Property and application of BACy-based functional hydrogels

Article abstract of DOI:10.1002/jccs.201300684

Conventional polyacrylamide hydrogels prepared from the free radical polymerization between acrylamide and N,N-methylenebisacrylamide (NMBA) have been frequently used in the biochemical technique like the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to resolve protein mixtures. In this study, we have prepared an alternative polyacrylamide hydrogel from the cross-linking of acrylamide and N,N-bisacrylylcystamine (BACy). In addition, we have compared the BACy-based hydrogel with the NMBA-based polyacrylamide hydrogel for their physical properties such as swelling ratio, shear modulus, crosslink density and morphology. Moreover, we further determined whether BACy-based polyacrylamide hydrogel could be applied to SDS-PAGE and proteomics research. The results showed that this type of hydrogel is capable of separating proteins and facilitates further in-gel protein digestion and the following protein identifications by mass spectrometry. In summary, our study provides a basis for the putative application of BACy-based hydrogels.

Full text of DOI:10.1002/jccs.201300684

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
Property and Application of BACy-Based Functional Hydrogels
Yan-Su Lin,a Yi-Long Huang,b Wen-Fu Leea* and Chao-Hsiung Linb*
aDepartment of Chemical Engineering, Tatung University, Taipei, Taiwan, Republic of China
bDepartment of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan,
Republic of China
(Received: Dec. 28, 2013; Accepted: Apr. 8, 2014; Published Online: May 6, 2014; DOI: 10.1002/jccs.201300684)
Conventional polyacrylamide hydrogels prepared from the free radical polymerization between acryl-
amide and N,N¢-methylenebisacrylamide (NMBA) have been frequently used in the biochemical tech-
nique like the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to resolve protein
mixtures. In this study, we have prepared an alternative polyacrylamide hydrogel from the cross-linking of
acrylamide and N,N¢-bisacrylylcystamine (BACy). In addition, we have compared the BACy-based
hydrogel with the NMBA-based polyacrylamide hydrogel for their physical properties such as swelling
ratio, shear modulus, crosslink density and morphology. Moreover, we further determined whether
BACy-based polyacrylamide hydrogel could be applied to SDS-PAGE and proteomics research. The re-
sults showed that this type of hydrogel is capable of separating proteins and facilitates further in-gel pro-
tein digestion and the following protein identifications by mass spectrometry. In summary, our study pro-
vides a basis for the putative application of BACy-based hydrogels.
Keywords: N,N¢-Bisacrylylcystamine (BACy); N,N¢-Methylenebisacrylamide (NMBA); Disulfide
linkage; Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
INTRODUCTION
SDS-PAGE has been frequently used method in bio-
linkers include the oxidation-sensitive N,N¢-1,2-dihydroxy-
ethylene bisacrylamide (DHEA),5,6 the alkaline-sensitive
ethylene glycol diacrylate (EGDA)7 and the reduction-sen-
sitive N,N¢-bisacrylylcystamine (BACy).8 Expansion of
pore size within the gel structure is anticipated when such
labile crosslinkers were put under conditions for cleavage.
Proteomics represents a large-scale investigation on struc-
tures and functions of total proteins in cells or tissues.9-12 A
typical proteomics approach includes the extraction of pep-
tides from in-gel protein digestion by proteases, and the
subsequent amino acid sequencing by mass spectrometry.
The pore expansion resulted from the cleavable poly-
acrylamide hydrogel upon cleavage conditions may in-
crease the amount of extracted peptides or proteins for the
subsequent analysis. Indeed, the base-labile EDGA was
previously used to prepare the reversible gel which facili-
tates collecting the sufficient amount of radio-isotope-la-
belled proteins for scintillation counting.7,13
chemical analysis and provides the capability to resolve
complex protein mixtures based on the difference of their
molecular weights.1 In addition, three-dimensional struc-
ture of polyacrylamide hydrogel is essential for SDS-
PAGE.2 The polyacrylamide hydrogel can be prepared
from free radical polymerization of acrylamide in the pres-
ence of diverse two-functional cross-linkers such as the
N,N¢-methylenebisacrylamide (NMBA), 1,2-diacrylamide
ethyleneglycol (DEG), ethyleneureabisacrylamide (EUB),
N,N¢-propylenebisacrylamide (PBA), N,N¢-diallyltartardi-
amide (DATD), diacrylamide dimethylether, and ethylene
diacrylate (EDA).3,4 The resulting polyacrylamide hydro-
gels exhibit diverse pore size within the three-dimensional
structures and provide distinct applications in molecular
separation and drug loading/release. Currently, bioanalyti-
cal methodology like proteomics frequently uses polyac-
rylamide hydrogels to separate proteins in the one- or two-
dimensional gel electrophoresis.
We herein investigate the characteristics of the reduc-
tion-cleavable polyacrylamide hydrogel as well as its ap-
plication potential in proteomics study. Since condition of
oxidation or base treatment causes damage and modifica-
tion to protein structure, the reducible BACy crosslinker
In addition, the cleavable polyacrylamide hydrogels
have also been developed by incorporating a cleavable
cross-linker into polymer structure. These cleavable cross-
* Corresponding author. Tel: +886-2-28267280; Fax: +886-2-28202449; Email: chlin2@ym.edu.tw (Chao-Hsiung Lin)
Tel: +886-2-21822928 ext 6267; Fax: +886-2-25861939; Email: wflee@ttu.edu.tw (Wen-Fu Lee)
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was used to prepare the cleavable polyacrylamide hydrogel
for the following study. The disulfide linkage of BACy can
be effectively and efficiently cleaved into two thiol groups
upon treatment of reducing agents like 1,4-dithiothreitol
(DTT) or 2-mercaptoethanol (b-ME).14 Therefore, such
reductive cleavage within the three-dimensional structure
of BACy-based polyacrylamide hydrogel provides a con-
trollable manner to expand the gel pore size. In the present
study, the swelling ratios and pore sizes of the BACy
polyacrylamide hydrogel were measured and compared to
those of the conventional NMBA polyacrylamide hydro-
gel. In addition to the protein separation efficiency of these
hydrogels, the drug release experiment of caffeine and bo-
vine serum albumin (BSA) from different hydrogels under
the treatment with or without reducing agents were carried
out to demonstrate the feasibility of reducible polyacryl-
amide hydrogel.
hydrogel used the same molar ratio of acrylamide (13.6
mmole) and crosslinker (BACy, 0.056 g, 0.216 mmole) as
in the NP hydrogel. The feed compositions as well as the
obtained physical and mechanical properties for all three
polyacrylamide hydrogels were summarized in Table 1. In
addition, the appearance of two BP hydrogels resembles
the NP hydrogel in the aspect of transparency, softness,
protein separation efficacy and effect of gel staining, as
shown in Figure 2. We further measured the swelling ratio
for each three hydrogels and the results shown in Table 1
indicated that the BPb hydrogel has the highest swelling ra-
tio (13.91 g/g) whereas NP and BPa hydrogels have similar
values, 9.78 g/g and 11.89 g/g respectively. This is because
the BPb hydrogel has the BACy crosslinker effect than NP
hydrogels, which led to a larger structure in BPb hydrogel.
Swelling kinetics profile of all three polyacrylamide
hydrogels in deionized water at 25 °C is shown in Figure 3.
The measured values of SReqs for polyacrylamide hydro-
gels indicated, i.e. BPb (13.91 g/g) > BPa (11.89 g/g) > NP
(9.78 g/g). As shown in Table 1, the G and rx values of the
RESULTS AND DISCUSSION
Characterization of the Polyacrylamide Hydrogels
BACy was synthesized at good quality according to
the modified procedure from our recent report (Figure 1).14
The corresponding polyacrylamide hydrogels were pre-
pared by mixing acrylamide with either NMBAor BACy as
crosslinker (as shown in Scheme 1) and the resulting hy-
drogels were respectively named as NP, BPa and BPb. NP
hydrogel was prepared following the conventional recipe
of acrylamide (0.967 g, 13.6 mmole) and NMBA ratio
(0.033 g, 0.216 mmole) for SDS-PAGE. In addition, BPa
hydrogel was prepared using the same weight ratio of
acrylamide (0.967 g, 13.6 mmole) and crosslinker (BACy,
0.033 g, 0.128 mmole) as in the NP hydrogel whereas BPb
Polymerization of the polyacrylamide hy-
drogels using acrylamide with NMBA (A)
and BACy (B) respectively
Scheme 1
Fig. 1. The BACy has m.p. of 126-127 °C (A) and pu-
rity more than 99% based on result of High
Pressure Liquid Chromatography (B).
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Table 1. Feed compositions and physical properties of the copolymeric hydrogels
wet gel[e]
Gel
code (mmole)
AAm[a]
NMBA[b]
(mmole)
BACy[b]
(mmole)
SReq[c]
(g/g)
G × 104[d]
(N/m2)
rx × 10-2
(mmole/cm3)
NP
13.6
13.6
13.6
0.216
-
9.78 ± 0.10
11.89 ± 0.12
13.91 ± 0.04
2.26 ± 0.30
2.07 ± 0.20
1.50 ± 0.11
2.10 ± 0.30
2.05 ± 0.20
1.58 ± 0.12
BPa
BPb
-
-
0.128
0.216
[a] AAm: Acrylamide. [b] NP: hydrogel with 0.216 mmole of NMBA; BPa: hydrogel with
0.128 mmole of BACy; BPb: hydrogel with 0.216 mmole of BACy. [c] SReq: equilibrium
swelling ratio. [d] G: shear modulus. [e] rx. effective cross-linking density.
polyacrylamide hydrogels are 2.26 × 104 N/m2, 2.10 × 10-2
for their ability to resolve protein mixtures. As shown in
Figure 5A, the BSA (M.W. 66 kDa), PhaZ (M.W. 34 kDa)
and mixture of standard protein weight markers (ranged
from 17 kDa to 170 kDa) were loaded into NP, BPa and BPb
hydrogels respectively for the SDS-PAGE analysis. Our
data showed that both BP hydrogels are capable to resolve
proteins and the separation efficacy is comparable to NP
hydrogel. Notably, all proteins exhibit slower mobility in
BPb hydrogel than in other two hydrogels. This phenome-
mmol/cm3 for NP gel, 2.07 × 104 N/m2, 2.05 × 10-2 mmol/
cm3 for BPa gel and 1.50 × 104 N/m2, 1.58 × 10-2 mmol/cm3
for BPb gel. These values indicated the higher the gel
strength (G) and the higher effective crosslinking density
(rx), the lower the swelling ratio of the hydrogels, i.e. NP >
BPa > BPb. In addition, the low effective crosslinking den-
sity (rx) of BPb indicated the less degree of crosslinking in
the hydrogel structure, which allowed more water absorp-
tion.
Scanning electron microscopy (SEM) was used to in-
vestigate the internal morphologies of the polyacrylamide
hydrogels and the resulting micrographs of SEM clearly
showed the pore sizes in these gels (Figure 4). Whereas BPa
and BPb gels have the larger pore size, which is explained
the difference of swelling ratios among these gels. i.e. BPb
> BPa > NP.
Effects of BACy on Protein Separation Efficacy
All three polyacrylamide hydrogels were examined
Fig. 3. Measurement of swelling kinetics of NP, BPa
and BPb hydrogels.
Fig. 2. Appearance of NP, BPa and BPb SDS-PAGE
gels. (A) Transparency of polyacrylamide hy-
drogels (B) Separation of pre-stained protein
markers without gel staining (C) Coomassie
blue staining of gels loaded with sample pro-
teins of PhaZ (lane 1) and BSA (lane 2).
Fig. 4. Morphology of NP, BPa and BPb hydrogels
with or without DTT treatment using SEM pho-
tography at 100x.
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Table 2. Relative mobility based on the RF value of BSA, PhaZ
and protein weight markers on NP, BPa and BPb hydro-
gels
non is likely due to the fact that BPb gel has a higher content
of crosslinker in the composition of polyacrylamide, which
results in denser structure and smaller space for protein
penetration. In addition, the retention factor (RF) which re-
flects the relative mobility of proteins in hydrogels during
electrophoresis was calculated and listed in Table 2. Fur-
thermore, plot of RF against molecular weight (M.W.) of
protein weight markers was shown in Figure 5B. In sum-
mary, proteins with higher molecular weight travel fast in
BPa hydrogel than in NP hydrogel whereas all proteins
move relatively slowly in BPb hydrogel. However, both BP
hydrogels exhibit good protein-separating ability compar-
ing to NP hydrogel.
Retention factor (RF)
Proteins
NP hydrogel BPa hydrogel BPb hydrogel
BSA (66 kDa)
PhaZ (34 kDa)
170 kDa marker
130 kDa marker
95 kDa marker
72 kDa marker
55 kDa marker
43kDa marker
34 kDa marker
26 kDa marker
17 kDa marker
0.43
0.73
0.13
0.17
0.25
0.34
0.43
0.55
0.68
0.81
0.95
0.50
0.76
0.16
0.22
0.29
0.38
0.47
0.56
0.66
0.79
0.93
0.38
0.64
0.11
0.15
0.21
0.29
0.36
0.45
0.57
0.70
0.95
Effects of BACy on Protein Identification Efficacy
In a typical proteomics study involving SDS-PAGE,
the in-gel trypsin digestion of selected protein band (or
spot) into peptides and the subsequent mass spectrometric
(MS) analysis are carried out to identify proteins within the
polyacrylamide hydrogel. Although the disulfide linkage
of BACy in polyacrylamide hydrogels may possibly react
with the free thiol group in any protein, whether the pres-
ence of the disulfide bond interferes with the trypsin diges-
tion of proteins or the MS analysis of tryptic peptides re-
main unknown. We next investigated effects of BACy on
the protein identification efficacy from NP, BPa and BPb
hydrogels by a MALDI-TOF mass spectrometry (MS)
(Figure 6). The BSA protein was chosen for the following
in-gel digestion and MS identification. BSA has 35 cy-
steines, but in its native state, 34 are in disulfide linkage. It
has a limited ability to potentially react with disulfide link-
age of BACy in hydrogels. As shown in Table 3, the identi-
fication of BSA was achieved from all three hydrogels with
similar identification score, sequence coverage and num-
ber of the identified peptides. These results indicated that
the BACy content or free cystein on BSA does not interfere
with the identification process of BSA proteins, including
the in-gel trypsin digestion procedure to generate peptides
and the MS-based analysis of peptide masses. In summary,
the BACy-based polyacrylamide hydrogels showed no
interference with the typical protein identification and
provide a good alternative for NP hydrogels.
Effects of BACy on Caffeine and BSA Release
As indicated in Table 3, release of caffeine from caf-
feine-incubated hydrogels was slightly increased upon the
elevation of BACy contents from BPa (3.20 mg/g) to BPb
(3.49 mg/g). Under DTT treatment, the released caffeine in
hydrogels was also slightly increased with an elevation of
BACy content in the hydrogels from BPa (3.27 mg/g) to
BPb (4.08 mg/g). These results indicate that higher swell-
ing ratio of BACy in hydrogels did not significantly affect
the release amount of caffeine.
Fig. 5. SDS-PAGE with NMBA or BACy as cross-
linker. (A) Protein separation results on NP,
BPa and BPb gels; lane 1, PhaZ (34 kDa); lane 2,
commercial MW markers; lane 3, BSA (66
kDa). (B) Retention factors (RF) of lane 2 MW
markers on NP, BPa and BPb gels were mea-
sured.
On the other hand, all three hydrogels exhibited the
clear increase of BSA released from hydrogels upon DTT
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Table 3. Effects of BACy on the protein identification and the swelling ratio as well as Caffeine and BSA release upon DTT reduction
Substance Release (mg/g)
BSA Protein Identification by MS
Swelling Ratios (g/g)
Bovine Serum Albumin
(BSA)
Caffeine
ID
Peptide
number
w/o DTT
w/ DTT
(HPLC)
w/o DTT
(WAKO)
w/ DTT
(HPLC)
Gel code ID Score
w/o DTT
w/ DTT
Coverage
(HPLC)
NP
1537.07
1456.28
1584.78
41.97%
42.77%
42.77%
28
27
28
9.78 ± 0.10
11.89 ± 0.12
13.91 ± 0.04
9.89 ± 0.16
29.56 ± 0.78
34.62 ± 0.99
2.61
3.20
3.49
2.75
3.27
4.08
1.10
2.05
3.05
13.90
21.25
32.64
BPa
BPb
treatment. As shown in Table 3, released amount of BSA
were increased from 2.05 mg/g to 21.25 mg/g for BPa gel
and from 3.05 mg/g to 32.64 mg/g for BPb gel. In addition,
more BACy content resulted in more increase of released
BSA upon DTT treatment, as BPb contains more BACy
than BPa (0.216 vs. 0.128 mmol). Although NP gel doesn’t
not contain disulfide bond for cleavage by DTT, it is antici-
pated that disulfide linkage of BSA proteins within NP
hydrogel was also reduced by DTT to adapt more linear
conformation, which allows BSAto be easily released from
gel network (increased from 1.10 to 13.90 mg/g in NP gel).
Notably, the BPb hydrogel which has more molar ratio of
Fig. 6. Mass spectra of the identified BSA from NP, BPa and BPb gels using a MALDI-TOF MS.
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BACy than BPa hydrogel released significant amount of
BSA proteins than other hydrogels upon DTT reduction,
which indicates the advantage of using BACy as cross-
linking reagents in polyacrylamide hydrogel. Currently, the
typical method to extract proteins from polyacrylamide
hydrogel needs technique like electroelution with limited
yield. Therefore, the BP hydrogels could provide an alter-
native choice for researchers that intend to extract proteins
of interest from protein-separating gel electrophoresis.
Further investigation on conditions of protein extraction
using BACy-based polyacrylamide hydrogel is suggested.
was determined using a sputter coater with gold (JEOL, JFC-
1200) and a scanning electron microscopy (SEM) (JSM-5300,
JEOL).
Synthesis of BACy: BACy was prepared according to our
previous protocol.14 Briefly, cystamine dihydrochloride (1 equiv.)
and TEA (4.2 equiv.) were first mixed in 80 mL of THF and then
added with acryloyl chloride (2.2 equiv.) dissolved in 20 mL of
THF dropwise at a rate of 1 mL/min. The resulting solution was
allowed to stir at 40 °C under nitrogen atmosphere for 24 h. After
evaporation of solvent, reaction product was purified by column
chromatography with hexane/ethyl acetate (Hex/EA) as mobile
phase and analyzed for purity by thin layer chromatography.
The obtained BACy was white crystals in 76% yield; Mp: 126-
127 °C.
EXPERIMENTAL
Materials: Acrylamide (AAm, Bio-RAD, 99.9%) was
used as monomer to prepare polymeric gels. N,N¢-methylene-
bisacrylamide (NMBA) (Sigma) and N,N¢-bisacrylylcystamine
(BACy) were used as cross-linking agents with AAm in the prep-
aration of three-dimensional polyacrylamide hydrogels. BACy
was synthesized by the coupling reaction of cystamine dihydro-
chloride (Cy·2HCl) (ACROS, 97%) and acryloyl chloride (AC)
(Alfa Aesar, 96%), as described in our previous report. Ammo-
nium persulfate (APS) (Sigma, 98%) as initiator and N,N,N¢,N¢-
tetramethylethylenediamine (TEMED) (Sigma, 99.5%) as an ac-
celerator were used as received. 1,4-Dithiothreitol (DTT) (USB,
99.5%) is used as reducing reagent to convert disulfide bond to
thiols. The PhaZ protein is purified from E. coli as described pre-
viously.15 The pre-stained protein molecular weight marker was
purchased from Thermo Scientific. The coomassie brilliant blue
(G250) was used for protein staining and purchased from BIO-
RAD. Protein Assay Rapid Kit (WAKO) is the high sensitive re-
agent to determine protein concentration at room temperature.
Sinapinic acid (SA) and peptide mass standard kit were purchased
from Bruker Daltonics for mass spectrometry. Tris(hydroxy-
methyl)aminomethane, sodium dodecyl sulfate (SDS), trifluoro-
acetic acid (TFA), glycine, imidazole, ethylenediamine tetra-
acetic acid (EDTA) were purchased from ACROS. Bovine serum
albumin (BSA) as a model protein and caffeine were used in drug
release experiment.
Preparation of Polyacrylamide Hydrogels: Polyacryl-
amide gels were prepared using acrylamide (0.967 g, 13.6
mmole) with varied amount of NMBA or BACy (0.216 mmole or
0.128 mmole, 0.216 mmole) as cross-linker, as indicated in Table
1. Briefly, acrylamide and the selected cross-linker were dis-
solved in 6 mL of deionized water at 40 °C and added with
tris-glycine and 0.1 mL of sodium dodecyl sulfate (SDS). The fol-
lowing polymerization was initiated when the 0.1 mL of 10% am-
monium persulfate (APS) and 0.004 mL of tetramethylethylene-
diamine (TEMED) in deionized water were added to make a final
volume of 10 mL. The resulting solution was loaded into a 7 × 7
cm2 gel cast with 0.5 cm spacer and allowed to reaction at room
temperature for 2 h.
Measurement of Equilibrium Swelling Ratios: Dry gels
were equilibrated in 10 mL of deionized water at different temper-
atures on 25 °C and weighed to calculate their equilibrium swell-
ing ratios (SReq).
SReq = (W¥ - Wd)/Wd
(1)
where W¥ and Wd were the weight of the wet gel at equilibrium
and the weight of the dry gel, respectively.
Measurement of mechanical properties: Dry gels were
equilibrated in 10 mL of deionized water at 25 °C and weighed.
The gel strength (t) was assessed with gel shear modulus (G) and
measured by uniaxial compression experiment with a Lloyd LRX
universal tester (JJ Lloyd, Poole, UK). The shear modulus (G)
was calculated from equation (2):16,17
Instrumentation: For protein identification, the MALDI-
TOF mass spectrometer (Ultraflex, Bruker Daltonics) was used.
High performance liquid chromatography (HPLC, Agilent 1100
dissolution analysis system G1312A binary pump, Minnesota,
USA) and DU 800 UV/VIS Spectrophotometer were used to de-
termine protein and caffeine concentration assay. Gel strength
measured by uniaxial compression experiment with a Lloyd LRX
universal tester (JJ Lloyd, Poole, UK). Morphology of dry gel
t = F/A = -G (l-l-2)
(2)
where t is the compression stress, F is the compression load, A is
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BACy-Based Hydrogels
the cross-sectional area of swollen gels, and l is the compression
strain (DL/Lo) where DL is the difference of the thickness of de-
formed gel and initial swollen gel Lo. At low strains, a plot of
shear stress versus (l-l-2) would yield a straight line whose slope
is shear modulus (G). The effective crosslink density (rx) could
be calculated from the shear modulus and polymer volume frac-
tion (n2) as follows:
gels exhibited comparable protein separation efficacy to
NP hydrogel in SDS-PAGE analysis. In addition, the con-
tents of BACy did not interfere with protein separation or
the subsequent protein identification for proteomics study.
Measurement of swelling ratios of NP, BPa and BPb hydro-
gels showed that BPb has the highest swelling ratio. In addi-
tion, the SEM analysis also verified that BPb hydrogel has
the largest pore size. Further treatment of DTT on these
gels showed that the swelling ratios of the reduced BP
hydrogels were increased but not for NP hydrogel. This
phenomenon is supported by the significant increase of
BSArelease upon DTT treatment in BP hydrogels. The BPb
hydrogel exhibited the most degree of BSA release by
DTT-mediated reduction of gel structure. Such protein re-
lease may be advantageous to researchers who use protein
extraction to obtain proteins from gel electrophoresis. In
summary, the current study provides a basis for the putative
application of BACy-based polyacrylamide hydrogels in
biochemical and proteomics study.
rx = G/n21/3RT
(3)
where R is the gas constant and T is the absolute temperature.
Gel Morphology and Pore Size: The dried polyacryl-
amide hydrogels were equilibrated in deionized water at 25 oC for
24 h, and the resulting swollen hydrogels were freeze-vacuum
dried on –50 oC for 48 h. The hydrogels were then immersed in
liquid nitrogen and fractured into pieces. The fractured speci-
mens were coated with a gold layer for proper surface conduc-
tion, and determined for their morphological details using a
scanning electron microscope (SEM, JEOL, JSM-5300, and To-
kyo, Japan).
In Vitro BSA and Caffeine release experiment: Dry
pieces of polyacrylamide hydrogel with fixed size were rehy-
drated in the aqueous solution of bovine serum albumin (BSA) (1
mg/mL) or caffeine (0.3 mg/mL) at 25 °C for 24 h. The resulting
BSA or caffeine-containing hydrogels were transferred into a vial
with DTT (1 mmole in 1 mL of deionized water), and controlled
not DTT, then maintained at 37 °C in a water bath for 24 h. A 0.1
mL of solution was then collected for measuring the concentra-
tion of released BSA or caffeine using high performance liquid
chromatography (HPLC, Agilent 1100 dissolution analysis sys-
tem G1312A binary pump, Minnesota, USA) and DU 800 UV/
VIS Spectrophotometer were used to determine protein and caf-
feine concentration assay.
ACKNOWLEDGEMENTS
This work was supported by the National Science
Council of Taiwan, R.O.C. (NSC101-2321-B-010-016,
101-2311-B-010-006 and 102-2321-B-010-014) and the
Ministry of Education for "Grant of Aim for Top Univer-
sity Project".
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J. Chin. Chem. Soc. 2014, 61, 945-952