purchased from Merck. Chloroform D1 and THF D8 (99.5% D) were purchased from Carl Roth (Karlsruhe, Germany). N,N′-Dimethyl-1,3-propanediamine and 1,3-propane sultone were acquired from Alfa-Aesar (Ward Hill, MA). Cadmium(II) acetate was purchased from ChemPur (Karlsruhe, Germany). Instrumentation. The photoluminescence spectra were collected using a Fluorolog-3 spectrometer equipped with a TBX photomultiplier and air-cooled CCD camera detectors (HORIBA Jobin-Yvon Inc., Edison, NJ). The time-resolved (TR) fluorescence decay profiles were collected and analyzed using a time correlation single photon counting system (TCSPCS), integrated into the Fluorolog-3 spectrometer above. A pulsed excitation signal, provided by a NanoLED440LH (λEX = 440 nm, 100 ps, fwhm) with a repetition rate of 1 MHz was used for sample excitation, and detection was collected on the same TBX photomultiplier tube. UV−vis absorption spectra were collected using a Shimadzu UV−vis absorption spectrophotometer (UV 2450 model). Ligand Synthesis (Polymers and Molecular Zwitterion Ligands). The synthesis of the polymers used for the QD coating/encapsulation was carried out following the procedures detailed in recent studies reported by Weller and co-workers. It involved the preparation of PI-b-PEO−OH (MN = 10 500 g/ mol) synthesized via living anionic polymerization,36 functionalization of PI-b-PEO−OH to yield PI-b-PEO-NH2 and PI-bPEO−COOH; the PI-b-PEO-NH2 was synthesized using 1,1′carbonyldiimidazole (CDI) reaction of PI-b-PEO followed by nucleophilic substitution with ethylenediamine, while PI-bPEO−COOH was synthesized by reacting PI-b-PEO−OH with succinic anhydride.37 In addition, we prepared lipoic acidmodified polymer, PI-b-PEO-LA, via activation of lipoic acid with thionyl chloride followed by coupling to PI-b-PEO−OH.21 Poly(isoprene)-diethylenetriamine (PI-DETA; MN = 1200 g/ 25733


Article


drop, ΔPLQD−FG, between the initial value (before adding the AuNPs), PL0, and the equilibrium value, PLf, at several reagent concentration yields a relationship between the PL drop and 1/ K (Figure S4 b).22,26

assembly of the PI-b-PEO-coated QDs with the AuNPs were collected using the following conditions. Aliquots of QD dispersions (10 nM) were mixed with varying amounts of AuNP dispersions to yield different molar ratios of AuNP-toQD. We used two sets of AuNPs: fully passivated (Au:LA-ZW = 1:1) and partially passivated (Au:LA-ZW = 50:1) NPs. After 30 min incubation, steady-state (excitation wavelength :350 nm) and time-resolved fluorescence spectra were collected. The experiments were repeated at least three times, and average values are shown. The inner filter effect of AuNPs, due to light absorption and scattering at the excitation wavelength, was corrected using eq 144,45 PLcorr = PLmeasured100.5A(λexcitation)

PL0 − PLf = PL0

kT 6πηri,hyd

k 0 = 4πNAR 0(DQD − FG + DAuNP)

+ [QD]

(4)

−1

Smaller values for K are measured for QD-LA, in comparison to QD-NH2 and QD-COOH, following the trend discussed above (Table 1). The measurements were repeated at least twice for several experimental conditions. Table 1. Binding Parameters Extracted from the Experimental Data for QD-FG and Partially-Passivated AuNPsa

(1)

where PLcorr is the corrected fluorescence intensity, PLmeasured is the measured PL intensity in the presence of the AuNP, and A(λexcitation) is the absorbance of the AuNP at the excitation wavelength in the absence of QD-FGs. Determination of the Diffusion-Controlled Biomolecular Rate Constant for QD-FG and AuNPs. To determine the diffusion-controlled biomolecular rate constant k0(NP) for QD-FG and AuNPs, the Stokes−Einstein eq 2 and the Smoluchowski eq 3 were used.45,46 The diffusion-controlled biomolecular rate constant determines the conditions where the dynamic PL quenching may be attributed to collisions in solution. When the bimolecular quenching constant exceeds k0, the binding between fluorophore and quencher is inferred.45 Here, Di is the diffusion coefficient, k the Boltzmann constant, T the temperature, η the viscosity of the medium, ri,hyr the hydrodynamic radius of the NP, NA the Avogadro number, and R0 the distance between successive collisions (or collision radius), which can be approximated by the Förster radius. Using the experimental conditions, rQD‑FG,hyr = 15 nm, rAuNP,hyr = 10 nm, T = 296.15 K, and R0 = 28.6 nm, determined by light scattering and the Förster expression,45,47 the rate constant derived for the QD-FG and AuNP is 1.4 × 1010 M−1 s−1; note that the high Förster radius compensates the slower diffusion of the NPs. Di =

[QD] 1 K

Stern−Volmer analysis sample QDCOOH QD-NH2 QD-LA

kinetic investigation

kq (1015 M−1 s−1)

1/KSV (nM)

1/K (nM) (from the drop in PL signal, SI)

1.2 ± 0.1

19.2 ± 1.5

41.2 ± 6.9

3.9 ± 0.4 45 ± 6

13.0 ± 1.0 1.6 ± 0.3

20.9 ± 4.9 0.62 ± 0.18

a

The bimolecular quenching constant kq and the dissociation constant 1/KSV were derived from the Stern−Volmer analysis of the timeresolved fluorescence data. The dissociation constant 1/K was derived from investigation of the self-assembly kinetics, as detailed in the Supporting Information and refs 22 and 26.

Hybrid Assembly and Affinity Chromatography Test. To further investigate the additional conjugation of the QDAuNP assembly to proteins, QD-FG-AuNP nanocomposites at AuNP:QD molar ratio of 1:1 were used. QD-FG and AuNPs solutions (20 mM PBS buffer, pH 7.2) were mixed for 30 min at room temperature in the dark before incubation (also for 30 min) with HIS7-MBP (14 equiv. per AuNP, final concentration of QDs = 1 μM, and final volume = 200 μL). No aggregation build up was observed under these conditions. To provide additional proof of the binding between the fluorescent/plasmonic assembly and MPB-His protein we relied on a visual assay, testing the specific binding of MBP to amylose, followed by competitive release by soluble maltose. Briefly, 1.5−2 mL of amylose stock gel was loaded onto a 10 mL capacity column and washed several times with 10 mL of PBS buffer (20 mM, pH 7.2). After loading the sample (QD ∼ 1 μM, total V = 200 μL), the column was washed several times with buffer (up to 25 × 2 mL was tested); the assembly stayed bound to the amylose column. Then, 10 mL of a Dmaltose solution (20 mM) was added, readily releasing the hybrid bioconjugates, which was monitored visually, either by tracking the QD emission under UV illumination using a handheld UV lamp, or the pink color of the AuNPs under white light exposure.26

(2) (3)

Investigation of the Kinetics of the Self-Assembly. To investigate the kinetics of the self-assembly between the QDFG and AuNP, we use a kinetic model, as done for the selfassembly between QDs and proteins promoted by metal− histidine coordination.22,26 The molar ratio between AuNP and QD-FG was maintained at 1:1 while the reagent concentration was varied from 1 to 20 nM. First the QD-FG were dispersed in 20 mM PBS buffer (pH 7.2) and loaded into a quartz cuvette. The PL was monitored over a 100 s period to ensure stability of the PL signal. The collection was paused, AuNPs were added, the content was homogenized, and data acquisition was immediately resumed (typically 1−3 s). The time-dependent PL signal decay for several reagent concentrations was recorded. Since the self-assembly events of a QD with one, two, or three AuNPs can be considered independent of each other and the PL loss involves one-to-one interactions at equilibrium, we can express the dissociation constant as 1/K = [QD]0[AuNP]/[bQD], where [bQD] is the concentration of the bound QDs.22,26 Further manipulation of the PL intensity

■

RESULTS AND DISCUSSION Quantum Dot and Gold Nanoparticle Design. The strategy employed here for promoting the transfer of QDs to buffer media relies on a ligand exchange of the native cap with polyisoprene-diethylenetriamine (PI-DETA) followed by encapsulation within a functional amphiphilic poly(isoprene)block-poly(ethylene oxide) (PI-b-PEO-FG) diblock copolymer.36,37,48 This route yields dispersions of QDs encapsulated within a cross-linked polymer that preserves high quantum 25734


Article


Figure 1. Normalized absorption (red) and emission (blue) spectra of QD-FG together with the normalized absorption of AuNPs (black). (b) Schematic representation of encapsulated QD-FG, with the three functional groups used. The gray region surrounding the QD represents the hydrophobic PI layer while the PEO are drawn in blue. (c) AuNP growth route: reduction of tetrachlorauric acid with sodium borohydride in the presence of LA-ZW ligand (additional passivation step not shown).

inset in Figure 1 d and Figure S1), indicating that direct surface access of the FG is necessary for binding between QD-FG and AuNPs (compare schematics in Figure 2).19,20,22 Figure 2a shows a plot of the QD PL quenching, QE, extracted from the steady-state florescence data for the three sets of QD-FG mixed with partially passivated AuNPs. The full set of PL spectra is provided in the Supporting Information, Figure S2. The QE was extracted from the spectra using the

yield (i.e., compared to that measured for the native hydrophobic materials). The cross-linking is provided by radical initiation reaction between neighboring poly(isoprene) moieties, schematized as a gray region around the QD in Figure 1.40 In addition, the ability to introduce terminal functional groups (e.g., amine and carboxylic acid) imparts reactivity to the final QD.37 Three sets of surface-functionalized QDs were prepared, namely, amine-functionalized (QD-NH2), carboxyfunctionalized (QD-COOH), and QDs bearing terminally exposed lipoic acid (QD-LA). The AuNPs used here were grown in a single aqueous phase using borohydride reduction of AuCl4− precursor in the presence of LA-ZW.49 This growth route yields AuNP dispersions that exhibit great colloidal stability, when compared to citrate-stabilized particles. After the initial growth step, the AuNPs are partially capped, while extra ligand addition yields fully capped AuNPs.26,50 To investigate the self-assembly of the functionalized QDs with AuNPs and its dependence on the AuNP surface coverage, we used two sets of AuNPs (d = 8.5 ± 1.5 nm): one set was prepared by limiting the reaction to the initial growth (i.e., no extra passivation).26 The second set was made of fully passivated NPs, where an extra passivation step was carried out (see the Experimental Section for further details).26 Both sets of AuNPs were used for self-assembly with the various QD-FGs. Photoluminescence Quenching Measurements. To investigate the hybrid system, real time determination of the geometry within the hybrids is desired. Such a study with the necessary accuracy is very demanding with techniques such as cryoelectron microscopy, while PL intensities and dynamics in such systems are very sensitive to interparticle distances.51 Thus, we employed fluorescence quenching measurements to probe the self-assembly between surface-functionalized QDFGs (acting as energy donors) and the LA-ZW-coated AuNPs (acting as fluorescence quenchers). We measured a net QD PL loss only when partially capped AuNPs were used. Control measurements showed that no PL quenching was measured when the QD-FG were mixed with fully passivated AuNPs (see

equation QE = 1 −

PL(Q) , PL0

where PL(Q) and PL0 designate the

PL intensities of the QDs in the presence and absence of the AuNP quenchers, respectively. Data show that the QD PL loss strongly depends on the nature of the functional groups presented on the QD-surrounding micelle: While modest quenching was measured for amine- and carboxy-functionalized QDs, the quenching efficiencies were substantially larger when lipoic acid (LA) groups were presented on the QDs. The time-resolved PL data collected from these mixtures support and complement the steady-state data. The shortening of the PL lifetimes was found to depend on the functional group, as expected with faster decays measured for samples prepared using LA-presenting QDs (details about the timeresolved PL decay fit are provided in the Supporting Information). The contribution of the fastest component is relatively small for assemblies prepared starting with QD-NH2 and QD-COOH (typically