European Polymer Journal 45 (2009) 550–556

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European Polymer Journal

journal homepage: www.elsevier .com/locate /europol j

Preparation of fluorescent polystyrene microspheres by gradual solventevaporation method

Qi Zhang, Yan Han, Wei-Cai Wang, Lei Zhang, Jin Chang *

Institute of Nanobiotechnology, School of Materials Science & Engineering, Tianjin University, Tianjin, PR China

a r t i c l e i n f o

Article history:Received 1 September 2008Received in revised form 7 November 2008Accepted 10 November 2008Available online 21 November 2008

Keywords:Seed polymerizationFluorescentMicrospheresPolystyreneSolvent evaporation method

0014-3057/$ - see front matter � 2008 Elsevier Ltddoi:10.1016/j.eurpolymj.2008.11.016

* Corresponding author. Tel.: +86 022 27401821.E-mail address: jinchang@tju.edu.cn (J. Chang).

a b s t r a c t

Highly cross-linked polystyrene beads of 9.2 lm were synthesized by seed polymerizationwith styrene as monomer and divinylbenzene as cross linker. Other sized monodisperse PSmicrospheres were also prepared by varying seed particle diameter and proportion ofswelling agents. Furthermore, the polystyrene beads were stained by gradual solvent evap-oration method using dyes such as rhodamine 101 and acridine orange. Gradual solventevaporation method facilitates a high concentration of fluorescent dyes on beads. This isthe key to obtain fluorescent beads with high intensity. The results showed that the fabri-cated fluorescent microspheres could be excited to various wavelengths (such as yellow,green, red and scarlet). Our synthesized microspheres offer high fluorescence emission effi-ciency compared to commercial fluorescent microspheres in the mean time have otherproperties in common.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Highly uniform fluorescent microspheres are widelyused in many fields of medicine, biotechnology and nano-technology [1–6]. The micro-scale fluorescent beads areoften used as a control particle to calibrate the absolutenumber of specific lymphocytic cluster of differentiation(CD) cell [7]. Flow cytometry, an important tool in biologyto count and sort the cells invariably rely on the use offluorescent microspheres. Though there are many com-mercial sources for fluorescent beads, the cost, limitedchoice in bead size and fluorophore limit the experimentaldesign of a researcher. Commercial fluorescent beads areless sensitive at single bead level detection since they haveonly few copies of fluorophore. Developing efficient andself-help preparation with control on size, scale and degreeof fluorescence labeling will allow researchers to use in-house tailored beads for their experimental need.

The fluorescent microspheres are prepared by pigmen-tation method where microspheres are stained with a solu-

. All rights reserved.

tion of appropriate fluorescent dyes [3,7–10]. This methodhas advantages over others [11–13] such as bead size andshape control and less fluorescence quenching. But fluores-cence beads prepared by pigmentation method and othermethods suffer from low emission efficiency due to solu-bility issues of dyes in the staining medium. Herein wereport an efficient procedure for the preparation of fluores-cent microspheres of various sizes with high degree offluorescence labeling using gradual solvent evaporationmethod. Gradual solvent evaporation process leads tosuper saturation of dyes in the staining medium and shiftthe diffusion equilibrium towards to bead.[14] Our meth-odology provides high emission efficiency per bead and isscalable. And the fabricated fluorescent beads find manyapplications in flow cytometric analysis, multiplex analysisand so on.

2. Experimental

2.1. Materials

2,2-Azobis(isobutyronitrile) (AIBN) was purchasedfrom Tianjin chemical reagents Corp. Styrene (99%),

Q. Zhang et al. / European Polymer Journal 45 (2009) 550–556 551

Poly(vinylpyrrolidone) (PVP K-30, Mw = 40,000 g/mol),divinylbenzene (DVB, 80%) and sodium dodecyl sulfate(SDS) were purchased from Aldrich. Styrene was washedwith 1 M NaOH and deionized water to remove inhibitor,purified through vacuum distillation and stored at �20 �Cbefore use. DVB was washed with 1 M NaOH and deionizedwater to remove inhibitor, added anhydrous CaCl2 to re-move residual water. Benzoyl peroxide (BPO) and Rhoda-mine (R101) were purchased from Fluca. Acridine orange(AO) was purchased from Invitrogen. Distilled-deionized(DDI) water was used in all experiments. All other chemi-cals were of analytical grade. They were obtained from lo-cal suppliers and used as such without any furtherpurification.

2.2. Seed particles by dispersion polymerization

The monodisperse polystyrene (PS) seed particles weresubjected to dispersion polymerization method in ethanolmedium with PVP K-30 as stabilizer and AIBN as initiator[15–18]. The polymerization was carried out in a 150-mLfour-necked round-bottom flask (under argon atmosphere,kept 70 �C in water bath). 40 g ethanol solution with3.6 wt% of PVP K-30 was first poured into the flask. Afterthe temperature increased to 70 �C, 15 g styrene contain-ing 2 wt% AIBN was charged. After the polymerization,the spheres were obtained by centrifugation and repeat-edly washed by centrifugation to remove residual styreneand PVP for three times. After that, the spheres were driedunder vacuum at ambient temperature.

2.3. Cross-linked beads by seed polymerization

The seed polymerization was also carried out in a four-necked glass reactor under argon atmosphere [19–21]. Thestirring speed was fixed at 200 rpm throughout the reac-tion process. Firstly, the seed particles (0.1 g) were redi-spersed in 0.25% sodium dodecyl sulfate (SDS) aqueoussolution (30 mL) by sonication (10 min, 100 W), and theswelling agents, like cyclohexane (0.3 g) was emulsifiedby ultrasonic in 0.25% SDS solution (10 g). The mixture ofseed particles solution and swelling agent solution wasadded into the reactor, followed by stirring for 10 h at30 �C. And then, the mixture of styrene (9 g), DVB (1 g,10 wt% to styrene) and BPO (0.1 g, 1 wt% relative to styreneand DVB) was poured into the reactor which had beenemulsified in 100 g of 0.25% SDS solution by sonication.The swelling stage of monomer was hold on for another6 h at 30 �C. Finally, 2.5 wt% PVA aqueous solution (80 g)was added into the reactor. Thereafter, the polymerizationwas carried out at 80 �C for 10 h with copper chloride andsodium nitrite (0.01 g totally) as inhibitor. The beads wereobtained by repeated centrifugation and washing for threetimes. The cleaned beads were dried under vacuum at30 �C.

2.4. Staining of the microspheres

The cross-linked PS beads could be swollen by chloro-form and isopropanol mixture. 5 ml chloroform and isopro-panol mixture (50:50, v/v), 0.05 g tween-20, 0.1 g PS beads,

1–10 mg R101 and 1–10 mg AO were charged into a 25 mlbeaker, dispersed by ultrasonic (10 min, 100 W) to form ahomogeneous solution. And then, the mixture of bead waskept in vacuum drying oven lasted for about 12 hr (30 �C,vacuum degree around 0.1 MPa), which is so-called gradualsolvent evaporation method. After exhaustive evaporationof the solution, the stained beads were repeatedly centri-fuged and washed by alcohol for three times to remove theresidual fluorescent dyes.

2.5. Characterization

Scanning electron microscopy (SEM, XL-30, Philips Corp.)was used for observation of the morphology of PS particles.In order to calculate the number-average particle diame-ter-E(D) and coefficient of variation (Cv) of PS microspherediameter, about 100 individual particles (

Pni = 100) were

counted from SEM photographs with formula (1) and (2).The maximum absorption of fluorescent dye was deter-mined by ultraviolet (UV) spectrophotometer, and fluores-cence emission spectrum was measured by fluorescencespectrophotometer. Fluorescence microscope (Olympus,CX-51) was used to visualize fluorescent beads. Flow cytom-etry was used to determine FSCH signal (forward light scat-ter, representing the size of beads), SSCH signal (side lightscatter, showing the interior details of beads), FL1, FL2 andFL3 fluorescent signals, simultaneously. The transmittedwavelength of filters in FL1, FL2 and FL3 channel are530 ± 15 nm, 582 ± 21 nm (bandpass filter) and >670 nm(longpass filter), respectively.

EðDÞ ¼P

nidiPni

ð1Þ

Cv ¼ðPðdi � EðDÞÞ2=

PniÞ1=2

EðDÞ � 100

¼ ðPðdi � ð

Pnidi=

PniÞÞ2=

PniÞ1=2

Pnidi=

Pni

� 100 ð2Þ

where ni is the number of particles with di.

3. Results and discussion

3.1. Cross-linked PS beads by seed polymerization

It has been shown that the complete separation ofnucleation and growth is critical for the successful synthe-sis of monodisperse particles, in other words, the inhibi-tion of additional secondary nucleation during particlesize growth is essential [22,23]. In seed polymerization,the monodisperse seed particle is previously synthesizedby other methods, such as dispersion polymerization andsoap-free emulsion polymerization, and then the reactionof particle size growth is mainly taken place at the seedparticle. It means that the nucleation (seed particle) andgrowth (seed polymerization) process is totally separated.Hence, the bead with narrow distribution can be easily fab-ricated by this method. As shown in Figs. 1 and 2, PS seedparticles (Fig. 1a) of 2.35 lm were synthesized by disper-sion polymerization with Cv less than 2.1%. Along withthe increase of seed particle size from 2.35 lm to3.07 lm, the size of final obtained bead was increased from

Fig. 1. The SEM microphotographs of (a) seed particle synthesized bydispersion polymerization (�5000) and (b) the final bead (�2000)synthesized by the microsphere in picture 1a as seed particle and 1 wt%DVB as cross linker.

Fig. 2. The SEM microphotographs of (a) seed particle with a diameter of3.07 lm (�5000) and (b) PS bead synthesized by seed polymerizationwith the microspheres in Fig. 2a as seed particle and 10 wt% DVB as crosslinker (�1000).

552 Q. Zhang et al. / European Polymer Journal 45 (2009) 550–556

6.34 lm (Fig. 1b) to 9.2 lm (Fig. 2b). Other sized micro-spheres were also available by varying seed particle diam-eter and proportion of swelling agents.

The polymer bead should be cross-linked to avoid con-glutination in staining process. Furthermore, the highcross-linked structure is contributed to resist diffusion ofdye molecules from polymer matrix to liquid in storage.However, the microsphere with high cross-linked structureis difficult to be swollen. Hence, it is hard to be stained dueto high diffusion resistance (Supplementary informationFig. s1). And the proper particle size suitable for flow cyto-metric analysis is around 10 lm due to visibility and dyesloading capability. The bead with bigger size should havehigher fluorescent dye loading capability, but this kind ofbead led to a higher value of SSCH in flow cytometric anal-ysis, resulting in a relative decrease of cell signal. It meansthat the cell signal may not be sensitive enough to analysis.Hence, in this work, the beads of 9.2 lm (Fig. 2b) wereused in the following pigmentation process.

3.2. The gradual solvent evaporation method

In order to fabricate highly bright fluorescent bead, thegradual solvent evaporation method was utilized to form a

high concentration (even super saturation) of fluorescentdyes solution in staining process [14]. It was critical toobtain bright fluorescence bead. For the high solubility offluorescent dyes in the mixture of chloroform and isopro-panol, it’s hard to drive the fluorescent dyes to diffusefrom solution to polymer matrix, resulting in difficulty offabricating fluorescent bead with high intensity for FCManalysis application [24]. As shown in Fig. 4a1–a5, the fluo-rescent microspheres synthesized by traditional stainingmethod emitted very weak light due to few copies of fluo-rophore absorbed by the beads.

The cross-linked PS beads can be easily swollen by thechloroform solvent, and the extent of swelling is deter-mined by the concentration of chloroform. At higher con-centration of chloroform, the cross-linked beads areswollen to be much bigger, which means the dyes canmore easily diffuse from medium to polymer matrix. Sincethe component of chloroform and isopropanol azeotrope is95.8% chloroform and 4.2% isopropanol, there is no con-stant boiling point in the 50% chloroform solution. Afterthe uniform mixture is placed into vacuum atmosphere,the chloroform is easier to be evaporated for lower boilingpoint, resulting in gradual increase of the concentration ofisopropanol and fluorescent dyes. As illuminated in Fig. 3,

SolventEvaporation

BeadsShrinkage

:PS beads :AO :R101

Fig. 3. Schematic diagram of gradual solvent evaporation method.

Fig. 4. Microphotographs of fluorescent microspheres (�400, scale bar is 20 lm). a1–a5 the beads synthesized by traditional method. b1–b5 the beadslabeled by single fluorescent dye-AO and dispersed by stirring, c1–c5 the beads labeled by R101 and dispersed by stirring, d1–d5 the beads labeled by AO andR101 with a ratio of 1:1 and dispersed by sonication at high concentration (98%) of chloroform, e1–e5 the beads labeled by AO and R101 with a ratio of 2:1and dispersed by sonication. a1–e1:bright field; The excitation light of other pictures was 350–380 nm (a2–e2), 420–480 nm (a3–e3), 520–550 nm (a4–e4)and 545–580 nm (a5–e5), respectively; the filter were >420 nm (a2–e2), >520 nm (a3–e3), >580 nm (a4–e4) and >610 nm (a5–e5), respectively. (Forinterpretation of color mentioned in this figure the reader is referred to the web version of the article.)

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with the evaporation of chloroform (faster) and isopropa-nol (slower, compared with chloroform), the concentrationof fluorescent dyes is increased and the bead is shrunk. The

dye molecules are exerted to diffuse from solution to beadand retained in polymer matrix with the shrinkage of bead.After totally evaporation of chloroform, only a little of

Table 1The fluorescence intensity of various kinds of beads detected by flow cytometry.

Signal Beads in Fig. 5a Beads in Fig. 5b Commercial fluorescent bead in Fig. 5c

Events Y mean X mean Y mean X mean Y mean X mean

SSCH vs. FL1 5 – – 153.68 7969.67 168.87 3221.51SSCH vs. FL2 288 77.19 1591.15 153.57 6480.14 170.32 6201.37SSCH vs. FL3 246 76.29 6019.02 153.43 7640.30 168.20 3925.97

Fig. 5. Flow cytometric analysis diagrams at kE of 488 nm in PBS buffer. a1–a4 the beads in Fig. 4-d. b1–b4 the beads in Fig. 4e. c1–c4 the commercialfluorescent beads (flow cytometry absolute count standard, Bangs Laboratories, Inc, Cat. No. 580) a1–c1: SSCH vs. FSCH, a2–c2: SSCH vs. FL1, a3–c3: SSCH vs.FL2, a4–c4: SSCH vs. FL3.

554 Q. Zhang et al. / European Polymer Journal 45 (2009) 550–556

residual solution is left which will be exhausted in the nextfew hours (total evaporation time is 12 h), thereafter, thehighly bright fluorescent beads are obtained. ComparedFig. 4e with Fig. 4a (Fig. 4a as negative control), the fluores-cence intensity increased a lot. And as shown in Table 1(Fig. 5b), the fluorescence intensities of FL1, FL2 and FL3were 7969, 6480 and 7640, respectively. The chart ofFSCH–SSCH (Fig. 5b1) showed the uniform dynamics sizedistribution and light scatter signal of fabricated fluores-cent bead. Compared with the corresponding signals ofcommercial bead, the fluorescent intensity was strongerthan those of commercial bead. And the SSCH and FSCHsignal had a similar property as commercial one (the fluo-rescent pictures of commercial bead were shown as Fig. s2in Supplementary information). In flow cytometric applica-tion, the signals (SSCH vs. FL1, FL2 or FL3) of beads shouldbe appeared at the upper right corner of diagram in order

to leave enough analysis space for cell’s signals. It meansthat the higher fluorescence intensity of bead is a betterbenefit. And a narrow distribution is also required to de-crease the space occupied by fluorescent bead.

The total volume of solution and initial concentration ofchloroform play an important role in the formation of ahigh concentration of fluorescent dyes in evaporation pro-cess. The total volume of mixture should not be over 10 mLin order to form a high concentration of dyes in final stage.The cross-linked bead can not be efficiently swollen bylower concentration of chloroform, but can be over dis-solved linear polymer resulting in formation of nanoporesin the inner of matrix, surface erosion and adhesion of twobeads. As shown in Fig. 4d, the beads adhered with eachother to form doublets when high concentration (98%) ofchloroform solution was used. The reason might be thatthe surface of beads became very soft at high chloroform

AO

Mixture

Wavelength (nm)

R101( )λE=565nmR101( )λE=565nm

317

215

113

11478nm 554.7nm 631.3nm 708nm

R101

).u.a(ytisnetnI

ecnecseroulF

Fig. 6. Spectra of R101 and AO in isopropanol solution. (a) Absorptionspectrum measured from 350 to 700 nm with a resolution of 1 nm. Theratio of AO to R101 in 1–5 is 6:0, 6:1, 1.5:1, 1.5:4 and 0:4, respectively (b)Fluorescence emission spectrum resolved with a bandwidth of 1 nm from400 to 700 nm (the excitation wavelength is 490 nm if not mentioned).(For interpretation of color mentioned in this figure the reader is referredto the web version of the article.)

Q. Zhang et al. / European Polymer Journal 45 (2009) 550–556 555

concentration, resulting in exchange part of polymer ma-trix between beads during swelling. As shown in Fig. 5a,the fluorescent beads containing doublets broadened SSCHsignal and FSCH signal, consequently disturbing the FL1,FL2 and FL3 signals in FCM analysis.

The morphology of bead is also impacted by the disper-sion method. As shown in Fig. 4b1–b5, c1–c5, part of beadwas fragmental when stirring dispersion method was uti-lized. For the swollen beads were fragile, when the beadswere sheared or crashed by the magnetic stirrer, the beadsmight be twisted, broken and cut into pieces. Fortunately,as shown in Fig. 4d1–d5, e1–e5, the morphology of beadwas complete during sonication dispersion. The resultshowed the flow excited by ultrasonic wave was not strongenough to break the polymer network even if the bead hadbeen softened.

3.3. The fluorescent properties of beads

The multicolor beads need to be labeled by a seriesof fluorescent dyes which are carefully selected basedon excitation energy transfer. Multiple fluorescent dyescomposed of two or more fluorescent dyes should haveoverlapping emission and absorption spectra. It allows effi-cient energy transfer from the emission wavelength of thefirst dye in the series, transferred through the dyes in theseries and re-emitted as an optical signal at the absorptionwavelength of last dye, resulting in a desired effectiveStokes shift for the multiple dyes that is controlled throughselection of appropriate dyes. The multiple fluorescentbeads are available by staining polymer bead with properproportion of multiple dyes. This strategy enables a muchboarder emission wavelength of bead, which makes mul-ti-signals analysis possible in flow cytometric analysis[24–26].

As it is shown in Fig. 6, excitation wavelength kE andfluorescence emission wavelength kF of AO are 490 nm,524 nm (blue line, in Fig. 6b) in isopropanol medium,respectively. As for R101, the values of kE and kF are564.5 nm and 592 nm (red line, in Fig. 6b), respectively.The fluorescent beads are partial to mono-color sincemono-fluorescent dye is used. As shown in Fig. 4, the beadslabeled by AO were excited to bright green light (Fig. 4b3),while the beads stained by R101 were excited to brightred–yellow light (Fig. 4c4 and c5), correspondingly.

In Fig. 6a, there was a slightly mutual influence ofabsorption spectrum between AO and R101. Compared(a)-3 with (a)-4, the intensity of AO was increased a littlefor R101 had a low absorption at absorption spectrum ofAO. When the ratio of AO to R101 was 1.5:1, the maximumabsorption of AO was equal to that of R101. In Fig. 6b, themaximum emission wavelength of R101 was very low at kE

of 490 nm due to low excitation efficiency. However, whenR101 was mixed with AO, the emission peak of R101 at592 nm was strengthened for nearly ten times. To the con-trast, the maximum AO emission was declined by approx-imately 35%.

The optimum ratio of AO to R101 for energy transfer inisopropanol medium may not be the optimum one for pig-mentation, due to the different partition coefficient of dyesbetween polymer matrix and liquid phase. In fact, R101

has a relative lower solubility than AO. it can be more eas-ily absorbed by beads. As shown in Fig. 4e, when the beadstained by dual fluorescent dyes (AO and R101) with a ratioof 2:1, more colors were observed for broader emissionspectrum. The beads were excited to bright yellow(Fig. 4e2), bright green (Fig. 4e3), bright red (Fig. 4e4) andscarlet (Fig. 4e5) lights, respectively. Moreover, in Fig. 5b,the signals of FL1 (Fig. 5b2), FL2 (Fig. 5b3) and FL3(Fig. 5b4) can be detected simultaneously, due to the effi-cient energy transfer from AO to R101 at kE of 488 nm.However, as shown in Fig. 5a (or Fig. 4-d), when muchmore R101 was used (the proportion was 1:1), R101 ab-sorbed most of emission light of AO, and the residual greenlight was too weak to be observed. It’s the reason why thecolors of beads in all pictures were red or red-yellow. Cor-respondingly, the FL1 (Fig. 5a2) signal was very weakwhich could not be separated from the background, theFL2 (Fig. 5a3) signal was not very strong and the FL3 signal(Fig. 5a4) was very bright (intensity is 6019). Hence, theoptimum ratio of AO to R101 was approximate 2:1 in orderto obtain a broader emission wavelength.

556 Q. Zhang et al. / European Polymer Journal 45 (2009) 550–556

4. Conclusions

The highly monodisperse fluorescent polystyrenemicrospheres were fabricated by first synthesizingcross-linked beads of 9.2 lm and then staining the blankPS beads with fluorescent dyes. The production with highfluorescence intensity and uniform distribution was suffi-ciently satisfied the requirement of flow cytometric anal-ysis, having potential application of fluorescentcalibration and immunoassay reagents. Furthermore, anew staining method-gradual solvent evaporation meth-od was developed to fabricate a high fluorescent inten-sity beads. It’s the key of this method that theevaporation speed and ratio of swelling agent to mediumshould be carefully selected in order to form a high con-centration of fluorescent dyes. This method could be ex-tended to fabricate other kind of fluorescent polymerbeads in fluorescent analysis field.

Acknowledgment

This work was supported by the key project of NationalHigh Technology Program (No:2007AA021808).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.eurpolymj.2008.11.016.

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