Highly Stable, Water-Soluble, Intrinsic Fluorescent Hybrid Scaffolds for

Jan 11, 2011 - A synthetic method is proposed for the preparation of micrometer-sized hybrid materials in the form of highly stable, water-soluble, po...
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Highly Stable, Water-Soluble, Intrinsic Fluorescent Hybrid Scaffolds for Imaging and Biosensing Ranjith Krishna Pai* and Mircea Cotlet* Center for Functional Nanomaterials, Brookhaven National Laboratory, 735 Brookhaven Avenue, Upton, New York 11973, United States

bS Supporting Information ABSTRACT: A synthetic method is proposed for the prep aration of micrometer-sized hybrid materials in the form of highly stable, water-soluble, porous, and intrinsic fluorescent vaterites. This is an easy, cost-effective, and polymer-free synthetic method, that is, free of any supplementary complex synthetic or natural macromolecular stabilizers. This method uses a double decomposition reaction to introduce fluorescence as an intrinsic property into the vaterite scaffold, through either organic dyes or dihydrolipoic acid coated core/shell CdSe/ZnS quantum dots. The resulting hybrid scaffold has excellent brightness, photostability, thermal stability, and pH stability. Combined with a large loading surface offered by the vaterite scaffold and the ease of chemical functionalization provided by the water-soluble quantum dots, the obtained hybrid scaffolds show promise in biological applications. Fluorescence imaging and fluorescence-resonance-energy-transfer-based sensing of proteins based on these hybrid materials is illustrated with these hybrid materials at the single-particle level.

1. INTRODUCTION The controlled synthesis of organic-inorganic hybrid materials of specific size, shape, and morphology has seen increased interest because of the applicability of such hybrids in a variety of fields, including medicine, biosensing, and advanced materials.1-3 These biomimetic methods are inspired from nature where biological organisms create organic-inorganic hybrid materials of myriad shapes and sizes and often high strength, through synthesis methods that use aqueous solutions that are free of toxic products and under ambient conditions. In the process of biomineralization, matrix proteins or other macromolecules or low-molecular weight organic molecules organize as nanostructures to provide frameworks for specific shapes and orientations of inorganic crystals, for example, calcium carbonate, hydroxyapatite, or silica, materials with perfect morphologies. Biomimetic synthesis of calcium carbonate has received considerable attention because of its application in a variety of industrial and biorelated fields.3-7 Calcium carbonate has been confirmed to increase enzyme activity and to penetrate the cell membrane, a property exploited toward its use as a carrier for drug delivery and genes.6,8,9 The application of biomimetically synthesized calcium carbonate particles strongly depends on physicochemical parameters such as morphology, size, shape, structure, and specific surface area.3,10 These parameters are dictated by the type of organic templates or additives used to r 2011 American Chemical Society

control the crystallization of calcium carbonate and by experimental parameters affecting the rate of the nucleation process, including pH, temperature, and rate of mixing or intensity of agitation of the reaction mixture.11-16 Calcium carbonate crystallizes into three different polymorphs: calcite (rhobohedral), which is the most thermodynamically stable form, aragonite (orthorhombic), and vaterite, which is the least thermodynamically stable form. Vaterite appears as 1-10-μm spherulitic crystals composed of nanoparticles 20-30 nm in size. Vaterite can be manufactured in laboratory either by mixing concentrated solutions of calcium- and carbonate-containing salts or by bubbling carbon dioxide through a calcium salt solution. Vaterite has attracted particular attention because it has some features such as high specific surface area, low density (gravity), high solubility compared to calcite and aragonite, and high dispersion. Although stable under dry conditions, vaterite transforms easily and irreversibly into a thermodynamically stable polymorph of calcite, thus limiting its biological applications. Water solubility and biocompatibility can be achieved by coating stabilized vaterite with polyelectrolytes,9,17-19 organosilica and silica,20 or functionalized nanoparticles 8 and by adding biologically relevant functions by the use of peptides, ligands, and proteins. 6,21 Many of these methods are expensive, require Received: October 6, 2010 Revised: December 13, 2010 Published: January 11, 2011 1674

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intense labor, and sometimes limit vaterite dispersibility15 and solubility.22 Herein, we introduce a sythetic method for the fabrication of highly stable, highly porous water-soluble fluorescent vaterite spherruloids of ∼2-μm size that is free of complex synthetic/ natural macromolecular stabilizers. This polymer-free synthetic method is based on a double decomposition reaction23 used to stabilize the vaterite structure by means of a small organic molecule, namely, rhodamine 101. The resulting vaterites are inexpensive and easy to prepare; highly dispersible over a large pH range; and highly fluorescent, thus providing an intrinsic fluorescence scaffold for biological purposes. We further extend this method to create water-soluble vaterites by using dihydrolipoic acid coated core/shell CdSe/ZnS semiconductor quantum dots (DHLA-Qdots) for stabilization and demonstrate model biosensors based on DHLA-Qdot-stabilized vaterites that exploit fluorescence resonance energy transfer (FRET) between DHLAQdots and biological targets such as intrinsic fluorescent proteins and streptavidin conjugated to Qdots.

2. EXPERIMENTAL SECTION 2.1. Vaterite Microparticle Fabrication. Fluorescent vaterite microspheres were formed through a double decomposition reaction by mixing two aqueous solutions in an ice bath. A sodium bicarbonate solution (0.125 mol/dm3) was added, at a rate of 20 mL/min, to a solution of CaCl2 (0.125 mol/dm3) and Tris buffer (0.200 mol/dm3) and containing either rhodamine 101 (Sigma-Alrich, 0.104 mol/dm3) or DHLA-coated core/shell CdSe/ZnS quantum dots of 525-nm emission (1.33  10-9 mol/dm3). DHLA-coated Qdots were prepared by ligand exchange from commercially available carboxyl-coated poly(ethylene glycol) (PEG) core/shell CdSe/ZnS. The presence of DHLA on the Qdots was confirmed by FTIR spectroscopy. All reactions were carried out under nitrogen. For biosensing experiments, biotinilation of DHLA-Qdot525 was achieved using commerially available kits (Pierce Biotechnology) by reacting the carboxylic acid of DHLA with an amine-based biotin through a zero-length cross-linking agent [1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), Pierce]. 2.2. Fluorescence Spectroscopy and Microscopy. Steadystate fluorescence spectroscopy was performed using a CaryVarian fluorimeter. Fluorescence spectra were recorded at a right angle using 488-nm excitation. Time-resolved fluorescence spectroscopy was performed at 488-nm laser excitation by using the frequency-doubled output of a pulse-picked (8 MHz) femtosecond Ti:sapphire laser (80 MHz repetition rate) and a FluoTime 200 lifetime spectrometer (Picoquant, 45-ps instrumental response time). Fluorescence decays were analyzed using the FluoFit Pro software from Picoquant. Confocal fluorescence lifetime imaging microscopy (FLIM) was performed with 488-nm pulsed light using a home-built scanning-stage inverted microscope equipped with a 1.2 NA 60 water-immersion objective lens. Fluorescence was collected with the same objective lens, filtered with a dichroic mirror (Chroma, 505 DRLP), spatially filtered with a 75-μm pinhole, and spectrally separated with a second dichroic mirror for two-color FLIM (Chroma 545DRLP) before being focused onto two single-photon-counting avalanche photodiodes (MPD Picoquant). Additional band-pass filters (donor, HQ525/30, acceptor, HQ580/40) were mounted in front of each detector to

Figure 1. Structural characterization of water-soluble vaterites: Scanning electron microscopy (SEM) images of (a-b) Rh-vaterites formed by double decomposition reaction, (c) crushed Rh-vaterite, and (d) Qdot525-vaterite. (e) SEM image of calcite microparticles grown in a control experiment without rhodamine dye and otherwise identical conditions to those used to fabricate microparticles shown in panels a-d. (f) Transmission electron microscopy (TEM) image of a crushed Qdot525-vaterite featuring loading of Qdots at the surface only. The arrow in the TEM image points toward Qdot nanocystals loaded at the vaterite surface.

further suppress the excitation light. FLIM images were acquired with a piezo-scanner (Physics Instrumente) interfaced with a TCSPC PicoHarp 300 multichannel analyzer. FLIM images and pixel-by-pixel fluorescence decays were analyzed using the Symphotime software (Picoquant). For rhodamine-stabilized vaterites, samples for FLIM imaging were prepared by dispersing vaterites on transparent coverglass from buffer solution. For DHLA-Qdot-stabilized vaterites, samples for FLIM were prepared as follows: Clean cover glasses were reacted with 3-aminopropyltrimethoxysilane (APTMS, Sigma) under vacuum and 1675

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then treated at low heat. DHLA-coated Qdot525-stabilized vaterites were immobilized to functionalized cover glasses by reaction with EDC. To avoid eventual nonspecific binding to the silanized surface of either fluorescent proteins or streptavidincoated Qdots, samples were incubated overnight, prior to FRET experiments, with blocking buffer (Pierce). Finally, fluorescent proteins or streptavidin-coated Qdots were incubated for 30 min, with vaterites deposited on the surface.

3. RESULTS AND DISCUSSION 3.1. Fabrication of Water-Soluble Fluorescent Vaterites. Rhodamine 101 dye molecules were introduced into an aqueous solution of buffered calcium chloride. Carboxylic acid groups present on the rhodamine 101 dye were deprotonated with a base in TRIS buffer and coupled with calcium ions in reasonably high yield (>80%). The resulting calcium-containing dye molecules were converted into vaterite microspheres by adding sodium bicarbonate solution. All of these derivatized dye molecules were successfully incorporated into vaterite microspheres by reaction with carbonate ions in a basic TRIS solution. A reaction scheme is presented in the Supporting Information (see Figure S1). The mixed solutions were stirred for 30 min, turning the mixture almost instantaneously opaque. The relatively small size (∼2 μm) and homogeneous size distribution of the vaterite microspheres (see Figure 1a,b and Figure S2, Supporting Information) was achieved by controlling the intensity of agitation of the reaction mixture (∼1100 rpm). Thereafter, the dispersions were aged for 24 h under stagnant conditions. The resulting microspheres were isolated by centrifugation and repeatedly washed with Milli-Q water and were thereafter dispersed in buffer for further characterization. This procedure helped remove unbound dye from porous particles to a level such that the supernatant solution became colorless, thus indicating very little or no dye leaching from isolated vaterites. All reactions were carried out under a nitrogen atmosphere. The obtained fluorescent vaterite microspheres showed good dispersibility in buffers of various pH values, including physiological pH (see Figure 2a). They were also dispersed when deposited from buffer on cover glass for observation by confocal fluorescence microscopy (see Figure 2c). Unlike other fluorescent inorganic particles prepared from synthetic or natural macromolecules, which precipitate in water at neutral and basic pH,24-26 no supplementary complex synthetic or natural macromolecules are present on the surface of the vaterite microspheres produced by us. We confirmed the presence of rhodamine 101 within the vaterite microparticles by laser-induced fluorescence (Figure 2b), fluorescence spectroscopy (Figure S3, Supporting Information), and confocal fluorescence microscopy (Figure 2c,d). To demonstrate the importance of rhodamine dye, in particular, its carboxyl acid function, we performed control experiments using conditions similar to those described above but without rhodamine. In the absence of the organic dye, we observed the formation of calcite particles instead of vaterites (see Figure 1e). The fluorescent vaterites introduced by us have good thermal stability, exhibiting both dispersibility and bright fluorescence even after being refluxed in water for one day (see Figure S4, Supporting Information). Stabilization of vaterite microspheres and achievement of intrinsic fluorescence was also demonstrated using DHLA-Qdots with 525-nm photoluminescence color, following a procedure similar to that for rhodamine 101. The presence of DHLA-Qdot-

Figure 2. Fluorescent properties of vaterites: Rh-vaterite microspheres dispersed in buffers of various pH values and observed under (a) natural light and (b) 488-nm light illumination (laser beam from an argon-ion laser coming from the side of the cuvettes). (c) confocal fluorescence image of Rh-vaterites dispersed on coverglass. High-resolution confocal fluorescence images of (d) single Rh-vaterite and (e) single Qdot525vaterite microspheres.

stabilized vaterites was confirmed by fluorescence spectroscopy (data not shown), confocal fluorescence microscopy (Figures 2e and 3b), and transmission electron microscopy (TEM, Figure 1f). 3.2. Structural and Optical Characterization of WaterSoluble Fluorescent Vaterites. Figure 1a shows a scanning electron microscopy (SEM) image of fluorescent vaterite microspheres stabilized with rhodamine 101, thus demonstrating the homogeneous size distribution of the obtained microparticles. Formation of the vaterite structure was confirmed by X-ray powder diffraction experiments (Figure S5, Supporting Information). Figure 1c displays an SEM image of a crushed vaterite microsphere, showing the channel-like interior structure characteristic to the vaterite polymorph. Such channels that go directly from the outermost surface to the center of the vaterite microspheres are suitable for loading drugs, genes, or other biological objects.6,18,27 The carboxylic acid groups present in the dye molecules are stabilized by the unique structure of vaterite. Rhodamine induces a smooth texture (see Figure 1a,b) and a reduction in vaterite crystal size that is markedly different in texture, shape, and size from the crystals produced without rhodamine (see Figure 1e). Rhodamine acts as an effective inhibitor to calcite nucleation. When conditions are such that the formation of calcite nuclei is significantly reduced, the nucleation of the less stable polymorph (vaterite) takes place (see Figure S1 in the Supporting Information). In the absence of rhodamine, the precipitate is calcite, the thermodynamically stable polymorph of calcium carbonate. We monitored the crystal growth process from the initial nanoparticles to the final vaterite microspheres, by examining the early stage of the crystallization (after 5 min) with differential interference contrast (DIC) optical microscopy, in view of the optically anisotropic nature of vaterite.20 When observed by DIC optical microscopy, microspheres are dark before crystallization but appear bright after crystallization (see Figure S6, Supporting Information), thus indicating the presence of an amorphous calcium carbonate (ACC) form at the early stage of crystallization, followed by a polymorph (vaterite microspheres) at later stages. In the initial 1676

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Figure 3. Confocal fluorescence lifetime microscopy (FLIM) of fluorescent vaterite microspheres. All data were recorded with 488-nm pulsed laser excitation. (a) FLIM image of single Rh-vaterites deposited on a cover glass (average fluorescence lifetime = 3 ns). (b) FLIM image of single Qdot525vaterites deposited on a cover glass (average fluorescence lifetime = 4 ns). (c) Fluorescence decay of an individual Rh-vaterite (black, raw data) and biexponential fit (green) having lifetimes of 1.2 ns (50 wt %) and 3.4 ns (50%). Shown in red is a single-exponential fit mismatching the raw data. The inset shows histograms of pixel-by-pixel fluorescence lifetimes estimated by high-resolution FLIM for a single Rh-vaterite (red) and from an area containing rhodamine dye adsorbed on coverglass. (d) Fluorescence decay of an individual Qdot525-vaterite (black, raw data) and multiexponential fit (green) yielding an average lifetime of 4 ns. The inset shows a histogram of pixel-by-pixel fluorescence lifetimes estimated by high-resolution FLIM from a single Qdot525-vaterite particle by high-resolution FLIM.

stages, large amounts of calcium and carbonate ions accumulate electrostatically on the carboxylate groups of the rhodamine dye, leading to sudden formation of a large number of ACC particles. The ACC particles aggregate by covalent interactions between calcium ions on the surface of CaCO3 and carboxylic groups on rhodamine (see Figure S1, Supporting Information). When the growth of CaCO3 particles reaches a critical size, the inorganic/ organic particles tend to adopt a spherical morphology, as this gives the minimum total surface energy for a given volume.15 X-ray diffraction data confirmed that the microspheres obtained at later stages of the crystallization were vaterites (see Figure S5, Supporting Information). DHLA-Qdot-stabilized vaterites have a size and homogeneous size distribution similar to those of rhodamine-stabilized vaterites, including a relatively smooth texture (see Figure 1d and Figure S7 in Supporting Information). The TEM image of crushed DHLA-Qdot-stabilized vaterites suggests loading of DHLA-Qdots at the external surface of the vaterites (see Figure 1f, with Qdots indicated by the arrow). The presence of Qdots within the vaterites was confirmed also by energy-dispersive spectroscopy (EDS; see Figure S8, Supporting Information). Rhodamine 101 stabilized vaterites, henceforth called Rh-vaterites, when dispersed in aqueous medium, produce bright photoluminescence (see Figure 2b). Their steady-state fluorescence is slightly blue-shifted (peak at 520 nm; see Figure S3, Supporting Information) when compared to that of rhodamine 101 in water (peak at 524 nm). The fluorescence decay measured from Rh-

vaterites in TRIS buffer (488-nm excitation) by the time-correlated single-photon-counting method28 is biexponential, with fluorescence lifetimes of 1.23 ns (50 wt %) and 3.4 ns (50%). This is in contrast to a single-exponential fluorescence decay profile for rhodamine 101 dissolved in water (4.3-ns lifetime). We used confocal fluorescence lifetime imaging (FLIM) microscopy29,30 to characterize fluorescent vaterites at the level of individual particles. High-resolution FLIM images were recorded from Rh-vaterites dispersed onto a transparent cover glass (Figure 2d). For a single Rh-vaterite, we detected, on average, brightness values of 630 kHz per particle for an average power of 1 kW/cm2 at the sample (488-nm excitation). As a comparison, individual DHLA-Qdots of 525-nm photoluminescence color, when deposited on cover glass and exposed to a similar laser light intensity, exhibited brightnesses as high as 5 kHz per Qdot, a value reflecting the low photoluminescence quantum yield of these water-soluble nanoparticles (5% in TRIS buffer). Confocal FLIM demonstrates efficient loading of rhodamine dyes by a single Rh-vaterite, both at the external surface and on the surface of the internal pores of the vaterite (Figure 2d). Fluorescence decays from single Rh-vaterites are biexponential, with lifetimes/contributions similar to those estimated at the ensemble level. Figure 3c shows an example of a fluorescence decay measured from a single Rh-vaterite. For a single Rh-vaterite, the distribution of fluorescence lifetimes estimated pixel-by-pixel by high-resolution FLIM is asymmetric, with a main peak at 2.7 ns and a contributing tail at long lifetimes (Figure 3c, inset, distribution in green). This is in contrast to the distribution of fluorescence lifetimes estimated 1677

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The Journal of Physical Chemistry C from rhodamine 101 dye deposited on cover glass which is symmetric, with a main peak at 4.3 ns, similar to that of the dye in water (Figure 3c, inset, distribution in red). This suggests that, for the case of Rh-vaterites, the dyes loaded at the external surface and onto the surface of the internal pores exhibit different photophysics. We speculate that dyes incorporated into the vaterite internal pores, although chemically bound to the particle lattice, might experience restricted motion and therefore conformational change. This might be associated with a decreased electronic conjugation that can result in spectrally blue-shifted emission and a shorter lifetime when compared to the free dye. Dyes adsorbed at the external surface will be less influenced by the vaterite scaffold, therefore having photophysics similar to that of the free dye. We hypothesize that rhodamine dyes incorporated into the internal pores will be less susceptible to photobleaching when Rh-vaterites are under intense illumination, making Rh-vaterites bright and photostable when used in conjunction with confocal fluorescence microscopy. We measured the photobleaching decay curve from a single Rh-vaterite exposed to 488-nm laser light in the confocal microscope (1 kW/cm2 average power; see Figure S9, Supporting Information). Irradiation over an extended period (600 s) led to minimal decrease in the fluorescence signal (about 2-fold) from an initial photon count rate of about 700 kHz. DHLA-Qdot stabilized vaterites, hereafter called Qdot525vaterites, emit around 525 nm, with a photoluminescence spectrum similar to that of free DHLA-Qdot525 nanocrystals in TRIS buffer (data not shown). They exhibit brightness values between 15 and 45 kHz per particle, on average, for an average laser excitation power of 1 kW/cm2 at 488 nm. This relatively low brightness of Qdot525-vaterites, when compared to that of Rhvaterites, is the result of at least two factors: the low quantum yield of the DHLA-Qdot525 nanocrystals (5%, compared to 100% for rhodamine 101) and the low amount of nanocrystals loaded onto the vaterite at the external surface only. Interestingly, by monitoring the photoluminescence intensity from a single Qdot525-vaterite particle, we did not observe blinking (on/off switching), an intrinsic property associated with individual quantum dots.31 This suggests that several nanocrystals were loaded on the external surface of a single Qdot525-vaterite, thus averaging out the photoluminescence blinking at the level of a single vaterite particle. Photoluminescence decays measured from individual Qdot525-vaterites dispersed onto cover glass are multiexponential (see Figure 3d), with average lifetimes similar to those of free DHLA-Qdot525 nanocrystals dispersed in TRIS buffer (4 ns). The distribution of photoluminescence lifetimes of a single Qdot525-vaterite estimated pixel-by-pixel by high-resolution FLIM is symmetric, with a mean peak at around 4 ns, suggesting that the vaterite scaffold has little or no influence on the photophysics of the DHLA-Qdot525 nanocrystals loaded on its external surface. The relatively high brigthness of Qdot525-vaterites, surpassing that of commercially available water-soluble CdSe/ZnS Qdots emitting around 525 nm, together with the lack of photoluminescence blinking, makes Qdot525-vaterites promising candidates for biological imaging. Adding the large loading surface of vaterites and the fact that core/shell CdSe/ZnS Qdots can be easily functionalized with biologically relevant ligands such as biotin or carbohydrates or with proteins, Qdot525-vaterites are promising scaffolds for biosensing applications (see below). Compared to other fluorescent colloidal particles, such as dyeloaded silica nanoparticles,32 our vaterites are thermally stable; highly photostable; and highly dispersible in water over a large

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Scheme 1. Concept of FRET-Based Biosensing Schemes Using Qdot525-vaterites: (a) Biosensor Based on Qdot525vaterites Binding Target Poly-His-Tagged Fluorescent Proteins (TdTomato) and Undergoing FRET upon Blue-Green Illumination of Qdot525-vaterites and (b) Biosensor Based on Biotinilated-Qdot525-vaterites Binding Target Streptavidin-Conjugated Qdots Emitting at 602 nma

a Q , DHLA-Qdot 525 quantum dot; Q0 , streptavidin-Qdot602 quantum dot; FP, fluorescent protein; FRET, fluorescence resonance energy transfer. Arrows indicate direction of FRET upon blue-green illumination.

pH range, including biologically relevant pH. In the case of silica nanoparticles, the smooth surface and nonporous structure limits dye loading to the level of a monolayer.33 An increase in dye loading is usually achieved by repetitive and tedious multilayer assembly procedures that involve the use of excess silane compounds as a stabilizer.22 This is also the case for fluorescent latex particles.34,35 An excess amount of stabilizers can adversely affect the size uniformity and solubility of the inorganic particles at biologically relevant pH values, while producing cytotoxicity by the terminal amine groups from the silane-treated nanoparticles.25,26 3.3. Fluorescence-Resonance Energy Transfer Model Biosensors Based on DHLA-Qdot-Loaded Vaterites. We engineered two model biosensors using the Qdot525-vaterite scaffold that are based on fluorescence resonance energy transfer (FRET)36,37 between DHLA-Qdot525 nanocrystals loaded on the vaterite surface (FRET donors) and a biological target, either poly-His-tagged TdTomato38 protein (FRET acceptor absorbing/emitting at 544/581 nm) or streptavidin-conjugated core/ shell CdSe/ZnS Qdot emitting at 602 nm (FRET acceptor). Scheme 1 describes the biosensing concept. The demonstration of FRET-based sensing for each particular case is discussed below. In all experiments, Qdot525-vaterites were attached to amine-functionalized cover glasses using EDC as a zero-length cross-linker (see details in the Experimental Section). Although more complex FRET-based sensing schemes can be envisioned, for example, biosensing of antibody-antigen interactions or DNA hydridization, the two models from Scheme 1 should be sufficient for the proof-of-concept of Qdot-vaterite biosensing. FRET between green-emitting core/shell CdSe/ZnS nanocrystals and poly-His-tagged fluorescent proteins has been proposed as a biosensing method for measuring in vitro enzymatic activity.39,40 Poly-His-tagged fluorescent proteins such as TdTomato binding coordinatively to core/shell CdSe/ZnS nanocrystals undergo sensitized emission by FRET when the nanocrystlas are irradiated with UV-blue light.40 Enzyme cleavage of the poly-His tag supposedly breaks the nanocrystalprotein connectivity, thus interrupting FRET and consequently 1678

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Figure 4. Demonstration of FRET-based biosensing of poly-His-tagged proteins using Qdot525-vaterites. All data were recorded with 488-nm pulsed laser excitation. Confocal FLIM images of (a) Qdot525-vaterites (donor channel) and (b) poly-His-tagged TdTomato proteins bound to vaterites (acceptor channel). (c) Fluorescence decays of a single Qdot525-vaterite (donor, decay in gray, fit in green, average lifetime = 1.3 ns) binding polyhistidine-tagged TdTomato proteins (acceptor, decay in black, fit in red, average lifetime = 3.4 ns). Binding of proteins to the vaterite scaffold quenches the average lifetime of the Qdot525-vaterite from 4 to 1.3 ns, corresponding to a FRET efficiency of 67% (see text for details). (d) Histograms of pixel-by-pixel fluorescence lifetimes estimated by high-resolution FLIM from a single Qdot525-vaterite binding TdTomato proteins. The green- and red-colored histograms correspond to Qdot525-vaterite quenched by FRET and FRET-sensitized TdTomato proteins, respectively.

protein emission. We followed a concept similar to that applied in ref 40 (see Scheme 1a) to demonstrate FRET-based sensing of poly-His-tagged TdTomato using Qdot525-vaterites. To a transparent cover glass containing immobilized Qdot525-vaterites immersed in TRIS buffer were added TdTomato proteins. Following incubation (30 min) and washing of excess protein, we monitored FRET activity by two-color confocal FLIM (Figure 4a, donor channel; Figure 4b, acceptor channel). Binding of poly-His-tagged proteins to the Qdot525-vaterites resulted in efficient quenching of Qdot525-vaterites accompanied by FRETsensitized emission of bound TdTomato proteins. The disk-like shape of the fluorescent objects from Figure 4b (acceptor channel), with sizes similar to that of the vaterite scaffold, indicates that binding of poly-His-tagged proteins happens exclusively to the Qdot525-vaterite scaffolds. Figure 4c further illustrates FRET biosensing of poly-His-tagged proteins. A single Qdot525-vaterite binding poly-His-tagged TdTomato proteins quenches its average photoluminescence lifetime by FRET from 4 ns (Figure 3d) to 1.3 ns (Figure 4c; decay in gray, fit in green). At the same time, FRET-sensitized emission of TdTomato is detected with an average lifetime of 3.4 ns (Figure 4c; decay in black, fit in red). An estimate of the FRET efficiency for this particular biosensor yields a value E = 1 - τDA/τD ≈ 67%, with τDA and τD being the lifetimes of Qdot525-vaterite in the presence and absence of TdTomato, respectively. FRET for this particular biosensor is also illustrated by the histograms of the pixel-by-pixel fluorescence lifetimes calculated

from the donor and acceptor channels corrresponding to a single Qdot525-vaterite-TdTomato pair (see Figure 4d). In particular, the lifetime distribution corresponding to the donor channel exhibits a peak at low values, indicating quenching of Qdot525-vaterite by FRET to bound TdTomato proteins. We engineered a second model biosensor in which DHLAQdot525 nanocrystals loaded on the vaterite scaffold were functionalized with biotin ligands so that they can bind target steptavidin-conjugated CdSe/ZnS nanocrystals emitting at 602 nm (Qdot602; see Scheme 1b for concept). To a transparent cover glass containing immobilized biotinilated-Qdot525-vaterites immersed in TRIS buffer were added streptavidinconjugated Qdot602 nanocrystals. Following incubation (30 min) and washing of excess streptavidin-conjugated nanocrystals, we monitored FRET activity by two-color confocal FLIM (Figure 5a, donor channel; Figure 5b, acceptor channel). Binding of streptavidin-conjugated Qdot602 to biotinilated Qdot525-vaterites resulted efficient quenching of Qdot525vaterites (Figure5a), accompanied by FRET-sensitized emission from streptavidin-conjugated Qdot602 (Figure5b). Figure 5c illustrates FRET-based biosensing at the single particle level: the fluorescence lifetime of an individual biotinilated Qdot525-vaterite quenches from 4 to 1 ns upon binding of streptavin-conjugated Qdot602. This FRET-induced quenching is accompanied by sensitized emission from bound streptavidinQdot602 with an average lifetime of 15 ns. The histograms of the 1679

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Figure 5. Demonstration of FRET-based biosensing of streptavidin-conjugated Qdot602 quantum dots using Qdot525-vaterites. All data were recorded with 488-nm pulsed laser excitation. Confocal FLIM images of (a) Qdot525-vaterites (donor channel) and (b) streptavidin-conjugated Qdot602 quantum dots bound to vaterites (acceptor channel). (c) Fluorescence decays of a single Qdot525-vaterite (donor, decay in gray, fit in green, average lifetime = 1.0 ns) binding streptavidin-conjugated Qdot602 quantum dots (acceptor, decay in black, fit in red, average lifetime = 15 ns). (d) Histograms of pixel-by-pixel fluorescence lifetimes estimated by high-resolution FLIM from a single Qdot525-vaterite binding streptavidin-conjugated Qdot602 quantum dots. The green- and red-colored histograms correspond to Qdot525-vaterite quenched by FRET and FRET-sensitized streptavidinconjugated Qdot602 quantum dots, respectively.

pixel-by-pixel photoluminescence lifetimes shown in Figure 5d further confirm FRET activity for this particular biosensor and at the single particle level. Note that part of the signal detected from the acceptor channel for this particular biosensor comes from direct excitation of the acceptor (streptavidin-Qdot602), which is unavoidable in the case of CdSe/ZnS nanocrystals.

4. CONCLUSIONS In summary, we have demonstrated a polymer-free synthetic method to prepare water-soluble, highly stable, porous fluorescent vaterite microspheres. Stabilization was achieved by the use of rhodamine 101, which also introduces fluorescence as an intrinsic property to the scaffold. We showed that these fluorescent vaterites are highly dispersible in water and over a large pH range, from neutral to acid pH, and that they have good thermal stability and amazing fluorescence properties, including excellent brightness and photostability. Taken together, these properties make rhodamine-based vaterites promising scaffolds for fluorescence imaging, in particular, timelapse microscopy or multicolor tracking microscopy using vaterites stabilized by carboxyl-rhodamine dyes of different colors. We demonstrated stabilization of vaterites by DHLA-coated core/shell CdSe/ZnS semiconductor quantum dots, resulting in intrinsic fluorescent scaffolds with better performance than commercially available water-soluble core/shell CdSe/ZnS quantum dots of similar emission color. Using DHLA-Qdot-stabilized vaterites, we illustrated FRET-based biosensing of biological targets including fluorescent proteins and streptavidin-conjugated Qdots.

’ ASSOCIATED CONTENT

bS

Supporting Information. Mechanism of vaterite formation, fluorescence spectroscopy, XRD data, optical (DIC) microscopy images of particle growth process, EDS data, and photostability data. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Tel.: 631-344-7778. Fax: 631-344-7765. E-mail [email protected] (R.K.P.), [email protected] (M.C.).

’ ACKNOWLEDGMENT This research was carried out at the Center for Functional Nanomaterials, Brookhaven National Laboratory, which is supported by the U.S. Department of Energy, Office of Basic Energy Sciences (Contract No. DE-AC02-98CH10886). RKP acknowledges financial support from the International Iberian Nanotechnology Laboratory (INL) in Braga, Portugal. We thank Professor Gang Bao and Dr. A. Dennis from Georgia Tech for providing us expressed TdTomato protein, Dr. Zhihua Xu, Dr. Lihua Zhang and Dr. Fernando Camino from Brookhaven National Laboratory for help with some characterization experiments. ’ REFERENCES (1) Mann, S.; Ozin, G. A. Nature 1996, 382, 313. (2) Pomogailo, A. D.; K., V. N. Metallopolym. Nanocompos. 2006, 81, 377. 1680

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