Functional-Group-Dependent Formation of Bioactive Fluorescent

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Functional Group Dependent Formation of Bioactive Fluorescent-Plasmonic Nanohybrids Jan-Philip Merkl, Christian Schmidtke, Fadi Aldeek, Malak Safi, Artur Feld, Hauke Kloust, Hedi Mattoussi, Holger Lange, and Horst Weller J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b05204 • Publication Date (Web): 21 Sep 2016 Downloaded from http://pubs.acs.org on September 25, 2016

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The Journal of Physical Chemistry

Functional Group Dependent Formation of Bioactive Fluorescent-Plasmonic Nanohybrids Jan-Philip Merkl,a,b,c* Christian Schmidtke,a Fadi Aldeek,b,¶ Malak Safi,b,# Artur Feld,a Hauke Kloust,a Hedi Mattoussi,b* Holger Langea,c and Horst Wellera,c,d,e* a

Institute of Physical Chemistry; University of Hamburg, Grindelallee 117, 20146 Hamburg (Germany) b

Department of Chemistry and Biochemistry, Florida State University, 95 Chieftan Way, Tallahassee, Florida 32306 (United States of America)

c

The Hamburg Center for Ultrafast Imaging, University of Hamburg, Luruper Chaussee 149, 22761 Hamburg (Germany)

d

Center for Applied Nanotechnology (CAN) GmbH, Grindelallee 117, 20146 Hamburg (Germany)

e

Department of Chemistry, Faculty of Science, King Abdulaziz University, P.O BOX 80203 Jeddah 21589 (Saudi Arabia) ¶ current address: Florida Department of Agriculture and Consumer Services Division of Food Safety, Chemical Residue Laboratory, 3125 Conner Blvd. Lab 3, Tallahassee, Florida 32399 (United States of America)

#

current address: Laboratoire Physique des Solides, UMR 8502, Université de Paris Sud bât 510, 91405 Orsay Cedex (France)

*corresponding authors: [email protected] (+49-40-42838-5021), [email protected] (+1 -850-645-8615), [email protected] (+49-40-428383449)

1 ACS Paragon Plus Environment


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ABSTRACT We detail the assembly, driven by metal-affinity coordination, of fluorescent-plasmonic 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 acidterminated 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 polyhistidine-appended 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, yielding easily bio-functionalized “superstructures.” The protein assembly with the fluorescent-plasmonic constructs 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 surface-modified 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.


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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 spuerstructure, 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 convenient, straightforward tool to test the self-assembly between the surface-functionalized, polymerencapsulated 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 acidmodified 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 metalcoordination 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 N-terminal polyhistidine sequence (His7-tag) appended proteins or 3 ACS Paragon Plus Environment


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peptides and metal-rich nanoparticle surfaces. Though the strategy was originally developed for conjugating various proteins and peptides onto fluorescent QDs with Zn-rich 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, 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, sButhylithium,

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). Trioctylphosphineoxide (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


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NanoLED-440LH (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 absorpXon 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= 10500 g/mol) synthesized via living anionic polymerization,36 functionalization of PI-bPEO-OH to yield PI-b-PEO-NH2 and PI-b-PEO-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-b-PEO-COOH was synthesized by reacting PI-b-PEO-OH with succinic anhydride.37 In addition, we prepared lipoic acid-modified 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/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 reference.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 and coworkers,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 zinc(II) acetate dissolved in TOP, TOP:Se and hydrogen sulfide. Additional details of the original growth method along with subsequent modifications can be found in references 39 and 38.



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Encapsulation of the above mentioned QDs within the PI-b-PEO diblock copolymer was carried out stepwise as described in reference 37,

36

and 39. Briefly, the hydrophobic QDs were

precipitated twice with ethanol, then dispersed in a solution of PI-DETA 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). 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 equivalent per PI-double bound) were added. Afterwards, 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, then heated to 80 °C for 4 h to initiate the crosslinking 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 exhibit 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 coworkers with ε(540nm) = 1.8*106 M-1 cm-1.41

Growth of LA-zwitterion-Capped AuNPs. LA-ZW-capped 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 reference.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 LAZW to the dispersions; here ligands were added to reach a final Au:LA-ZW 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-assembly of the PI-b-PEO-coated QDs with the AuNPs were collected using the


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following conditions. Aliquots of QD dispersions (10 nM) were mixed with varying amounts of AuNP dispersions to yield different molar ratios of AuNP-to-QD. 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, due to light absorption and scattering at the excitation wavelength of AuNPs, was corrected using equation 1:44,45

PLcorr = PLmeasured 100.5 A(λexcitation )

(1)

where PLcorr is the corrected fluorescence intensity and 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 equation (2) and the Smoluchowski equation (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, then 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 is 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, 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.



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kT 6 π η ri , hyd

(2)

k 0 = 4 π N A R 0 ( D QD − FG + D AuNP )

(3)

Di =

Investigation of the kinetics of the self-assembly. To investigate the kinetics of the selfassembly between the QD-FG and AuNP, we a kinetic model, as done for the self-assembly 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 homogenized and data acquisition was immediately resumed (typically 1-3 seconds). 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 oneto-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 drop, ∆PLQD − 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 PL0 − PL f PL0

=

[QD] 1 + [QD] K

(4)

Smaller values for K-1 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 the several experimental conditions.


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Table 1: Binding parameters extracted from the experimental data for QD-FG and partially-passivated AuNPs. The bimolecular quenching constant kq and the dissociation constant 1/KSV were derived from the Stern-Volmer analysis of the time-resolved fluorescence data. The dissociation constant 1/K was derived from investigation of the self-assembly kinetics, as detailed in the supporting information and references 22 and 26.

Stern-Volmer analysis Sample

kinetic investigation

kq

1/KSV

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

[1015 M-1 s-1]

[nM]

QD-COOH

1.2±0.1

19.2± 1.5

41.2± 6.9

QD-NH2

3.9±0.4

13.0± 1.0

20.9± 4.9

QD-LA

45±6

1.6± 0.3

0.62± 0.18

Hybrid assembly and affinity chromatography test. To further investigate the additional conjugation of the QD-AuNP 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 HIS7MBP (14 eq. 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 25x 2 mL was tested); the assembly stayed bound to the amylose column. Then, 10 mL of a D-maltose solution (20 mM) was added, readily releasing the hybrid bio-conjugates, which was tracked by visualizing the QD emission using either a hand held UV lamp, or by simply tracking the pink color of the AuNPs under white light exposure.26 9 ACS Paragon Plus Environment


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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-bPEO-FG) diblock copolymer.36,37,48 This route yields dispersions of QDs encapsulated within a cross-linked polymer that preserves high quantum 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 grey 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), carboxy-functionalized (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 LAZW.49 This growth route yields AuNP dispersions that exhibit great colloidal stability, when compared to citrate-stabilized particles. Afer the initial growth step the AuNPs are partially capped, while extra ligand addition yields fully capped AuNPs.26,50 To investigate the selfassembly 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 fullypassivated 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.


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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 grey 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).

Photoluminescence Quenching Measurements To investigate the hybrid system, time-resolved determination of the geometry within the hybrids is desired. Such a study with the necessary accuracy is very demanding with techniques such as cryo-electron 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 QD-FGs (acting as energy donors) and the LA-ZW caoted 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 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



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Figure 2: Top row: Schematic representation of the interaction between QD-FG and AuNPs, some of them lead to hybrid formation. (Left) Incubation of QD-FG with fully-passivated AuNPs; no binding occurs. (Right) Self-assembly of QD-FG with partially-passivated AuNPs: nanocomposites are formed as indicated by PL quenching. a) PL quenching efficiency vs. AuNP concentration (fitted to eq. 1); the inset shows the quenching efficiency extracted from the average fluorescence lifetime, (panels b–d). The concentration of QD-FG is c(QD-FG)= 10 nM (b-d) Normalized time-resolved PL decay at the designated AuNP concentrations for QD-COOH (b), QD-NH2 (c) and QD-LA (d), c(QD-FG)= 10 nM. The inset in d shows the fluorescence lifetime of QD-NH2 incubated with fully-passivated AuNPs, as a representative sample.

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 12 ACS Paragon Plus Environment

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spectra using the equation QE = 1 −

PL( Q ) , where PL(Q) and PL0 designate the PL intensities of PL0

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 QDsurrounding micelle: While modest quenching was measured for amine- and carboxyfunctionalized 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 time-resolved 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 < 10% of the overall decay), while it dominates assemblies prepared with QD-LA.51 These findings indicate distinct different binding behaviors between the three sets of QDs (see Figure 2 and Table S1). To gain additional insights into the nature of the interactions between the QDs and AuNPs in these assemblies, we analyzed the PL-quenching within the Stern-Volmer formalism (Eq. 2-4).45 This formalism can be used to identify interactions promoted by binding vs. those resulting from collisions in a solution sample. The ratio between PL0 and PL(Q) is plotted as function of quencher concentration, [Q], using: PL0 = 1 + K SV [Q ] PL(Q)

(5)

Expressing equation 5 in terms of changes in average PL-lifetimes in the absence of the quencher, 0, and in the presence of the quencher, (Q), yields an estimate for the dynamic Stern-Volmer constant (associated with the average PL-lifetime), Kdyn, as shown in equation 6 and the supporting Information: < τ >0 = 1 + K dym [Q ] < τ > (Q )

(6)



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Comparison between Kdyn and KSV indicates that these constants are equal for three set of QDFG used (Supporting Information, Figure S3), which implies that quenching is fully evaluated by tracking the shortening of the PL lifetime. This further proves the absence of any static quenching, which accounts for the formation of a non-fluorescent complex between QD-FG and AuNP. Its’ absence can be attributed to the protecting polymer coating on the QD.39,52 If shortening in the PL lifetime is due to dynamic encounters, or binding between the QDs and AuNPs can be analyzed by calculating the bimolecular quenching constant, kq, defined as:45

K dyn = k q < τ >0 If kq exceeds the rate constant of diffusion limited reactions, binding between the Donor and the quencher is deduced.45 For small molecules this limit is 1 × 1010 M-1 s-1 and can – for bigger particles – be calculated with the Stokes-Einstein and the Smoluchowski equation, as described in the experimental section. Using these equations, the limit for diffusion controlled reaction of QD-FG and AuNP pair is 1.4 × 1010 M-1 s-1. The values of the bimolecular quenching constant kq for the three sets of QD-FG/AuNP are shown in Table 1; all exceed the diffusion limit by at least five orders of magnitude.45,53 This indicates that all QD-FG bind the AuNPs and confirms the differences in the binding behaviors between QD-COOH, QDNH2 and QD-LA.

Evaluation of the Self-Assembly Kinetics In order to develop a better understanding of the binding between the QDs and AuNPs in these mixtures, we investigated the kinetics of the self-assembly between the various QD-FGs and the partially passivated AuNPs. We followed previously discussed rationales by maintaining the molar ratio between QD and AuNP fixed at 1:1, but varied the reagent concentrations from 1 to 20 nM.22,26 Then, the kinetics of the self-assembly was monitored by tracking the timedependent changes in the QD PL immediately after reagent mixing. We found, that most of the drop in the PL intensity occurs during the “dead time”, the time elapsed between mixing and the start of the PL signal collection (see Figure S4 a). This indicates very fast binding, much faster than in recent studies. Indeed, it’s essentially too fast to resolve with this experimental


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procedure and is therefore explained in more detail in the experimental section and, respectively, the supporting information.22,26

Hybrid Assembly and bio-functionalization Having shown that partially-passivated AuNPs bind surface-functionalized QDs, we opted to explore whether the AuNPs immobilized on the QD can still interact with polyhistidine proteins via metal-affinity interactions.26 Indeed, we have found recently that proteins expressing a terminal polyhistidine tail can easily self-assemble onto these partially-passivated AuNPs.26 A key finding was that direct access of the imidazole groups in the polyhistidine tail to the metalrich surface of the NPs for coordination is required.26 Due to the small QD-FG:AuNP ratio, we reasoned that within the QD-AuNP self-assemblies the AuNPs would still have exposed surfaces that can interact with polyhistidine-tagged proteins. For this, a dispersion of QD-FG:AuNP assemblies was prepared at a molar ratio of 1:1 and using 1 µM reagent concentration. The samples were equilibrated for 30 min and no aggregation or precipitation was observed, which is in contrast to reports on similar, citrate based systems.21 After that a solution of maltose binding protein appended with an N-terminal poly-histidine sequence (His7-MBP) was added and left to incubate for 20-30 min (see scheme in Fig. 3); we used a His7-MBP-to-AuNP molar ratio of 14 in the mixture. Conjugation between the QD-AuNP hybrids and His7-MBP was tested using affinity chromatography, where we probed the competition between the binding of MBP to amylose, or to its substrate maltose; this allowed us both: the verification of the functionalization as well as to test the structural integrity of the hybrid material. Following conjugation the QD-AuNP/His7-MBP mixture was loaded onto a column filled with amylose gel. The QD-AuNPs/His7-MBP hybrid formed a tightly bound layer on top of the amylose gel column, as shown in Figure 3. This layer stayed bound to the gel after multiple washes with PBS buffer. Due to its bimodal character the hybrid complexes can easily be identified via both the QD PL signal as well as the pinkish color of the AuNPs. No sign of free QDs (no fluorescence) or AuNPs (no reddish color) were observed in the eluted buffer, which was also confirmed with optical methods (e.g. investigation of the eluted buffer with fluorescence and absorption measurements). Addition of 2-3 mL of 20 mM maltose solution readily eluted the colored layer



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from the column. In comparison, when QD-AuNPs (without His7-MBP) were loaded no binding was measured, as the content (colored) was readily eluted with the addition of 1-2 mL PBS buffer (see Supporting Information, Figure S6). These findings unequivocally prove that His7MBP interacted with the exposed AuNP surface within the self-assembled QD-AuNP and that the entire hybrid is sable and well connected. In addition following binding, the MBP maintained its biological activity within the QD-AuNP/His7-MBP hybrid.23,26

Figure 3: a) Scheme of the metal-affinity driven self-assembly of HIS7-MBP on the AuNPs in the hybrid nanocomposite. Photographs of the amylose column loaded with binding assay of QD-NH2-AuNP hybrids alone (b), or loaded with AuNPs premixed with His7-MBP (c). The nanocomposites can be identified by both the pinkish color of the AuNPs (top row) as well as the fluorescent band of the QDs visible under UV excitation (bottom). Binding to amylose and release by maltose of mixture shown in panel b indicate that His7-MBP directly accessed the exposed surface of the AuNPs within the preformed hybrid assembly.


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Discussion Overall the ideas descried in this study to assemble a hybrid nanostructure that combines fluorescent, plasmonic and biological properties relying on metal-coordination driven selfassembly. The obtained binding constants measured for the metal-coordination driven selfassembly are similar to those reported for simpler structures (NP + protein-His and NP + peptide-His conjugates, with equilibrium constants in the nM regime).22,26 However, the formation of the presented system is faster, which is likely due to the high amount of available functional groups on the micelle-encapsulated QDs, in comparison to the number of imidazole groups on the polyhistidine tags appended on proteins or peptides.22,26,37 The use of zwitterionic-stabilized AuNPs is highly beneficial as it improve the colloidal stability of the prepared hybrid systems; the latter have hydrodynamic radii close to those anticipated from simply summing the value measured for the starting individual components used to form the larger hybrid nanostructures (see Figure S7). Most importantly, no aggregation build up was observed, even when high concentrations were used, which contrasts with structures prepared using citrate-stabilized AuNPs.21 The obtained hybrids stayed colloidally stable for several weeks. We attribute this result to the strongly stabilizing interparticle interactions imparted by the LA-ZW ligands. This result is a highly beneficial feature that bodes well for using these constructs in biology, where absence of aggregation is a desired feature, e.g. intracellular uptake.54



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Figure 4: Schematic depiction of the interaction and binding leading to the PL-quenching data collected for a 1:1 mixture of QD-FG and AuNPs. The AuNPs bind to all the QD-FG. However, the internal structure and the relative local fluctuations within the ligand shell affects the separation distances between the AuNPs and the QD-FG (i.e., QD-LA, QD-NH2 and QD-COOH). The grey circle represents the distance where the quenching efficiency if 50% (Förster Radius). On average more LAs are coordinated to the AuNP surface, bringing the QD and AuNP in closer proximity. Weaker coordination with COOH and NH2 allow more flexibility and larger separation distances.

Although the data discussion within the Stern-Volmer kinetic model suggests that there is chemical equilibrium between bound QD and free QD-FG, the absence of QDs and AuNPs in the eluting buffer during the amylose gel assay as well as the PL decay traces do not support a ‘simple’ chemical equilibrium between bound and free QDs in solution. The above experiments and corresponding data indicate absence of free (unbound) QDs. We thus describe the constructs as made of QDs self-assembled with AuNPs in a “more loosely” manner, likely associated with the long-range interactions between QDs and AuNPs.55 We attribute the differences in the PL quenching measured for the three sets of QD-FG to differences in the metal-coordination of the functional groups to the AuNP surfaces.56,57 This produces different time-average distances between QD-FG and AuNP, taking the distance dependence of the AuNP and QD interaction into account.14,17–20,30–33,55 The range of distances extracted for each set of QD-FG in the nanohybrids may be attributed to differences in the average number of FG bound


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to each AuNP, as schematically depicted in Figure 4. Due to the higher affinity exhibited by lipoic acid towards Au surface, higher number of FGs are bound to each AuNP, which results in lower average distance between QD and AuNP in the construct, and vice versa. We attribute this to the flexibility and softness of the outer PEO block in the polymer micelle. Within our description the structure of the QD-FG-AuNP hybrid is controlled by a process similar to geminate recombination in molecular species, where the products of a dissociation reaction preferentially recombine instead of dissociating and moving away from each other. The rather large number of functional groups per QD suppresses complete dissociation, similar to chelating ligands in molecular complexes,58and the average amount of FG bound to the AuNP surface determines the average separation distance and the resulting photo physical properties.

Conclusion In this study we have probed the self-assembly between polymer encapsulated QDs and plasmonic AuNPs, resulting in nanoscale luminescent-plasmonic hybrid composites. Due to sparse surface coverage of the AuNPs, polyhistidine-appended protein could be easily coupled to the hybrid system via metal-histidine coordination. The binding-process was analyzed with a combination of steady-state and time-resolved fluorescence measurements, along with an investigation of the kinetics of the assembly. We probed the effects of varying the nature of the functional group on the QDs micelle, using amine-, carboxy-, and lipoic acid-functionalized polymers to encapsulate the QDs. Pronounced differences in the PL quenching indicate differences metal-coordination interactions between the functional groups and the gold surface. Due to zwitterionic ligands on the AuNPs, the hybrid system exhibits excellent colloidal stability. We anticipate that the information extracted from this system will help in future design of hybrid constructs for biological application, where controlled binding conditions along with colloidal stability are essential part for use of multifunctional hybrid systems.



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SUPPORTING INFORMATION DESCRIPTION Fluorescence spectra of QD-FG mixed with partially passivated AuNPs, fluorescence quenching of QD-FG mixed with fully-passivated AuNPs and AuNP-His7-MBP conjugates, Stern-Volmer analysis, kinetic evaluation of QD-COOH and AuNPs, absorption, light scattering measurements and amylose affinity chromatography control experiments. This material is available free of charge via the Internet at http://pubs.acs.org. ACKNOWLEDGMENTS This work was supported by the EU within the FP 7 program (Vibrant, EU 228933), the State Excellence Initiative “Nanotechnology in Medicine” from the Free and Hanseatic City of Hamburg, the DFG by the Cluster of Excellence CUI and by the US National Science Foundation (grants NSF-CHE #1508501 and #1058957). J.-P.M., H.M. and H.W. acknowledge the support of the Chemical Industry Fund, VCI: German Chemical Industry Association and the GermanAmerican Fulbright Program. We also thank Goutam Palui, Xin Ji (at FSU) and Ning Fang (Iowa State University) for helpful discussions. REFERENCES (1) Sailor, M. J.; Park, J.-H. Hybrid Nanoparticles for Detection and Treatment of Cancer. Adv. Mater. 2012, 24, 3779–3802. (2)

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SUGGESTION COVER ART


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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 functionalgroups used. The grey 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). 366x203mm (96 x 96 DPI)

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Top row: Schematic representation of the interaction between QD-FG and AuNPs, some of them lead to hybrid formation. (Left) Incubation of QD-FG with fully-passivated AuNPs; no binding occurs. (Right) Selfassembly of QD-FG with partially-passivated AuNPs: nanocomposites are formed as indicated by PL quenching. a) PL quenching efficiency vs. AuNP concentration (fitted to eq. 1); the inset shows the quenching efficiency extracted from the average fluorescence lifetime, (panels b–d). The concentration of QD-FG is c(QD-FG)= 10 nM (b-d) Normalized time-resolved PL decay at the designated AuNP concentrations for QD-COOH (b), QD-NH2 (c) and QD-LA (d), c(QD-FG)= 10 nM. The inset in d shows the fluorescence lifetime of QD-NH2 incubated with fully-passivated AuNPs, as a representative sample. 381x331mm (96 x 96 DPI)


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a) Scheme of the metal-affinity driven self-assembly of HIS7-MBP on the AuNPs in the hybrid nanocomposite. Photographs of the amylose column loaded with binding assay of QD-NH2-AuNP hybrids alone (b), or loaded with AuNPs premixed with His7-MBP (c). The nanocomposites can be identified by both the pinkish color of the AuNPs (top row) as well as the fluorescent band of the QDs visible under UV excitation (bottom). Binding to amylose and release by maltose of mixture shown in panel b indicate that His7-MBP directly accessed the exposed surface of the AuNPs within the preformed hybrid assembly. 241x190mm (150 x 150 DPI)



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Schematic depiction of the interaction and binding leading to the PL-quenching data collected for a 1:1 mixture of QD-FG and AuNPs. The AuNPs bind to all the QD-FG. However, the internal structure and the relative local fluctuations within the ligand shell affects the separation distances between the AuNPs and the QD-FG (i.e., QD-LA, QD-NH2 and QD-COOH). The grey circle represents the distance where the quenching efficiency if 50% (Förster Radius). On average more LAs are coordinated to the AuNP surface, bringing the QD and AuNP in closer proximity. Weaker coordination with COOH and NH2 allow more flexibility and larger separation distances. 292x276mm (96 x 96 DPI)


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TOC graph 213x219mm (96 x 96 DPI)



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Suggestion Cover-Page 297x297mm (96 x 96 DPI)


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Functional-Group-Dependent Formation of Bioactive Fluorescent

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