Journal of Colloid and Interface Science 363 (2011) 137–144

Contents lists available at ScienceDirect

Journal of Colloid and Interface Science www.elsevier.com/locate/jcis

Dye-labeled polystyrene latex microspheres prepared via a combined swelling-diffusion technique Jung-Hyun Lee a, Ismael J. Gomez a, Valerie B. Sitterle b, J. Carson Meredith a,⇑ a b

School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst Drive, Atlanta, GA 30332, USA Georgia Tech Research Institute, Georgia Institute of Technology, 250 14th Street NW, Atlanta, GA 30332, USA

a r t i c l e

i n f o

Article history: Received 11 May 2011 Accepted 15 July 2011 Available online 27 July 2011 Keywords: Polymer composites Colloids Fluorescent dyes Molecular assembly Combined swelling-diffusion Block copolymer surfactant

a b s t r a c t A series of water-insoluble, biologically compatible dyes, meso-tetraphenylchlorin, meso-tetraphenylporphyrin and chlorophyll-a, were successfully incorporated into beads composed of linear polystyrene (PS) via a tunable combined swelling-diffusion process. Dyed PS beads were prepared by the addition of a dye solution in tetrahydrofuran to an aqueous suspension of 10 lm PS beads in the presence of a poly((ethylene glycol)-b-(propylene glycol)-b-(ethylene glycol)) block copolymer surfactant. The presence of surfactant was found to be beneficial to prevent particle aggregation, especially at tetrahydrofuran contents above 30%. Dye loading was shown to be tunable by simple adjustments in dye composition. Confocal fluorescence microscopy indicated that dyes were distributed uniformly throughout the entire PS bead, but heterogeneously with 500 nm diameter droplets, indicative of a separate dye phase within the PS matrix. The stability of dyed beads, indicated by resistance to dye leaching in solvent, was found to be governed by the degree of swelling of PS in the solvent medium. Hence, no leaching was observed even when a good solvent for the dye was used (ethanol), as long as that solvent did not swell the carrier particle, PS. No leaching of dyes from the beads was observed during long-term (2 years) storage in water. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction Fluorescent dye-doped polymer particles are of interest to many applications such as light-emitting diodes [1,2], photonic crystals [3–5], cell labeling [6,7], and optical biosensors [8]. Polymers, often in the form of submicrometer- to micrometer-sized beads, can serve as an excellent carrier particle for optical dyes because they are inexpensive to make, biologically inert, optically transparent, and provide a convenient method for storage and delivery. A number of techniques for the preparation of organic dyeincorporated polymers have been reported, including both in situ and ex situ methods. In situ methods incorporate dye during synthesis of the polymer bead [9]. For example, an emulsion polymerization technique can be used to combine polymer monomers, such as styrene and methyl methacrylate, with fluorescent dye comonomers to yield polymer particles labeled with dyes [3,10]. Sudan IV dye was entrapped in polystyrene/silica composite particles by using a sol gel process in which the dye incorporates into the triethoxysilane phase [11]. The dispersion polymerization of monomers in the presence of nonpolymerizable dyes successfully produced dyed polymer particles as well [12]. Although in situ ⇑ Corresponding author. Fax: +1 404 894 2866. E-mail address: [email protected] (J.C. Meredith). 0021-9797/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2011.07.047

incorporation techniques are effective for incorporating a variety of organic dyes into polymer carriers, they are time-consuming and inherently complex due to the simultaneous reaction and phase separation occurring, hence requiring control over numerous parameters such as composition, reaction conditions and solvent system. Ex situ methods incorporate dyes into preformed polymer particles through surface attachment (external labeling) or dye entrapment (internal labeling). Various chemical interactions between dyes and polymers have been employed for external labeling. For example, electrostatic and hydrophobic interactions were used to drive the adsorption of a positively charged dye such as Rhodamine 6G onto negatively charged poly(styrene sulfate) latex beads [13]. Cibaron blue F3G-A dye ligands were covalently bound to the surface of poly(vinyl alcohol)-stabilized poly(styrene-co-divinylbenzene) particles through a sodium hydroxide treatment [14]. Ester-functionalized cyanine dyes were covalently attached inside the core of poly(N-isopropylacrylamide) microparticles as a part of the post-polymerization step [15]. While external labeling techniques broaden the spectrum of types of dyes that can be incorporated into polymers, they unfortunately require additional procedures for surface modification. In contrast, internal labeling of dyes into polymeric microspheres via diffusion and entrapment is a relatively facile method [16,17]. This technique involves solvent-induced swelling of the

138

J.-H. Lee et al. / Journal of Colloid and Interface Science 363 (2011) 137–144

polymer bead, diffusion of dye from solution into the particle and entrapment of dye after the solvent removal. Many commercially available dyed beads are prepared by the internal labeling technique. However, details of the incorporation process (procedure, composition and mechanism) are not usually reported because of its proprietary nature. This makes it difficult to predict candidates for novel dyes and/or polymers for applications requiring specific spectral characteristics. Recently we reported a combined swelling–heteroaggregation (CSH) method for the preparation of metal nanoparticle (NP)coated polystyrene (PS)–divinylbenzene (DVB) beads [18,19]. A swelling solvent is used to control the deposition of polymercapped metal NPs of different chemistries and geometries onto polymer microspheres initially dispersed in water. The antisolvent composition controls the swelling of the beads and the heteroaggregation of NPs with the beads. Subsequently, NPs are trapped near the bead’s surface by the removal of swelling solvent. In the present study, we expanded the CSH technique to the incorporation of water-insoluble fluorescent dyes into PS beads. A series of dyes, meso-tetraphenylchlorin (TPC), meso-tetraphenylporphyrin (TPP) and chlorophyll-a (CP), were successfully loaded into unfunctionalized PS beads via a combined swelling-diffusion (CSD). The dyes used in this study were selected because literature suggests their low toxicity, and they are soluble in organic solvents, not in water. They also exhibit strong spectral absorption and/or emission in the visible to near infrared region above 600 nm wavelength, where there is very little optical interference from tissue and other biological substrates. This combination of factors makes them potential candidates for contrast agents in biological imaging applications [20,21]. In addition, porphyrins and their related compounds (chlorin and chlorophyll dyes) have attracted immense interest due to applications as photosensitizers for photodynamic therapy and solar cells [20,22–24]. Dyed PS beads were obtained by the addition of dye solution in tetrahydrofuran (THF) to an aqueous suspension of 10 lm PS beads in the presence of block copolymer surfactant (Pluronic F127), followed by the removal of THF. The role of the surfactant used in the CSD method was studied. THF composition was optimized on the basis of dye loading efficiency, and dye loadings were controlled by adjusting dye concentrations. The optical properties of the resulting dye-loaded polymers were characterized by various microscopic tools. The stability of dye-incorporated beads (e.g. prevention of dye leaching) was also evaluated.

2. Experimental 2.1. Materials The three fluorescent dyes, meso-tetraphenylchlorin (TPC, Frontier Scientific), meso-tetraphenylporphyrin (TPP, Sigma– Aldrich) and chlorophyll-a (CP, Sigma–Aldrich) were used as received. Their chemical structures and optical properties are given in Table 1. The triblock copolymer surfactant, poly((ethylene glycol)-b-(propylene glycol)-b-(ethylene glycol)) (Pluronic F127: (PEG)97-(PPG)56-(PEG)97) was obtained from BASF. THF (P99.9%) was used as purchased from EMD Chemical Inc. Linear PS microspheres (10.0 ± 1.0 lm, 2.63 wt.%, in water), with sulfonate groups on their surface, were purchased from Polyscience Inc. Linear PS beads were used because of their higher swelling nature, compared to cross-linked beads. Sulfonate groups were selected to facilitate the dispersion of PS beads in an aqueous medium due to electrostatic stabilization arising from their highly negatively charges. Deionized (DI) water (18.2 MO cm) was prepared in a Millipore Milli-Q Plus 185 purification system.

2.2. Dye incorporation A 150 lL aqueous suspension of PS beads, as-received, was put into a 1.7 mL centrifuge tube, to which 300 lL of DI water and 150 lL of 2 wt.% Pluronic F127 aqueous solution were added and stirred for 1 min. A volume of 300 lL of a known concentration of dye in THF was added dropwise to the bead suspension until the THF content reached 33 vol.%. UV–vis spectra of dyes in pure THF and THF-water mixtures in the absence of F127 were identical, indicating that dyes were soluble in THF-water mixtures. Dye loading (wt.%) into the PS bead was calculated from a mass balance based on the known masses of PS and dye added, assuming complete incorporation of all dye added and complete removal of THF from the beads (confirmed for 33 vol.% THF by UV–vis spectroscopy). For example, 0.25 mg dye/mL THF resulted in a dye loading of 1 wt.% in the THF-free PS. The mixture was mixed using a VWR Analog Vortex Mixer for 10 min, followed by agitation using a rotational shaker (Barnstead Thermolyne Labquake Shaker Rotisserie) for 10 min. The mixture was then washed with DI water and centrifuged in a microcentrifuge (VWR Micro 1207) for at least four cycles. The total mixture volume and amounts of PS and THF were kept constant for all samples. 2.3. Dye stability test THF, acetone, and ethanol were used to assay the resistance of dye-incorporated beads to leaching of dye. A series of 500 lL volumes of 0.2 wt.% dyed PS bead (loaded with 4 wt.% CP) aqueous suspensions were prepared, to which were added various concentrations of the solvents (33–92 vol.% for THF, and 92 vol.% for ethanol and acetone, respectively). The mixtures were vortexed for 2 min, followed by agitation using a rotational shaker for 2 h. Supernatant solutions were collected by centrifugation. The amount of dye leaching from the beads was evaluated qualitatively by visual inspection of the color and UV–vis spectroscopy of the supernatant solution. 2.4. Characterization UV–vis spectra of dyes in solutions were measured with an Agilent 8453 spectrometer. UV–vis microabsorbance spectra of single PS beads loaded with dye were measured using an SEE1100 microspectrometer under 50 magnification using a spot size of 3 lm. The spectra measured from dye-loaded PS beads were referenced to that of a plain PS bead. An Olympus BX51 upright microscope equipped with a mercury lamp and a Chroma U-N41002 TRITC filter cube (535 nm excitation and 610 nm emission filter) was used for fluorescence microscopy of dye-loaded PS beads under 50 magnification. Bright field optical images of dyed PS beads were taken with a Leica DMRX optical microscope at a magnification of 50. SEM was performed with a LEO 1530 instrument for characterizing the surface morphology of dye-loaded PS beads. A Zeiss LSM 510 VIS confocal microscope using a green channel was used for the fluorescence microscope image of CP-loaded PS beads cross sections under 63 magnification. 3. Results and discussion 3.1. Composition and controlling parameters of the CSD technique Starting with PS beads and Pluronic F127 surfactants dispersed in water, a series of dyes (TPC, TPP and CP) were successfully incorporated into 10 lm unfunctionalized, linear PS beads by the addition and removal of 33 vol.% of THF-dye solution, as illustrated in Scheme 1. In analogy with previous studies of the CSH process

139

J.-H. Lee et al. / Journal of Colloid and Interface Science 363 (2011) 137–144 Table 1 Structures and optical properties of dyes used in this study. Dyes

Structure

Absorption (kmax)

Meso-tetraphenylchlorin (TPC)

Emission (kmax)

a

420

660b

419c

654c

430/660d

666e

N H N

N H N

Meso-tetraphenylporphyrin (TPP)

N H N

N H N

Chlorophyll-a (CP)

CH2

H3C

H3C

CH3

N

N Mg N

N CH3

H3C

O

O O

H3C

O O CH3

CH3

CH3 CH3 a b c d e

In In In In In

CH3

benzene [25]. chloroform [26]. methylene chloride [27]. ether [28]. ether [29].

Scheme 1. Proposed dye incorporation process using the CSD method.

140

J.-H. Lee et al. / Journal of Colloid and Interface Science 363 (2011) 137–144

for loading PS beads with metals, the dye incorporation process is thought to occur in three steps: (1) THF-induced swelling of the PS beads, (2) diffusion and penetration of dyes, and (3) shrinking of the beads by removal of THF. Upon the addition of the THF-dye solution into a PS bead aqueous suspension, PS beads swell because THF is a good solvent of PS. The size of PS increases continuously with THF composition as shown in Fig. 1a–d. Simultaneously, the dyes preferentially diffuse into PS due to a decrease in solubility of the dyes in the THF-water mixture. Subsequent deswelling of the PS by the removal of THF densifies the polymer chains and traps the dyes in the polymer matrix. Compared to the incorporation of metal NPs onto PS–DVB beads via the CSH method [16,17], dyes are incorporated much faster (completed within 20 min) and adsorbed dyes penetrate deeply into the swollen PS beads. Confocal microscopy on the CP dyeloaded PS beads, shown in Fig. 2, reveals the penetration of the dye into the PS beads. Furthermore, looking at a cross section of the PS bead along an equator, it is evident that the CSD method promotes dye incorporation throughout the entire sphere. Interestingly, dye appears to be heterogeneously-distributed throughout the sphere as a speckle pattern of dots with a size of approximately 0.5 lm, which suggests formation of a separate dye phase within the PS matrix. In the absence of Pluronic F127, the water-suspended linear PS beads can swell and shrink reversibly by the addition and removal of up to 33 vol.% THF, but the PS beads cannot recover from swelling at higher THF fractions (P33 vol.%). Rather, above 33 vol.% THF PS particles fuse together and form larger PS clumps (Fig. 1f). However, in the presence of Pluronic F127, the swelling was reversible and aggregation was suppressed at a THF fraction of 50 vol.% (Fig. 1d). 50% was the maximum THF vol.% where PS particles could be stabilized by Pluronic F127 surfactant. Even though swelling was reversible in the absence of surfactant at 33 vol.% THF, the particles exhibited some stickiness and appeared to fuse at the end of the dyeing procedure (Fig. 3b). However, the use of the polymeric surfactant at 33 vol.% THF appeared to suppress the particle– particle adhesion and minimize the aggregation of the dyed beads,

Fig. 2. Confocal microscope image of a center cross-section of 4 wt.% CP-loaded PS beads. The scale bar is 10 lm.

as illustrated in Fig. 3a. The Pluronic appears to act as a steric stabilizer within the THF-water mixtures, where the electric double layer repulsion of the charged PS would be compromised relative to pure water. This idea is supported by previous literature that has shown that swelling PS (in chloroform/methanol) is an effective method to anchor Pluronic F127 to its surface, producing stable coverage of the surfactant [30]. Polymer surfactants grafted to PS particles appear to increase the stability of the microspheres by providing a steric barrier to aggregation [31]. The primary reason for using F127 was to reduce the coalescence between swollen beads or onto sample containers through

Fig. 1. Optical microscopy images of PS beads in THF-water solutions at various THF concentrations (vol.%) in the presence of Pluronic F127: (a) 0%, (b) 17%, (c) 33%, (d) 50% and (e) 70% THF and in the absence of Pluronic F127: (f) 50% THF. All images were taken after 20 min mixing and the scale bars are 25 lm.

J.-H. Lee et al. / Journal of Colloid and Interface Science 363 (2011) 137–144

141

Fig. 3. Optical microscope images of 2 wt.% (of the PS bead weight) TPC-loaded PS beads obtained from 33 vol.% THF-water solution after drying on a glass substrate: (a) in the presence of Pluronic F127 and (b) in the absence of Pluronic F127. The scale bars are 25 lm.

the mechanism of steric hindrance. The Pluronic concentration in our system is above the critical micelle concentration, so that THF and dye could potentially go into micelles. If so, then the distribution of dye between micelles and solvent would influence dye diffusion and equilibrium loading in PS beads. However, we did not observe any evidence of dye in the supernatant solution containing F127 after centrifugation. In addition, UV–vis spectra indicated that nearly all dye is incorporated into PS using 33% THF. Although this is not direct evidence that dyes are not going into micelles, it indicates that the presence of F127 has a minor effect on the dye diffusion into PS beads. The solvent composition for optimal dye incorporation was investigated by varying THF vol.% and holding other compositions (PS bead, dye and surfactant) constant. The amount of dye incorporated was monitored qualitatively by UV–vis absorption spectroscopy of supernatant solutions (Fig. 4). Using 33 vol.% THF as the swelling medium resulted in the least observed UV–vis absorbance of the dye in the supernatant, indicating a maximum amount of the dye incorporated into beads at 33 vol.% THF, compared to 17 and

2.5 Dye solution 17 % 2.0

33 %

Absorbance

50 % 1.5

1.0

0.5

0.0 450

500

550

600

650

700

Wavelength (nm) Fig. 4. UV–vis absorbance spectra (with a 10 mm path length) of TPC dye in supernatant solutions after dye incorporation into PS beads using various THF vol.% for 20 min (colored lines) and TPC dye dissolved in THF before the incorporation (black line). The dye fractions in the total solution volume were kept constant (0.083 mg dye/ml solution) for all samples.

50 vol.%. This result suggests that the mass balance approach used to estimate the amount of dye incorporated into the beads is valid to the detection limit of UV–vis spectroscopy near 33 vol.% THF. The minimum dye content in the supernatant at 33 vol.% THF indicates a trade-off in dye incorporation as a function of THF content. An increase in THF composition is expected to initially facilitate dye diffusion into PS beads, due to an increased degree of PS swelling. However, dye incorporation is expected to decrease above some THF concentration, as the dye becomes increasingly soluble in the medium.

3.2. Optical properties of dyed PS beads Based on the above dye optimization experiments, the dyes of interest, TPC, TPP, and CP, were successfully incorporated into PS beads using 33 vol.% THF with a minimal amount of dye present in the supernatant solution. Dye loading based on the PS weight was adjusted by the fraction of dye present in the initial solution. Fig. 5 shows UV–vis spectra of a single dye-loaded PS bead (TPC (Fig. 5a) and TPP (Fig. 5b)) with different levels of dye loading. Dyed beads retain the characteristic absorbance peaks, identical to those of dyes in THF solution. The intensity of the characteristic peaks of dye-containing beads (425 nm (Soret) and 515, 550, 600 and 650 nm (Q-bands) for TPC, and 425 nm (Soret) and 515, 580 and 650 nm (Q-bands) for TPP, respectively [20]) increased as dye loading increased. The maximum dye loading into the PS bead was approximately 2 wt.% of the PS weight for both TPP and TPC. Further increasing the dye fraction in the solution resulted in large portion of dye remaining in the supernatant solution with a dense red color. This is probably due to limited dye solubility into PS beads, which is related with the chemical affinity of the dyes with the PS matrix and their solubility in the solution medium. Bright field and fluorescence microscope images of unmodified PS beads and dye (TPC and TPP)-loaded PS beads can be seen in Fig. 6. Contrary to the unmodified plain beads (Fig. 6d), red1colored fluorescence images of the dyed beads under a green channel (a filter cube containing a 535 nm excitation and a 610 nm emission filter) further confirm the successful incorporation of dyes into PS beads (Fig. 6e and f). Upon further SEM examination of the dried PS particles, shown in Fig. 7, the beads retain their size and shape even after incorporating dye through the swelling/deswelling procedure. Large 1 For interpretation of color in Figs. 2, 4–6, 8–10, and Scheme 1, the reader is referred to the web version of this article.

142

J.-H. Lee et al. / Journal of Colloid and Interface Science 363 (2011) 137–144

(b)

1.2

1.2

0.9

0.9

Absorbance

Absorbance

(a)

0.6

0.3

0.0 400

0.6

0.3

450

500

550

600

650

700

Wavelength (nm)

0.0 400

450

500

550

600

650

700

Wavelength (nm)

Fig. 5. UV–vis absorbance spectra of dye-loaded PS beads with different levels of dye loading (1 wt.% (black) and 2 wt.% (red) of the PS weight, respectively): (a) TPC, (b) TPP. UV–vis spectrum was measured from a single PS bead loaded with dye and obtained by subtracting that of a plain PS bead.

Fig. 6. (a)–(c) Bright field microscope images and (d) and (e) fluorescence microscopy (green channel) images: (a) and (d) plain PS beads, (b) and (e) 2 wt.% TPC-loaded PS beads, (c) and (f) 2 wt.% TPP-loaded PS beads. The scale bars are 20 lm.

Fig. 7. SEM images of (a) plain PS beads, (b) and (c) dye-loaded beads obtained from 33 vol.% THF: (b) 2 wt.% TPC and (c) 10 wt.% CP. The scale bars are 2 lm.

aggregates of dyes were not observed on the PS surface, although Fig. 2 indicates some dye aggregation within the PS sphere. CP-containing PS beads were prepared up to 10 wt.% dye loading with increasing dye concentration. The higher maximum dye loading of CP compared to TPC and TPP dyes is presumably attributed to CP’s better solubility in the PS matrix originating from its

chemical nature. Dye incorporation was verified by bright field and fluorescence (green channel) optical microscope images, as shown in Fig. 8. More intense green images of the CP dyed beads were observed in bright field microscopy as dye loading increased (Fig. 8a–d). The fluorescence intensity and wavelength of the CPloaded PS beads are dependent on dye loading. Fig. 8e–h show that

143

J.-H. Lee et al. / Journal of Colloid and Interface Science 363 (2011) 137–144

Fig. 8. (a)–(d) Bright field and (e)–(h) fluorescent (green channel) microscopy images of CP-loaded PS beads with different levels of dye loading: (a) and (e) 1 wt.%, (b) and (f) 2.5 wt.%, (c) and (g) 4 wt.%, (d) and (h) 10 wt.%. The scale bars are 20 lm.

as dye loading within the beads increases, the fluorescent color changes from red–orange to redder-orange to red, indicating the red-shift of emission wavelength with increasing dye loading. Further, emission intensity dropped off significantly at 10 wt.% dye loading (Fig. 8h). It is well known that the formation of dye aggregates results in the red-shift of emission wavelength and the reduction in fluorescence efficiency [32]. Increasing dye loading and the dye aggregation within the beads is responsible for the red-shift and decrease in the intensity of fluorescent emission. This red-shift to eventual inhibition of fluorescent emission seen in Fig. 8h indicates the highest extent of dye aggregation at the highest dye loading (Fig. 8h). The UV–vis absorbance spectra of the CP-loaded PS beads are shown in Fig. 9. An absorption spectrum with three detectable peaks around 435 (Soret band), 486, and 665 nm was observed for the CP-loaded PS bead with the lowest dye loading (1 wt.%). As dye loading increased, other characteristic absorbance peaks corresponding Q-bands appeared at 531, 578 and 620 nm [32], and all peak intensities continued to increase. For the highest dye loading at 10 wt.%, the absorbance was significant and near

1.5

Absorbance

1.2

0.9

0.6

10

4

2.5

3.3. Stability (resistance to dye leaching) of dyed PS beads The stability of dyed PS beads in various organic solvent–water solutions was estimated by analyzing supernatants after rinsing the dye-containing beads with selected solutions for 2 h. The results of dye leaching tests of the 4 wt.% CP-loaded beads are summarized in Table 2. Solvent mixtures that minimally swell PS beads, 0 and 33 vol.% THF and 92 vol.% ethanol, are unable to remove the dye from the beads, which was confirmed by clean supernatants after washing dyed beads with these mixtures. In particular, the dyed beads showed no loss of the dyes during long-term (2 years) DI water storage. Rinsing the beads with solvent mixtures that significantly swell (50 and 67 vol.% THF) or dissolve (92 vol.% THF or acetone) PS resulted in green-colored supernatants, indicating that some of the dye escaped from the beads. These results were further verified by UV–vis spectroscopy analysis on the supernatant solution from each trial, shown in Fig. 10. The absorbance spectra of the supernatant solutions after washing the dyed PS beads with 0 and 33 vol.% THF and 92 vol.% ethanol, respectively, did not show any detectable peaks (curves A, B and G). In contrast, the continuous increases in the absorbance peaks were observed with increasing THF concentration from 50 to 92 vol.% (curves C–E), and the significant absorbance peaks were detected with 92 vol.% acetone (curve F). The stability of the dyed beads appears to be determined by the solubility (degree of swelling) of PS in medium: a more effective swelling solvent for PS leads Table 2 Dye leaching results (supernatant analysis) of 4 wt.% CP-loaded PS beads in various organic solvent-DI water mixtures.

0.3 1 0.0 400

the maximum dynamic range of the spectrometer. However, absorbance characteristics were preserved across all dye loadings evaluated. Together with the continuous increases in all absorbance peaks, the slight red-shift of peaks (in particular around 486 nm) was observed with increasing dye loading. This red-shifting in the absorbance peak is attributed to the increased number of dye aggregates as dye loading increases [32].

450

500

550

600

650

700

750

Wavelength (nm) Fig. 9. UV–vis absorbance spectra of CP-loaded PS beads with different levels of dye loading (1, 2.5, 4, and 10 wt.%). UV–vis spectrum was measured from a single PS bead loaded with dye and obtained by subtracting that of a plain PS bead.

Solvents

Supernatant color

0 vol.% THF (A) 33 vol.% THF (B) 50 vol.% THF (C) 67 vol.% THF (D) 92 vol.% THF (E) 92 vol.% Acetone (F) 92 vol.% Ethanol (G)

Clear Clear Green Green Green Green Clear

144

J.-H. Lee et al. / Journal of Colloid and Interface Science 363 (2011) 137–144

1.8 A

E

B

F

C

G

determined by the degree of swelling of PS beads in the solvent medium. Even when the medium is a good solvent (ethanol) for dye, leaching does not occur at a level observable by UV–vis spectroscopy if the medium does not swell PS. In addition, dye-loaded beads are stable with no loss of the dye during long-term (2 years) water storage. The dye-incorporated beads were characterized by fluorescence microscopy and UV–vis spectroscopy. It was shown that the PS beads inherited the absorbance and fluorescence properties of the incorporated dye molecules. This validated that the methods used to incorporate the dye did not negatively impact the desired optical characteristics of the dye species.

D

Absorbance

1.2

0.6 Acknowledgment

0.0 400

450

500

550

600

650

700

The authors would like to thank Prof. M. Srinivasarao for the use of UV–vis microspectrometer facilities. We also thank Minsang Park for assisting with experiments. This work was supported by KaVo Dental Corp., GmbH, and by the Air Force Office of Sponsored Research under Award No. FA955010-1-0555.

Wavelength (nm) References Fig. 10. UV–vis absorbance spectra of CP dye in the supernatants after washing 4 wt.% CP-loaded PS beads with various solutions.

to more dye loss from the beads. Dyes were not leached out from the beads in the ethanol–water solution, which is significant because this solvent is commonly used to extract CP dyes [33]. This result indicates that the leaching of dye from PS beads is controlled primarily by the degree of swelling in the solvent, and that the solubility of dye in the solvent medium has a minor effect on dye leaching. 4. Conclusions In summary, fluorescent dye (TPC, TPP, and CP)-loaded PS latex beads have been prepared by a solvent (THF)-controlled, combined swelling-diffusion technique. This approach offers a facile custom fabrication of dye-loaded PS beads. The resulting dye loaded-polymer microspheres are of interest for applications in biomedical imaging, sensors, optical devices and photonic crystals. Different dye loadings of TPC (1 and 2 wt.%), TPP (1 and 2 wt.%), and CP (1–10 wt.%) can be incorporated into commercially-available, unfunctionalized PS beads by adjusting the dye concentration in the presence of a polymeric surfactant. THF appears to play two major roles in this technique: (1) as a solvent of PS, it plasticizes the PS and swells the PS matrix, allowing the incorporation of the dye molecules, and (2) as a solvent of the dye molecules, it allows diffusion of the dye molecules through the medium to the PS beads. Aggregation of beads at higher THF content (above 33 vol.%, as the electric double layer is thinned in the lower dielectric water-THF mixture) is suppressed by the addition of Pluronic F127, a block copolymer surfactant that acts as a steric stabilizer of the PS beads. Dye incorporation efficiency is determined by a balance between PS bead swelling and the dye solubility in the medium. A solvent environment of 33 vol.% THF maximizes dye incorporation. Incorporated dyes are distributed throughout the whole volume of the PS bead, but show a speckle pattern that indicates that dye is phase separated into regions approximately 0.5 lm in diameter. The stability of dyed beads (resistance of dye leaching) is

[1] J. Kido, K. Hongawa, K. Okuyama, K. Nagai, Appl. Phys. Lett. 64 (1994) 815. [2] P.I. Shih, C.F. Shu, Y.L. Tung, Y. Chi, Appl. Phys. Lett. 88 (2006) 251110. [3] M. Muller, R. Zentel, T. Maka, S.G. Romanov, C.M. Sotomayor Torres, Chem. Mater. 12 (2000) 2508. [4] D. Nagao, N. Anzai, Y. Kobayashi, S. Gu, M. Konno, J. Colloid Interface Sci. 298 (2006) 232. [5] S.H. Park, B. Gates, Y.N. Xia, Adv. Mater. 11 (1999) 462. [6] M.R. Lorenz, V. Holzapfel, A. Musyanovych, K. Nothelfer, P. Walther, H. Frank, K. Landfester, H. Schrezenmeier, V. Mailänder, Biomaterials 27 (2006) 2820. [7] V. Holzapfel, A. Musyanovych, K. Landfester, M.R. Lorenz, V. Mailänder, Macromol. Chem. Phys. 206 (2005) 2440. [8] H.A. Clark, M. Hoyer, M.A. Philbert, R. Kopelman, Anal. Chem. 71 (1999) 4831. [9] R. Arshady, Biomaterials 14 (1993) 5. [10] F. Tronc, M. Li, J. Lu, M.A. Winnik, B.L. Kaul, J.-C. Graciet, J. Polym. Sci., A: Polym. Chem. 41 (2003) 766. [11] H. Sertchook, D. Avnir, Chem. Mater. 15 (2003) 1690. [12] D.-G. Yu, J.H. An, J.-Y. Bae, S.D. Ahn, S.-Y. Kang, K.-S. Suh, Macromolecules 38 (2005) 7485. [13] M.-T. Charreyre, P. Zhang, M.A. Winnik, C. Pichot, C. Graillat, J. Colloid Interface Sci. 170 (1995) 374. [14] Camli, J. Biomater. Sci., Polym. Ed. 10 (1999) 875. [15] C.D. Jones, J.G. McGrath, L.A. Lyon, J. Phys. Chem. B 108 (2004) 12652. [16] J.M. Brinkley, R.P. Haugland, V.L. Singer, US Patent No. 5,326,692, 1994. [17] B.P. Wittmershaus, J.J. Skibicki, J.B. McLafferty, Y.-Z. Zhang, S. Swan, J. Fluoresc. 11 (2001) 119. [18] J.-H. Lee, M.A. Mahmoud, V. Sitterle, J. Sitterle, J.C. Meredith, J. Am. Chem. Soc. 131 (2009) 5048. [19] J.-H. Lee, M.A. Mahmoud, V.B. Sitterle, J.J. Sitterle, J.C. Meredith, Chem. Mater. 21 (2009) 5654. [20] M. Ethirajan, Y.H. Chen, P. Joshi, R.K. Pandey, Chem. Soc. Rev. 40 (2011) 340. [21] X. Peng, F. Song, E. Lu, Y. Wang, W. Zhou, J. Fan, Y. Gao, J. Am. Chem. Soc. 127 (2005) 4170. [22] W.M. Campbell, K.W. Jolley, P. Wagner, K. Wagner, P.J. Walsh, K.C. Gordon, L. Schmidt-Mende, M.K. Nazeeruddin, Q. Wang, M. Gratzel, D.L. Officer, J. Phys. Chem. C 111 (2007) 11760. [23] R.K. Pandey, L.N. Goswami, Y.H. Chen, A. Gryshuk, J.R. Missert, A. Oseroff, T.J. Dougherty, Laser. Surg. Med. 38 (2006) 445. [24] X.F. Wang, O. Kitao, E. Hosono, H.S. Zhou, S. Sasaki, H. Tamiaki, J. Photochem. Photobiol., A 210 (2010) 145. [25] A.M. Stolzenberg, S.W. Simerly, B.D. Steffey, G.S. Haymond, J. Am. Chem. Soc. 119 (1997) 11843. [26] Y. Sakakibara, S. Okutsu, T. Enokida, T. Tani, Appl. Phys. Lett. 74 (1999) 2587. [27] A. Rosa, G. Ricciardi, E.J. Baerends, A. Romeo, L. Monsù Scolaro, J. Phys. Chem. A 107 (2003) 11468. [28] C.L. Comar, F.P. Zscheile, Plant Physiol. 17 (1942) 198. [29] F.P. Zscheile, D.G. Harris, J. Phys. Chem. – US 47 (1943) 623. [30] Y. Su, L. Wang, X. Yang, Appl. Surf. Sci. 254 (2008) 4606. [31] A.J. Kim, V.N. Manoharan, J.C. Crocker, J. Am. Chem. Soc. 127 (2005) 1592. [32] Y. Kuroiwa, Y. Kato, T. Watanabe, J. Photochem. Photobiol., A 202 (2009) 191. [33] D.P. Sartory, J.U. Grobbelaar, Hydrobiologia 114 (1984) 177.