High-performance liquid chromatographic determination of triclosan

Article
Poly(styrene-acrylamide-acrylic
acid) copolymer fluorescent
microspheres with improved
hydrophilicity: preparation and
influence on protein immobilization
High Performance Polymers
23(3) 255–262
ª The Author(s) 2011
Reprints and permission:
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DOI: 10.1177/0954008310391824
hip.sagepub.com
Xinghua Pan, Jianhui Ju, Jianjun Li and Daocheng Wu
Abstract
Poly(styrene-acrylamide-acrylic acid) copolymer fluorescent microspheres (PSAAFMs) with improved surface
hydrophilicity were synthesized through an improved soap-free emulsion copolymerization method, in which the proportion of acrylamide on the surface of the microspheres was increased. Azidocarbonyl groups, which can be rapidly coupled
with proteins under mild conditions, were introduced onto the PSAAFMs using an azido reaction. The PSAAFMs were
characterized using a fluorescence microscope, an ultraviolet/visible spectrometer, a Fourier transforms infrared spectrometer, a transmission electron microscope (TEM), a size analyzer, and a fluorescence spectrophotometer. Furthermore,
covalent linking through the azidocarbonyl groups and physical nonspecific attachments of bovine serum albumin (BSA),
trypsin, and human chorionic gonadotropin (HCG) onto the surface of the microspheres were also determined to evaluate the influence of improved surface hydrophilicity on nonspecific protein adsorption. Results from the TEM and size
analyzer showed that the PSAAFMs maintained spherical shapes with an average diameter of 2.5 + 0.22 mm. Fluorescence
measurement indicated that the maximum emission wavelength underwent a slight blue shift from 514 to 512 nm. Environmental factors, such as pH value, imposed certain effects on fluorescence intensities. The linear relationship between
fluorescence intensity and microspheres’ concentration, which ranged from 1 103 to 10 103 g L1, suggest their
quantitative application. The significant decrease in the physical nonspecific adsorption of BSA, trypsin, and HCG in comparison with the microspheres without improved hydrophilicity suggest the increased amount of acrylamide on the surface of the microspheres. The protein covalent immobilization experiments revealed significant increases in BSA and HCG
immobilization in comparison with the nonspecific physical attachment. The combination of high hydrophilicity and electrostatic repulsion could severely inhibit nonspecific protein attachment onto the surface of the microspheres.
Keywords
Fluorescence characteristic, hydrophilic microspheres, protein immobilization, physical nonspecific adsorption
Introduction
Fluorescent copolymer microspheres have recently become
powerful tools in biological and chemical research, including biochemical analysis, immune detection, and disease
diagnosis among others.1–5 In comparison with free fluorescent dyes, copolymer microspheres comprising fluorescent materials exhibit significant improvements in terms
of surface functionality, mobility, and maneuverability, as
well as the promotion of fluorescent characteristics.6–10
Biomolecules such as proteins, enzymes, and antibodies
attached to the surface of fluorescent microspheres have
Key Laboratory of Biomedical Information Engineering of Ministry of
Education, School of Life Science and Technology, Xi’an Jiaotong
University, Xi’an, People’s Republic of China
Corresponding Author:
Professor D. Wu, Key Laboratory of Biomedical Information Engineering
of Ministry of Education, School of Life Science and Technology, Xi’an
Jiaotong University, Xi’an 710049, People’s Republic of China
Email: [email protected]
256
been used in a wide range of applications such as clinical
diagnosis and cell separation. There are generally two
common approaches to immobilize biomolecules onto
microspheres, covalent binding and physical absorption.
Chemical covalent coupling is a specific binding approach
that requires functional groups, such as amino and carboxyl, onto the surface of the copolymer microspheres,
whereas nonspecific physical absorption relies on hydrophobic and electronic interactions, as well as hydrogen
bonding.11,12 In most circumstances, the covalent binding
of proteins is associated with a certain degrees of
nonspecific protein attachments due to the presence of
hydrophobic surfaces. Nonspecific adsorption of proteins
may cause sample contamination or purity loss during
application.13–15 Similarly, most of the biological and
chemical applications are involved in an aqueous
environment, such as blood and urine, and the surface
hydrophilicity of microspheres plays a key role in particle
monodispersity and avoidance of nonspecific aggregation
among microspheres and biomolecules.16 Thus, introducing hydrophilic surfaces can be an effective approach to
inhibit nonspecific attachments of proteins, as well as to
maintain particle stability. Existing methods used to introduce hydrophilic surfaces mainly employ hydrophilic
materials as a whole matrix or a coating layer on the surface
of the microspheres. Typical hydrophilic materials used in
the preparation of hydrophilic microspheres include silica17
and polysaccharides.18 Hydrophilic monomers such as
acrylamide, acrylic acid, and N-isopropylacrylamide have
been extensively studied in biological applications.19
Available approaches that use hydrophilic monomers to
prepare hydrophilic microspheres include block or graft
copolymerization of hydrophobic and hydrophilic monomers,20,21 water-in-oil reverse polymerization of hydrophilic microspheres,22 seeded polymerization for core-shell
structure with hydrophilic monomers as the shell layer,23,24
and grafting and modification of the hydrophilic monomer
onto a hydrophobic core in a core-shell structure.25 Among
these methods, copolymerization of hydrophobic and
hydrophilic monomers in one particle is a simple and effective method to prepare microspheres with rigid, hydrophilic
surfaces. Studies have shown that hydrophilic monomers
tend to accumulate on the outside of the particle during the
copolymerization process, which could maintain hydrophilicity. However, the copolymerization of hydrophilic and
hydrophobic monomers creates hydrophilic surfaces by
generating a gradient of increasing hydrophilicity from the
inside to the outside layers, which cannot produce a purely
hydrophilic outside layer.26 As the typical fluorescein dye,
fluorescein is highly absorbed, highly fluorescent, has good
quantum yield, and shows good biocompatibility, enabling
it to be widely used in protein labeling.27
In the present study, the hydrophilic monomer addition
method was improved to increase the hydrophilic monomer
content on the outside layer of the microspheres to promote
High Performance Polymers 23(3)
surface hydrophilicity. This approach mainly involves the
semi-continuous addition of hydrophilic monomers
during the soap-free emulsion copolymerization process.
At the same time, fluorescein was encapsulated into the
polymer matrix to achieve a fluorescent property, and the
fluorescence characteristic was studied using a fluorescence spectrometer and a fluorescence microscope.
Azidocarbonyl groups, which could increase covalent linking between microspheres and proteins, were introduced
onto the surface of the microspheres. These hydrophilic
fluorescent microspheres possess increased hydrophilicity
and sensitive fluorescence, as well as reduced nonspecific
protein immobilization. The linear relationship between the
concentrations of the hydrophilic fluorescent microspheres
and the fluorescence intensities show their potential applications for the quantitative determination of water-based
proteins.
Experimental
Materials
Styrene (St) was purchased from the Chinese Medical
Chemicals Company Limited (Shanghai, China) and
distilled under reduced pressure to remove the inhibitor.
Acrylamide (AAM) was purchased from Amresco Fraction
Inc. (America). Acrylic acid (AA) and azobisisobutyronitrile (AIBN) were obtained from the Third Chemical Plant
(Tianjin, China). Fluorescein was ordered from the Chemical Reagent Corporation (Shanghai, China). Bovine serum
albumin (BSA) was from Roche Fraction Inc. (Germany).
Trypsin was purchased from Amersco Inc. (USA).
Human chorionic gonadotropin (HCG) was provided by
Zhengzhou Autobio Co., Ltd of China. All chemicals
were of analytical reagent grade. Distilled deionized water
(Academic Milli-Q Millipore) was used for the preparation
of all aqueous solutions, and phosphate-buffered saline
(PBS) was prepared initially and used directly.
Preparation of PSAAFMs with improved
hydrophilicity
Poly(styrene-acrylamide-acrylic acid) copolymer fluorescent microspheres (PSAAFMs) with improved hydrophilicity were prepared through an improved soap-free
emulsion polymerization (Scheme 1). To remove oxygen,
deionized water was boiled first and then transferred into
a three-neck flask. Afterwards, 0.8 g of acrylamide was
added to the deionized water and 0.15 g of AIBN together
with 30 mg of fluorescein were dissolved in 3 mL of ethanol. Subsequently, the ethanol solution was mixed with
8 mL of styrene and the mixture was shaken for 5 min until
it was uniformly dispersed and then transferred into the
flask. The reaction was continued with vigorous stirring.
The temperature was increased to 70 C to initiate
Pan et al.
polymerization. Sodium hydroxide (1.0 mol L1) was
added for pH adjustment. Two hours later, 0.5 g of
acrylamide and 470 mL of acrylic acid solution were added
dropwise into the system every 15 min until the fifth hour
after initiation. The reaction continued for another 4 h
under a N2 atmosphere. After completion of the reaction,
the temperature was increased to 80 C to remove the ethanol. The obtained hydrophilic fluorescent microspheres
were washed thrice with water and ethanol, collected
through centrifugation, and stored at 4 C prior to use.
Preparation of PSAAFMs
PSAAFMs without improved surface hydrophilicity were
prepared as a contrast. At the beginning of the reaction,
1.3 g of acrylamide was added into the system, and this was
not repeated at later stages. Other conditions remained the
same as the procedures in the section above entitled
‘Preparation of PSAAFMs with improved hydrophilicity’.
Fabrication of hydrazide-hydrophilic PSAAFMs
The hydrazide groups were introduced onto the hydrophilic
fluorescent microspheres through hydrazinolysis. Hydrophilic fluorescent microsphere suspension (5%, 10 mL)
was added to a flask. The hydrazide reaction was allowed
to proceed for 8 h at 50 C, followed by the addition of
hydrazine hydrate (80%). After cooling to room temperature, the products were washed with distilled water several
times until the pH reached 7.0. The obtained hydrazide–
hydrophilic fluorescent microspheres were dialyzed, collected, and stored at 4 C prior to use.
Preparation of azidocarbonyl-hydrophilic PSAAFMs
and covalent binding through azidocarbonyl groups
Azidocarbonyl groups on the hydrophilic fluorescent
microspheres were introduced through the azido reaction.28
The pH of the hydrazide–hydrophilic fluorescent microsphere suspension (9.2 mg mL1, 5 mL) was adjusted to
2.0 with continuous stirring. Then, 0.1 mol L1 of NaNO2
was added dropwise until the potassium iodine–starch test
paper changed color. After the reaction has proceeded for
1 h at 4 C, 1 mL each of BSA (3.5 mg mL1), trypsin
(3.5 mg mL1), and HCG (3.5 mg mL1) solution were
added separately to the azidocarbonyl-hydrophilic fluorescent microsphere suspension and the pH value was then
adjusted to 7.0–7.2. The reaction was carried out at 4 C for
5 h and then terminated through the addition of glycine.
The product was dialyzed and separated, and all the
supernates were collected for the analysis of BSA
concentration.
The immobilization capacity of BSA, trypsin, and HCG
was determined using ultraviolet spectrophotometry. The
total unbound protein collected in all supernates was
257
measured according to a calibration curve. The amount of
protein immobilized onto the functional azidocarbonylhydrophilic fluorescent microspheres was then determined
by measuring the initial and unbound protein concentrations. The immobilization capacity of the hydrophilic
fluorescent microspheres was calculated as follows:
Z¼
C1 V1 C2 V2
100%
C1 V1
ð1Þ
where C1 and V1 are the concentration and volume of the
total protein added, respectively; C2 and V2 are the concentration and volume of the whole supernates, respectively.
The experiment was repeated three times.
Physical nonspecific adsorption of protein
The physical nonspecific adsorption of protein onto the
hydrophilic fluorescent microspheres was performed in
an aqueous solution at pH 7.0. About 1 mL of BSA
(3.5 mg mL1 and 0.1 mg mL1), trypsin (3.5 mg mL1,
0.1 mg mL1), and HCG (3.5 mg mL1, 0.1 mg mL1)
were added separately into 5 mL of hydrophilic fluorescent
microsphere suspension (9.2 mg mL1). The reaction was
continued under pH 7.0 with vigorous stirring for 2 h. The
unattached proteins collected in the supernates were determined using the method described in the section entitled
‘Fabrication of hydrazide-hydrophilic PSAAFMs’. The
nonspecific adsorptions of proteins onto the fluorescent
microspheres without improved hydrophilicity were performed in the same way. The experiment was repeated
three times.
Morphology and size distribution
The morphology of hydrophilic fluorescent microspheres
was examined under a transmission electron microscope
(TEM; JEM-100SX, Japan). The size distribution of the
microspheres was measured using a particle size analyzer
(Mastersizer 2000; UK).
Functional groups on the surface of hydrophilic
fluorescent microspheres
Functional groups on the surface of hydrophilic fluorescent
microspheres were evaluated using a Fourier transform
infrared spectrometer (FTIR; IR Prestige-21, Japan). In a
typical procedure, 0.25 mg of dry hydrophilic fluorescent
microspheres was mixed with IR-grade KBr (0.1 g) and
pressed (10 ton) into tablet form and its spectrum was then
recorded. The FTIR spectrum of fluorescent microspheres
without improved hydrophilicity was also recorded.
258
Scheme 1. Preparation of fluorescent microspheres with
improved hydrophilicity.
High Performance Polymers 23(3)
Figure 1. TEM image of fluorescent microspheres (a) without
and (b) with improved hydrophilicity.
Fluorescence characteristics
The fluorescence characteristics of the hydrophilic fluorescent microspheres were studied using a fluorescence microscope (OLYMPUS CX41; Japan) and a fluorescence
spectrophotometer (Hitachi F4500; USA). The concentration of the aqueous solutions of hydrophilic fluorescent
microspheres, hydrazide–hydrophilic fluorescent microspheres, and protein-immobilized hydrophilic fluorescent
microspheres were set at 15 103 g L1. The experiment
was repeated three times.
Results and discussion
Preparation of fluorescent microspheres with
improved hydrophilicity
Several approaches are currently proposed to increase the
hydrophilicity of microspheres, including the block copolymerization of hydrophobic and hydrophilic monomers, the
water-in-oil reverse polymerization of hydrophilic microspheres, seeded polymerization of core–shell structures
with hydrophilic monomers as shell layers, and grafting
and/or modification of hydrophilic monomers onto hydrophobic cores in a core–shell structure. The copolymerization of hydrophilic and hydrophobic monomers requires
fewer operation steps and has a high yield. However, the
typical copolymerization of hydrophilic and hydrophobic
monomers does not bind purely hydrophilic monomers in
the outside layer of the microspheres, but generates a
gradient of increasing hydrophilicity from the inside to the
outside layers. Soap-free polymerization of styreneacrylamide-acrylic acid was used in the present study to
prepare successfully monodispersed nanoparticles. To
increase the proportion of hydrophilic monomers in the
outside layer of microspheres, the semi-continuous addition of acrylamide and acrylic acid was adopted. During the
early stage of polymerization, styrene formed into droplets
due to the presence of unstable acrylamide; the copolymerization of styrene and acrylamide that occurred inside the
styrene droplet was initiated by the styrene-miscible initiator, AIBN. As the polystyrene chain grows, the acrylamide
in the styrene-acrylamide copolymer tends to form onto the
Figure 2. Size distribution of fluorescent microspheres with
improved hydrophilicity.
outside layer of the particles to maintain the particle stability. Subsequent semi-continuous addition of another portion of acrylamide could generally increase the portion of
acrylamide on the outside layer. Therefore, the rough surface of the microspheres at the final stage of copolymerization indicates that the polymerization occurs only
between acrylamide, rather than copolymerization with
styrene.
Particle morphologies and size distribution
TEM micrographs of the fluorescent microspheres with and
without improved hydrophilicity are presented in Figure 1,
which demonstrates the basic and regular sphericity of the
fluorescent microspheres. In Figure 1(b), the outer layer of
hydrophilic fluorescent microspheres exhibits a rough
surface. In comparison with the microspheres without
improved hydrophilicity (Figure 1(a)), the rough surface
of hydrophilic fluorescent microspheres may be attributed
to the presence of a large proportion of acrylamide and
reduced surface tension, which would greatly increase the
hydrophilicity of the microspheres. As shown in Figure 2,
the hydrophilic fluorescent microspheres exhibit an average diameter of 2.5 + 0.22 mm and a narrow distribution
(polydispersity index 0.046 + 0.009).
Pan et al.
259
Figure 3. FTIR spectra of fluorescent microspheres (a) without
improved hydrophilicity and (b) with improved hydrophilicity.
FTIR spectrum and functional groups
The FTIR spectrum of fluorescent microspheres with
improved hydrophilicity is shown in Figure 3. In Figure
3(a), the absorption peaks of the benzene ring appear: the
wide peak at 3462 cm1 was attributed to the superimposed
stretching vibrations of O–H and N–H bonds and the strong
peak at 1652 cm1 is attributed to the C¼O bond vibration,
indicating the presence of carboxyl and amino groups.
Therefore, the clear characteristic signals for the amide and
benzene ring groups indicate that both acrylamide and styrene have participated in the polymerization reaction.
Figure 4. Fluorescence microscope image of fluorescent
microspheres.
Fluorescence characteristics and protein
immobilization capacity
Figure 4 shows the fluorescence image of the fluorescent
microspheres with improved hydrophilicity when excited
under a fluorescence microscope. The fluorescein was
homogeneously embedded into the microspheres. Figure 5
shows the fluorescence emission spectra of the
hydrophilic fluorescent microspheres and fluorescein, in
which the maximum emission wavelength of the fluorescein is 512 nm and the maximum excitation wavelength
is 480 nm. After the fluorescein was embedded into the
copolymer matrix, a slightly visible blue shift (from
514 to 512 nm) was observed in the emission wavelength.
Hydrophilic fluorescent microspheres, hydrazide-hydrophilic fluorescent microspheres, and azidocarbonyl-hydrophilic
fluorescent microspheres exhibited the same fluorescence
emission and excitation spectra (data not shown). The
fluorescence intensities of the hydrophilic fluorescent microspheres (lex =lem , 480 nm/512 nm), hydrazide-hydrophilic
fluorescent microspheres (lex =lem , 480 nm/512 nm), and
protein-immobilized hydrophilic fluorescent microspheres
(lex =lem , 480 nm/512 nm) were linearly related to the concentrations, which ranged from 1 103 to
Figure 5. Fluorescence emission spectra of (a) fluorescein and
(b) fluorescent microspheres.
10 103 g L1. The linear equations are as follows:
IF ¼ 1:85 þ 11:58xðR2 ¼ 0:9916Þ for the hydrophilic
microspheres; IF ¼ 6:208 þ 3:48xðR2 ¼ 0:9966Þ for the
hydrazide-hydrophilic fluorescent microspheres; and
IF ¼ 17:1754 þ 6:285xðR2 ¼ 0:9984Þ for the proteinimmobilized hydrophilic fluorescent microspheres. Hence,
the fluorescence intensity can be considered as a function
of the concentration, which in turn can be used in quantitative determination.
During copolymerization, the fluorescence spectrum
had a slight visible blue shift (from 514 to 512 nm) in comparison with pure fluorescein. This phenomenon may be
attributed to the structure of the hydrophilic fluorescent
microspheres due to the semi-continuous addition of monomers, which deposits additional hydrophilic acrylamide
260
High Performance Polymers 23(3)
Figure 7. Protein immobilization of BSA, trypsin, and HCG
through covalent binding and nonspecific attachment on fluorescent microspheres with and without improved hydrophilicity.
Figure 6. Effect of pH on fluorescence property of (a) fluorescein
and (b) fluorescent microspheres.
and acrylic acid monomers onto the surface of the poly
(styrene-acrylamide) microspheres. The outer layer was a
transparent hydrogel consisting of acrylamide and acrylic
acid copolymer, which rarely has a block effect on the
fluorescein inside the microspheres.
Effects of pH on the fluorescence property
The effect of the pH of the fluorescein and hydrophilic
fluorescent microspheres is illustrated in Figure 6. The
fluorescence intensity of fluorescein shows a dramatic
increase at pH 6.0, indicating the instability of pure fluorescein, whereas that of the hydrophilic fluorescent microspheres slightly increased at pH ranging from 6.0 to 8.0.
The difference in the fluorescence property may be attributed to the specific structure of the hydrophilic fluorescent
microspheres, which can prevent the leakage of the fluorescein and can resist pH changes.
The effect of temperature on the fluorescence between
20 and 40 C was also investigated. No clear change in the
fluorescence spectra was found with respect to the emission
wavelength, except for the slight decrease in the fluorescence intensity when the temperature was increased (date
not shown).
Immobilization of protein: chemical covalent binding
and physical nonspecific adsorption
In practical applications, the immobilization of proteins
and other macromolecules could be accomplished through
several approaches such as physical adsorption, entrapment, and chemical binding. In comparison with physical
adsorption and entrapment, chemical binding provides a
specific linking site and steady bonding to the target protein. However, covalent binding requires that the supports
have suitable functional groups on their surface. Suitable
covalent binding groups promise modest linking conditions
such as mild temperature, little ionic concentration, specific linking sites of the molecules, and a small amount
of catalyst. Coupled with chemical binding, the physical
attachment of protein, which is mostly based on hydrophobic and electrostatic interactions, as well as hydrogen
bonding, accounts for a certain percentage of the total
immobilization of the protein. The physical attachment of
the protein would decrease the purity of the protein on the
surface during practical applications and cause the aggregation of the microspheres because of the protein–protein
interaction. To assess the influence of improved hydrophilicity on the physical adsorption of protein, model protein
(BSA), enzyme (trypsin), and antibody (HCG) at high
concentration were immobilized onto the surface of the
hydrophilic fluorescent microspheres, as shown in
Figure 7. The physical adsorption of protein onto the fluorescent microspheres without improved hydrophilicity was
also performed to show contrast. The amount of immobilized proteins through physical adsorption at high and low
concentrations is shown in Table 1, wherein hydrophilic
microspheres with improved hydrophilicity demonstrate
an overall decrease in the physical adsorption of protein.
As shown in Table 1, different proteins exhibited reduced
amounts of physical adsorption at different extents. BSA,
trypsin, and HCG showed significant decrease in the physical attachment onto the hydrophilic surface of 73.7, 35.8,
and 82.6%, respectively, compared with the hydrophilic
surface that was not improved. Trypsin still demonstrated
high adsorption efficiency even if it showed a 35.8%
decrease. The high nonspecific adsorption of trypsin can
be attributed to the electrostatic interaction greatly affecting the physical adsorption of protein. The zeta potential
of the fluorescent microspheres with and without improved
hydrophilicity and the different proteins at pH 7.0 are listed
in Table 2. The slightly positive charge of trypsin at pH
7.0 has significantly reduced the repulsing effect between
Pan et al.
261
Table 1. Nonspecific attachment of protein onto fluorescent microspheres.
Samples/proteins
PSAAFMs with hydrophilicity
High concentration (3.5 mg)
Low concentration (0.1 mg)
PSAAFMs without hydrophilicity
High concentration (3.5 mg)
BSA
Trypsin
HCG
6.46% (0.22 mg)
21.22% (0.02 mg)
51.90% (1.81 mg)
42.20% (0.04 mg)
2.89% (0.11 mg)
34.73% (0.03 mg)
24.58% (0.86 mg)
80.87% (2.83 mg)
16.57% (0.58 mg)
Table 2. Zeta potential of proteins and fluorescent microspheres.
Parameters/proteins
BSA
Isoelectirc point
Zeta potential (mv) (pH 7.0)
4.7
10.5
Trypsin
10.8
0.96
HCG
2.94
16.4
the negatively charged microspheres and protein, further
strengthening the attraction between the microspheres and
proteins due to their opposite charges. Thus, it is speculated
that preliminary electrostatic interaction accounts for the
nonspecific adsorption of protein onto microspheres. In
addition, the significant reduction in the physical nonspecific adsorption onto the microspheres is due to the combined
effects of increased hydrophilicity and electrostatic interaction. The zeta potential on the surface of the microspheres
plays a key role in maintaining microspheres monodispersity and the electrical neutrality on the microsphere surface.
Therefore, the relationship between the zeta potential of
microspheres and proteins at different pH conditions would
greatly affect the nonspecific adsorption of protein. BSA
and HCG, which have significantly decreased nonspecific
adsorption, were selected to undergo chemical covalent
binding through azidocarbonyl groups to assess the total
protein immobilization capacity of the hydrophilic fluorescent microspheres. The results showed that the azidocarbonyl hydrophilic fluorescent microspheres were heavily
coated with BSA and HCG at the immobilized portion of
45 and 75%, respectively, which are significantly higher
compared with nonspecific binding. The fluorescence characteristics remain unchanged. After the proteins were
immobilized onto the hydrophilic fluorescent microspheres, they did not affect the fluorescence characteristics
of PSAAFMs. Given the linear relationship between fluorescence intensity and PSAAFM concentration, the former
could be used as an indicator of immobilized protein
concentration.
Conclusions
Fluorescent PSAAFMs with amino groups and improved
hydrophilicity were prepared through improved soap-free
emulsion polymerization. Physical nonspecific adsorption
showed improved hydrophilicity would significantly
reduce the unspecific binding of proteins onto the surface
PSAAFMs with hydrophilicity
PSAAFMs without hydrophilicity
9.86
10.9
of the microspheres. Covalent binding between
microspheres and proteins exhibited a significant increase
toward nonspecific binding. The zeta potential and isoelectric point play key roles in determining the nonspecific
binding between microspheres and proteins. The results
show that the fluorescent microspheres with reduced nonspecific binding of proteins were excellent candidates for
the quantitative determination of water-based proteins.
Acknowledgements
This study was supported in part by the grants of the National
Nature Science Foundation of China (No. 30772658, 20905060
and 30970712) and Natural Science Foundation of Shaanxi Province of China (2006B10).
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