Preparation of polystyrene fluorescent microspheres based on some fluorescent labels

Article abstract of DOI:10.1039/b816963b

Five fluorescent labels were synthesized and used in the preparation of polystyrene fluorescent microspheres by the dispersion copolymerization and absorption method. Spectral properties of copolymerization fluorescent microspheres in tetrahydrofuran indicated these individual characteristics of labels should be maintained in the fluorescent microspheres. The differences of the fluorescent spectra between five fluorescent microspheres and their corresponding parent labels in ethanol have been investigated. These fluorescent microspheres were characterized by environmental scanning electron microscopy, laser scanning confocal microscopy and fluorescence spectrophotometry. They exhibited good dispersion and stable and high fluorescence intensity. Furthermore, copolymerization fluorescent microspheres were functionalized with amino groups. This means that a method for the preparation of copolymerization fluorescent microspheres was developed as a platform for the generation of functional fluorescent microspheres for diverse applications. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b816963b

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PAPER
www.rsc.org/materials | Journal of Materials Chemistry
Preparation of polystyrene fluorescent microspheres based on some
fluorescent labels
Qing-Hao Liu,a Jia Liu,b Jin-Chun Guo,c Xi-Long Yan,a Dong-Hua Wang,b Lei Chen,b Fan-Yong Yand
a
*
and Li-Gong Chen
Received 29th September 2008, Accepted 6th January 2009
First published as an Advance Article on the web 11th February 2009
DOI: 10.1039/b816963b
Five fluorescent labels were synthesized and used in the preparation of polystyrene fluorescent
microspheres by the dispersion copolymerization and absorption method. Spectral properties of
copolymerization fluorescent microspheres in tetrahydrofuran indicated these individual characteristics
of labels should be maintained in the fluorescent microspheres. The differences of the fluorescent
spectra between five fluorescent microspheres and their corresponding parent labels in ethanol have
been investigated. These fluorescent microspheres were characterized by environmental scanning
electron microscopy, laser scanning confocal microscopy and fluorescence spectrophotometry. They
exhibited good dispersion and stable and high fluorescence intensity. Furthermore, copolymerization
fluorescent microspheres were functionalized with amino groups. This means that a method for the
preparation of copolymerization fluorescent microspheres was developed as a platform for the
generation of functional fluorescent microspheres for diverse applications.
microspheres were prepared by dispersion polymerization.25
Introduction
Moreover, unsymmetrical rhodafluor and pyrrolidinyl rhoda-
mine S were incorporated into polystyrene microspheres with
sulfonic groups to prepare fluorescent microspheres. Spectral
properties of copolymerization fluorescent microspheres in
tetrahydrofuran were studied. And the differences of the fluo-
rescent spectra between five fluorescent microspheres and their
corresponding parent labels in ethanol had been investigated,
respectively. These fluorescent microspheres were observed by
environmental scanning electron microscopy, laser scanning
confocal microscopey and fluorescence spectrophotometry.
Furthermore, the introduction of amino groups to copolymeri-
zation fluorescent microspheres was carried out using silane
coupling reagents with vinyl, amido and ethoxy by a sol–gel
process.
Fluorescent microspheres are widely used in the standarization
of fluorescent-based instruments as well as the detection and
analysis of biological and synthetic molecules.1,2 Surface func-
tionalized fluorescent microspheres in particular have received
much attention because of their present and foreseen applica-
tions in the biomedical field.3–5 However, the application field
of fluorescent microspheres has been limited by their difficult
preparation, limited visibility, tendency to clump together and
background fluorescence. Thus, a number of fluorescent micro-
spheres designed to overcome these problems have been reported
recently.1,6–11
Normal fluorescent labels, rhodamine, fluorescein and Nile
red, are often used to prepare fluorescent spheres.12–16 Rhoda-
mine and fluorescein have good biocompatibility, high quantum
yields and large extinction coefficients.17–20 Nile red is well-
known as an environment-sensitive fluorescent probe for label-
ling and sensing biomolecules.21,22 In addition, micro-size
polystyrenes have high active surface areas, and excellent
hydrophobicities and affinities, which are widely used in
biological medicine and colloidal science.23,24
Experimental
1H NMR and 13C NMR spectra were recorded on an INOVA 500
and BRUKER AV400, respectively. High resolution mass
spectra (HRMS) were determined on an IonSpec 7.0T FT-ICR
mass spectrometer. UV-vis absorption spectra were measured on
a computer-controlled Shimadzu UV-2450 spectrometer. Fluo-
rescence spectra were recorded at room temperature on a Cary
Eclipser fluorescence spectrometer. The F measurement of dyes
was performed on an Edinburgh FLS920 spectrometer.
In this paper, we synthesized allyl rhodamine B, allyl Nile red,
allyl fluorescein, unsymmetrical rhodafluor and pyrrolidinyl
rhodamine S for the preparation of polystyrene-based fluores-
cent microspheres. Three kinds of copolymerization fluorescent
The average diameter and size distribution of microspheres
were measured using a Brookhaven Particle Size Analyzer (BI-90
plus). The environmental scanning electron images (ESEMs)
were obtained using a XL30 environmental scanning electron
microscope. The confocal microscopic images were obtained
using an OLYMPUS FV1000 laser scanning confocal micro-
scope. FT-IR spectroscopy was performed on a NICOLET
380 Fourier transform infrared spectrometer.
aSchool of Chemical Engineering and Technology, Tianjin University,
Tianjin, 300072, China. E-mail: lgchen@tju.edu.cn; Fax: +86 22
27406314; Tel: +86 22 27406314
bSchool of Pharmaceuticals and Biotechnology, Tianjin University, Tianjin,
300072, China
cQinghai Institute of Salt Lakes, Chinese Academy of Sciences, Xining,
810008, China
dSchool of Material Science and Chemical Engineering, Tianjin
Polytechnic University, Tianjin, 300160, China
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All of the chemicals used in this study were of analytical grade
or purified according to standard procedures. Two kinds of
polystyrene microspheres with diameters of 1.81 ꢀ 0.03 and 2.23
ꢀ 0.02 mm were purchased from Tianjin Baseline ChroTech
Research Centre (China).
7.5, 1.0 Hz, 1H), 7.58 (t, J ¼ 7.5 Hz, 1H), 7.66 (t, J ¼ 7.5 Hz, 1H),
8.03 (d, J ¼ 8.0 Hz, 1H).
Unsymmetrical rhodafluor 5. Compound 4 (0.50 g, 1.6 mmol)
was added to a solution of resorcinol (0.20 g, 1.8 mmol) in 10 mL
methanesulfonic acid. The resulting suspension was heated under
N2 at 85 ꢁC for 14 h. The mixture was poured into 50 volumes of
ice water, and then neutralized with saturated aqueous Na2CO3,
followed by filtration. The cake was dried to give crude product.
The title compound was separated by column chromatography
on silica (0.31 g, 73.8%) (methanol/dichloromethane ¼ 1 : 20, Rf
¼ 0.2). 1H NMR (400 MHz, CDCl3): d 1.12 (t, J ¼ 7.2 Hz, 6H),
3.60 (q, J ¼ 7.2 Hz, 4H), 6.31 (d, J ¼ 2.2 Hz, 2H), 6.62 (d, J ¼ 7.4
Hz, 2H), 6.89 (d, J ¼ 2.4 Hz, 1H), 7.11 (d, J ¼ 9.6 Hz, 1H), 7.32–
7.35 (m, 1H), 7.73–7.76 (m, 2H), 8.17 (d, J ¼ 7.6 Hz, 1H).
Synthesis of fluorescent labels
Allyl rhodamine B 126. A mixture of rhodamine B (2.40 g, 5.0
mmol), allyl bromide (0.73 g, 6.0 mmol), Na2CO3 (2.65 g, 25.0
mmol), hydroquinone and iodine (trace), and dry DMF (50 mL)
ꢁ
was heated and stirred at 71 C in the dark under N2 for 25 h.
Vacuum evaporation of the solvent yielded the crude product. The
title compound was isolated by column chromatography on silica
(2.00 g, 77.5%) (methanol/dichloromethane ¼ 1 : 40, Rf ¼ 0.2).
1H NMR (500 MHz, CD3OD): d 1.31 (t, J ¼ 7.0 Hz, 12H), 3.69
(q, J ¼ 7.0 Hz, 8H), 4.46 (d, J ¼ 8.5 Hz, 2H), 5.05–5.12 (m, 2H),
5.59–5.66 (m, 1H), 7.00 (d, J ¼ 2.5 Hz, 2H), 7.05 (dd, J ¼ 9.5, 2.5
Hz, 2H), 7.11 (d, J ¼ 9.0 Hz, 2H), 7.46 (d, J ¼ 7.5 Hz, 1H),
7.82 (t, J ¼ 8.0 Hz, 1H), 7.89 (t, J ¼ 7.5 Hz, 1H), 8.32 (d, J ¼
8.0 Hz, 1H).
Unsymmetrical rhodafluor 6. Thionyl chloride (0.55 mL) was
added to a solution of compound 5 (0.60 g, 1.5 mmol) in
ꢁ
n-butanol (5 mL) at 0 C. The mixture was stirred for 1 h, and
then heated at 70 ꢁC for 7 h. The solvent was evaporated under
reduced pressure. The residual material was purified by column
chromatography on silica to afford compound 6 (0.62 g, 90.0%)
(methanol/dichloromethane ¼ 1 : 80, Rf ¼ 0.3).
Allyl fluorescein 227. A mixture of fluorescein (2.00 g,
6.0 mmol), allyl bromide (2.42 g, 20.0 mmol), K2CO3 (4.97 g,
36.0 mmol), hydroquinone and iodine (trace), and dry DMF
(60 mL) was heated and stirred at 71 ꢁC in the dark under N2 for
25 h. The solvent was removed under reduced pressure. The
crude product was recrystallized from carbon tetrachloride, and
the resulting product was separated by column chromatography
on silica (0.91 g, 40.5%) (dichloromethane, Rf ¼ 0.1).
1H NMR (500 MHz, CD3OD): d 0.72 (t, J ¼ 7.25 Hz, 3H),
0.96–1.04 (m, 2H), 1.18–1.2 (m, 2H), 1.22 (t, J ¼ 7.0 Hz, 6H),
3.46 (q, J ¼ 7.0 Hz, 4H), 3.93 (t, J ¼ 6.5 Hz, 2H), 6.54–6.61 (m,
4H), 6.83–6.86 (m, 2H), 7.24–7.27 (m, 1H), 7.63 (t, J ¼ 7.5 Hz,
1H), 7.68 (t, J ¼ 7.5 Hz, 1H), 8.20 (d, J ¼ 7.5 Hz, 1H).
13C NMR (400 MHz, CD3OD): d 184.93, 167.22, 160.95,
158.01, 157.36, 155.44, 135.20, 133.82, 132.21, 132.14, 132.09,
131.68, 131.56, 131.12, 126.49, 115.02, 113.43, 112.92, 105.01,
97.27, 65.52, 46.38, 31.50, 20.20, 14.09, 12.87.
1H NMR (500 MHz, CDCl3): d 4.46 (d, J ¼ 6.5 Hz, 2H), 4.69
(d, J ¼ 4.5 Hz, 2H), 5.09–5.12 (m, 2H), 5.37–5.49 (m, 2H), 5.56–
5.62 (m, 1H), 6.03–6.09 (m, 1H), 6.72 (d, J ¼ 9.5 Hz, 2H), 6.84 (d,
J ¼ 8.0 Hz, 1H), 6.95–7.03 (m, 3H), 7.32–7.34 (m, 1H), 7.69–7.76
(m, 2H), 8.28 (d, J ¼ 7.0 Hz, 1H).
HRMS (ESI) C28H29O4N: calcd. 444.2169; found m/z
444.2172 [M]+.
Pyrrolidinyl rhodamine S 7
Allyl Nile red 3. Allyl Nile red was synthesized according to the
method of Martin-Brown et al.28 in the yield of 19.3%.
1H NMR (500 MHz, DMSO-d6): d 1.23 (t, J ¼ 8.5 Hz, 6H),
3.46 (q, J ¼ 7.0 Hz, 4H), 4.74 (d, J ¼ 5.5 Hz, 2H), 5.30 (dd, J ¼
10.5, 1.5 Hz, 1H), 5.45 (dd, J ¼ 17.5, 1.5 Hz, 1H), 6.06–6.11 (m,
1H), 6.20 (s, 1H), 6.64 (s, 1H), 6.82–6.85 (m, 1H), 7.27 (dd, J ¼
8.5, 2.5 Hz, 1H), 7.61 (d, J ¼ 9.0 Hz, 1H), 7.95 (s, 1H), 8.03 (d,
J ¼ 9.0 Hz, 1H).
3-Pyrrolidinylphenol was prepared according to the method of
Lee et al.29 in the yield of 58.7%.
1H NMR (500 MHz, DMSO-d6): d 1.89–1.92 (m, 4H), 3.12–
3.16 (m, 4H), 5.91 (t, J ¼ 2.0 Hz, 1H), 5.95–6.02 (m, 2H), 6.88 (t,
J ¼ 8.0 Hz, 1H), 8.94 (s, 1H).
The mixture of 3-pyrrolidinylphenol (0.49 g, 3.0 mmol),
crushed succinic anhydride (0.35 g, 3.5 mmol) and ZnCl2 (0.47 g,
3.5 mmol) was heated at 160 ꢁC under N2 for 4 h. After cooling,
the solid was pulverized and diluted with 2.5 mL of ethanol.
4.6 mL of HClO4 (60%) and 92 mL of water were added
Unsymmetrical rhodafluor 6
2-Carboxyl-40-diethylamino-20-hydroxy benzophenone 4. 3-
Diethylamino phenol (3.30 g, 20.0 mmol) and phthalic anhydride
(3.00 g, 21.0 mmol) in toluene (20 mL) were refluxed under
nitrogen for 4 h. The mixture was cooled to 50–60 ꢁC. 20 mLꢁof
35% aqueous NaOH (w/w) was added and then heated at 90 C
for 6 h. The resulting mixture was poured into 200 mL of H2O,
acidified with HCl (10.0 mol Lꢂ1), and allowed to stand at room
temperature for 2 h. The suspension was filtered, and the solid
was recrystallized from the mixture of water and methanol, then
dried to afford the desired product (4.75 g, 73.1%).
ꢁ
consecutively to the solution, which was kept at 5 C for 72 h.
The precipitate was collected and dried. The crude product was
isolated by column chromatography on silica (0.33 g, 55.9%)
(methanol/dichloromethane ¼ 1 : 100, Rf ¼ 0.2).
1H NMR (500 MHz, DMSO-d6): d 2.02 (t, J ¼ 6.5 Hz, 8H),
2.49 (t, J ¼ 2.0 Hz, 2H), 2.59 (t, J ¼ 7.5 Hz, 2H), 3.54 (t, J ¼
7.5 Hz, 8H), 6.62 (d, J ¼ 2.5 Hz, 2H), 7.0 (dd, J ¼ 9.5, 2.0 Hz,
2H), 8.05 (d, J ¼ 9.5 Hz, 2H).
13C NMR (400 MHz, DMSO-d6): d 172.60, 156.35, 153.96,
129.57, 115.07, 112.26, 96.24, 48.62, 34.81, 24.71.
HRMS (ESI) C24H27O3N2: calcd. 391.2016; found m/z
391.2010 [M]+.
1H NMR (500 MHz, CD3OD: DMSO-d6 ¼ 5 : 1): d 1.15 (t, J ¼
7.0 Hz, 6H), 3.40 (q, J ¼ 7.0 Hz, 4H), 6.09 (d, J ¼ 2.5 Hz, 1H),
6.15 (d, J ¼ 9.0 Hz, 1H), 6.84 (d, J ¼ 9.5 Hz, 1H), 7.35 (dd, J ¼
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Preparation of polystyrene fluorescent microspheres by the
dispersion copolymerization30–35
An appropriate amount of allyl rhodamine B, or allyl fluorescein
or allyl Nile red, styrene (910 mg), 2,20-azobis(isobutyronitrile)
(AIBN, 4.20 mg) and polyvinylpyrrolidone (PVP-K30, 160 mg)
were dissolved in ethanol (3.6 mL) and 2-methoxyethanol
(2.4 mL) or in ethanol (5.4 mL) and water (1.68 mL). The sealed
reaction vessels were immersed into a thermostated water bath
ꢁ
and set to rotate for 48 h under N2 at 70 C. The solution was
poured into ethanol to precipitate and filter fluorescent micro-
spheres. They were washed with ethanol and water until no free
dye was removed from microspheres, and then dried under
vacuum at 50 ꢁC .
Preparation of fluorescent microspheres with amino groups36
The above fluorescent microspheres (200 mg), triethoxy-
vinylsilane (50 mL) and 2,20-azobis(isobutyronitrile) (AIBN, 4.00
mg) were added to ethanol (8 mL) and water (1.6 mL). The sealed
flask was immersed and set to rotate in a water bath at 70 ꢁC for
24 h. The microspheres were rinsed and centrifuged to remove
unreacted reagents and contaminants.
The microspheres were again suspended in the mixture of
ethanol (8 mL) and water (1.6 mL) in the presence of ammonia
(40 mL) and tetraethoxysilane (50 mL) and stirred for 30 h at
room temperature.
After being washed several times, the microspheres were
redispersed in ethanol (8 mL) and water (1.6 mL). Ammonia
(40 mL) and g-aminopropyltrioethoxysilane (50 mL) were added
to the reaction, which was stirred for 30 h at room temperature.
The modified microspheres were rinsed and centrifuged with
ꢁ
ethanol, and then dried at 50 C under vaccum.
Preparation of polystyrene fluorescent microspheres with sulfonic
sodium
Polystyrene microspheres were sulfonated by sulfuric acid.
Polystyrene microspheres with sulfonic groups in 2 mol Lꢂ1
aqueous NaOH were stirred at room temperature for 2 h and
rinsed to neutralization.37 A solution of compound 6 or 7 dis-
solved in ethanol was added to the vigorously stirred suspension
of the corresponding polystyrene microspheres with sulfonic
sodium in ethanol and water (v : v ¼ 1 : 1). The resulting
suspension was filtered and dialyzed in ethanol to remove any
residual dye.
Results and discussion
Syntheses and photophysical properties of fluorescent labels
As shown in Scheme 1, simple chemical reactions yielded the
target compounds. Commercially available reagents were used
throughout and the overall preparative yields of compounds at
the milligram level were satisfactory.
Photophysical properties of five fluorescent labels and their
parent dyes are summarized in Table 1. The absorption
maximums of 2 are 455 and 473 nm in 0.1 mol Lꢂ1 NaOH. They
are close to the absorption maximums (459 and 483 nm) of
1-[2-(6-methoxy-3-oxo-3H-xanthen-9-yl)-benzoyl]-piperidine-4-
Scheme 1 Synthetic scheme for fluorescent labels 1, 2, 3, 6, 7.
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Table 1 Photophysical properties of five labels and their parent dyes
RhB
7.85
1
2
Flu
8.23
3
Nile red
6
7
3/mol cmꢂ1 L ꢃ 104
lab/nm
6.85
2.54
1.60
3.40
542
3.52
2.39
548
555
542
455d
473
455
520
80
489
543
525d
490
500
553
35
lex/nm
lem/nm
500
586
33
500
579
32
455
534
39
543
629
57
543
627
61
500
573
30
eFWHM/nm
F (%)
37.4a
50.2a
41.8b
72.9b
46.7c
35.9c
80.8c
83.5c
a
1, rhodamine B, 6 and 7 were excited at 500 nm in ethanol. b 2 and fluorescein were excited at 455 nm in 0.1 mol Lꢂ1 NaOH. c 3 and Nile red were
excited at 543 nm in ethanol. d labmax nmꢂ1 which represents lab of the maximal absorption value. F denotes the fluorescent quantum yield. e FWHM
denotes full width at half-maximum.
Table 2 The chromophore contents of fluorescent microspheresa
a1
b2
c3
d6
e7
(% w/w) 2.377 ꢃ 10ꢂ3 1.541 ꢃ 10ꢂ2 8.498 ꢃ 10ꢂ3 1.214 1.458
a
a1, b2, c3, d6, e7 denote fluorescent microspheres of 1, 2, 3, 6, 7,
respectively.
corresponding solvent, respectively. The amount of residual dye
was determined, using as references the maximum-absorption
coefficients of the corresponding 6 and 7 in the corresponding
solvent. The chromophore contents of a1, b2, c3, d6 and e7 are
listed in Table 2. The a1, b2 and c3 were obtained by copoly-
merization between dye and styrene, while d6 and e7 depend on
physical action to the absorbing dye. So contents of 1, 2 and 3 are
much lower than those of 6 and 7 in microspheres.
Fig. 1 Fluorescence and UV spectra of 2 in 0.1 mol Lꢂ1 NaOH (lex
¼
455 nm).
carboxylic acid reported by Gao et al.38 In Fig. 1, the inset shows
the UV spectrum of 2. The broadness of its UV spectrum
matches the broadness of its emission spectrum. In addition, the
absorption maximums of 6 are 490 and 525 nm. Following
comparisons with their parent dyes, the fluorescent spectra of 1, 2
and 3 produce red shifts with the attachment of the allyl
substituent.
The absorption and fluorescent properties of 1, 2, 3, a1, b2, c3
and the corresponding equimolar mixtures of 1, 2, 3 and PS in
tetrahydrofuran are listed in Table 3, respectively.26,40,41 Obvi-
ously, the absorption and fluorescent spectra of the corre-
sponding monomers and corresponding equimolar mixtures
have very similar features as those of the fluorescent micro-
spheres in tetrahydrofuran. This indicates that these individual
characteristics of 1, 2 and 3 should be maintained in the
fluorescent microspheres. It can also be suggested that spectral
properties of 1, 2 and 3 in tetrahydrofuran are not affected by the
aromatic environment, or the rigid nature of the PS matrix.42,43
The comparisions of the emission values of parent labels and
five microspheres dispersed in ethanol are listed in Table 4.
Undoubtedly, the spectral emissions of labels bonded or incor-
porated into microspheres were not greatly changed in compar-
ison with those of the labels.
The F measurement of dyes was performed on an Edinburgh
FLS920 spectrometer with an integrating-sphere attachment
under excitation of 500, 455 and 543 nm, respectively.39 The
quantum yields of 6 and 7 are higher than those of 1 and
rhodamine B under excitation of 500 nm in ethanol. The
quantum yield of 2 is lower than that of fluorescein under
excitation of 455 nm in 0.1 mol Lꢂ1 NaOH. The quantum yield of
3 is higher than that of Nile red under excitation of 543 nm in
ethanol.
Spectral properties of fluorescent microspheres
The chromophore contents of a1, b2 and c3 were determined by
spectroscopy of tetrahydrofuran solutions of the microspheres.
The maximum-absorption coefficients of the corresponding 1, 2
and 3 were used as references in the same solvent. However,
d6 and e7 cannot be dissolved in most organic solvents, such as
tetrahydrofuran and trichloromethane, because of the large
number of sulfonic groups of microspheres. Therefore, a certain
amount of 6 or 7 was added to the suspension of 100 mg of the
corresponding polystyrene microspheres with sulfonic sodium in
ethanol and water (v : v ¼ 1 : 1). And the resulting suspension
was filtered and dialyzed in ethanol to remove any residual dye.
Then the filtrate and dialysate were diluted to 50 mL with the
Table 3 lab/nm and lem/nm of 1, 2 and 3, fluorescent microspheres and
mixtures of 1, 2 or 3 and PS in THFa
1
2
3
a1
b2
c3
M1
559
M2
M3
524
lab
558
455
432
455
525
524
558
455
430
455
527
524
455
432
455
526
lex
lem
558
580
524
595
558
580
524
593
558
580
524
597
a
a1, b2, c3 denote fluorescent microspheres of 1, 2, 3, respectively. M1,
M2, and M3 are the equimolar mixture model systems containing 1, 2,
3 and PS corresponding to a1, b2 and c3, respectively.
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Table 5 lab/nm and lem/nm of 1, 2 and 3 in ethanol and toluene
Table 4 Comparison of lem/nm of parent labels and five microspheresa
1
a1
2
b2
3
c3
6
d6
7
e7
Ethanol
Toluene
lab
a
lem
a
Dyes
lab
lem
lab
lex
555
458
488
543
525
548
1
2
555
458b
488
578
515
564
590
533
555 555 458 458 543 543 525 525 548 548
460b
436
523
lem 578 595 515 555 629 606 546 560 566 586
Dl
17
40
23
14
20
3
543
629
595
a
a1, b2, c3, d6, e7 denote fluorescent microspheres of 1, 2, 3, 6, 7,
respectively. Labels (10ꢂ7 mol Lꢂ1 in ethanol) and fluorescent
microspheres (3% w/w dispersed in ethanol).
Excited at the absorption maxima of 1, 2 and 3 in ethanol. b labmax/nm
which represents lab of the maximal absorption value.
a
From Tables 4 and 5, the fluorescent spectra of 1 in toluene
and a1 in ethanol have a similar extent of red shift corresponding
to 1 in ethanol. This demonstrates that the shift of fluorescent
spectrum of a1 in ethanol is mainly due to its aromatic envi-
ronment. The fluorescent spectra of 2 in toluene and b2 in
ethanol have a different extent of red shift corresponding to 2 in
ethanol. This shows that the change of the fluorescence spectra of
As shown in Fig. 2, the fluorescent spectra of a1, b2, c3, d6 and
e7 produced different shifts in comparison with those of their
parent labels. In order to explain the shifts of the fluorescent
spectra of a1, b2 and c3, 1, 2 and 3 were dissolved in toluene,
which has a similar chemical composition to polystyrene, but is
a liquid. The absorption and fluorescence spectra of 1, 2 and 3 in
ethanol and toluene are tabulated in Table 5 and shown in Fig. 2.
Fig. 2 The emission spectra of labels in ethanol and toluene (1, 2 and 3) and corresponding microspheres dispersed in ethanol (a) 1, a1, rhodamine B
were excited at 555 nm in ethanol. 1 was excited at 555 nm in toluene. (b) 2, b2, fluorescein were excited at 458 nm in ethanol. 2 was excited at 458 nm in
toluene. (c) 3, c3, Nile red were excited at 543 nm in ethanol. 3 was excited at 543 nm in toluene. (d) 6, d6 were excited at 525 nm in ethanol. (e) 7, e7 were
excited at 548 nm in ethanol. a1, b2, c3, d6, e7 denote fluorescent microspheres of 1, 2, 3, 6, 7, respectively. Labels (10ꢂ7 mol Lꢂ1 in ethanol) and
fluorescent microspheres (3% w/w in ethanol).
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Finally, the fluorescent spectra of d6 and e7 offered red shifts
compared with those of 6 and 7 in ethanol, which are mainly due
to the hydrophobic environment of microspheres and the elec-
trostatic interactions between 6 or 7 and the sulfonic sodium of
microspheres.
Preparation of copolymerization fluorescent polystyrene
microspheres and fluorescent microspheres with amino groups
The average diameter and size distribution of microspheres are
given in Table 6.
It is found during the preparation of a1 that the size distri-
bution of microspheres was narrow when the ratio of 1 to styrene
is below 3 : 455, otherwise, the size distribution was wide as in
Fig. 4 and 5. It is highly probable that steric hindrance of the
rhodamine group and self-resisting polymerization of rhodamine
monomers prevent chain growth,26,44 which influences the
uniformity of the microspheres. This result has also confirmed
that the monomer of 1 had indeed been copolymerized with
styrene in spite of its low proportion. In addition, a1 with the
average diameter of 2.553 mm was prepared in the mixture of
ethanol and 2-methoxyethanol as in Fig. 4 (a). While the
microspheres of 1 with an average diameter of 1.128 mm were
prepared in aqueous ethanol as in Fig. 4 (b). Moreover, 1 does
not significantly change the net surface charge and hydrophobic
parking area of polystyrene microspheres owing to the small
amount of l.
Fig. 3 The emission spectra of 3 in different solvents (lex ¼ 510 nm).
b2 in ethanol is due to both the aromatic environment and the
rigid nature of the PS matrix. In addition, the spectral shift of 3
occurs when dissolved in different solvents22 as in Fig. 3 or
encaplused in hydrophobic microspheres as described by Haug-
land et al.1 The fluorescent spectra of 3 in toluene and c3 in
ethanol have a different extent of blue shift corresponding to 3 in
ethanol. This indicates that the fluorescent spectrum shift of c3 is
attributed to a hydrophobic environment, aromatic environment
and the rigid nature of the PS matrix.
Table 6 The average diameter and size distribution of microspheres
(in mm)a
The same phenomena occurred in the preparation of b2 and
c3. The ratio of 2 to styrene was only 1 : 910 to maintain
uniformity of the microspheres as in Fig. 6. However, b2 still has
a wider size distribution than those of the other two copoly-
merization microspheres, which is because of the two allyl groups
of 2 being involved in copolymerization. Only by keeping the
ratio of 3 to styrene as 1 : 1820 can the fluorescent microspheres
have good fluorescent intensity as shown in Fig. 7.
a1
b2
c3
d6
Blank (d6) e7
Blank (e7)
Mean 2.553 2.236 2.218 1.806 1.809
D
2.228 2.227
0.013 0.015
0.010 0.047 0.012 0.029 0.031
a
a1, b2, c3, d6, e7 denote fluorescent microspheres of 1, 2, 3, 6, 7,
respectively. Mean denotes average diameter. D denotes polydispersity
index. Blank (d6) and Blank (e7) denote blank microspheres of d6 and
e7 with sulfonic groups, respectively.
Fig. 4 ESEM images of fluorescent polystyrene microspheres of 1. (a) The ratio of 1 to styrene is below 3 : 455 (3000ꢃ) (fluorescent microspheres with
an average diameter of 2.553 mm); (b) the ratio of 1 to styrene is below 3 : 455 (10 000ꢃ) (fluorescent microspheres with an average diameter of 1.128 mm);
(c) the ratio of 1 to styrene is above 3 : 455 (6000ꢃ).
Fig. 5 Confocal microscopic images of fluorescent polystyrene microspheres of 1 (600ꢃ). (a) The fluorescent images; (b) their corresponding trans-
mission images (c) the overlays of fluorescent and transmission images. Confocal microscopy images were obtained with laser excitation at 555 nm.
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Fig. 6 Confocal microscopic images of fluorescent polystyrene microspheres of 2 (600ꢃ). (a) The fluorescent images; (b) their corresponding trans-
mission images; (c) the overlays of fluorescent and transmission images. Confocal microscopy images were obtained with laser excitation at 455 nm.
Fig. 7 Confocal microscopic images of fluorescent polystyrene microspheres of 3 (600ꢃ). (a) The fluorescent images; (b) their corresponding trans-
mission images; (c) the overlays of fluorescent and transmission images. Confocal microscopy images were obtained with laser excitation at 543 nm.
Fig. 8 The emission spectra of the different ratio of dye to styrene in THF. (a) The emission spectra of a1 (lex ¼ 558 nm); (b) the emission spectra of c3
(lex ¼ 524 nm).
As shown in Fig. 8, the fluorescent intensity of a1 and c3 was in
proportion to the ratio of 1 or 3 to styrene. There are no fluo-
rescence self quenching labels because of copolymerized with
styrene in low proportions.
In order to overcome the problem of no hydroxy group on
polystyrene microspheres, silanol groups were firstly attached to
the surface of fluorescent polystyrene microspheres by reacting
with triethoxyvinylsilane and tetraethoxysilane, respectively.
Amino groups were then introduced by reacting with g-amino-
propyltrioethoxysilane, which was confirmed by the IR. In the
range of 3448–3376 cmꢂ1, the absorption peak, which was the
Fig. 9 Conductometric titration curve of fluorescent microspheres of
characteristic one of amino groups, was weak and broad.
allyl rhodamine B coating with amino groups.
The amount of amino groups was determined by the
conductometric titration of the anti-titrimetric method due to
the amino groups being weak bases. As described in Fig. 9, the
microspheres with sulfonic groups. This indicated that absorbing
amount of amino groups attached on the surface of microspheres
dye had little effect on uniformity of microspheres.
was about 105 mmol gꢂ1
.
The amount of sulfonic groups of microspheres was about
114 mmol gꢂ1 determined by conductometric titration. There are
two actions involved in the absorption between microspheres and
labels, one is the hydrophobic action, the other is the ionic
action. The microspheres with sulfonic sodium have an excellent
Preparation of absorbing fluorescent microspheres
From Table 6, the average diameter and size distribution of d6
and e7 are in line with those of their corresponding blank
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J. Mater. Chem., 2009, 19, 2018–2025 | 2025