Colloids and Surfaces B: Biointerfaces 83 (2011) 220228

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Colloids and Surfaces B: Biointerfaces
j our nal homepage: www. el sevi er . com/ l ocat e/ col sur f b
Non-specic and specic interactions on functionalized polymer surface
studied by FT-SPR
Jizheng Wei
a,b
, Lesan Yan
a,b
, Xiuli Hu
a
, Xuesi Chen
a
, Yubin Huang
a
, Xiabin Jing
a,
a
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,
5625 Renmin Street, Changchun 130022, PR China
b
Graduate School of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e i n f o
Article history:
Received 20 April 2010
Received in revised form 8 November 2010
Accepted 10 November 2010
Available online 18 November 2010
Keywords:
Polymer lm
Fourier transform
Surface plasmon resonance
Protein adsorption
Biotinavidin interaction
a b s t r a c t
Fourier transform surface plasmon resonance (FT-SPR) was utilized to study specic and non-specic
interactions between proteins and a biotinylated polymer lm by monitoring adsorptions of strep-
tavidin (SAv) and bovine serum albumin (BSA) on the polymer lms. The biotinylated polymer,
poly(lactide-co-2,2-dihydroxymethyl-propylenecarbonate-graft-biotin) [P(LA-co-DHC/biotin)], was pre-
paredby ring-opening copolymerizationof lactide anda OH-bearing cyclic carbonate monomer, followed
by biotinylation of the OH groups. The copolymer was coated onto the FT-SPR chip and vacuum-dried,
hydrated at 70

C, and treated with a blocking agent respectively to achieve different surface status. The
FT-SPR results showed that the vacuum-dried lmhad the most BSAadsorption; hydration treatment led
to migration of the biotin moieties from inner lm to surface and thus resulted in less BSA adsorption;
blocking layer on the polymer surface saturated the active sites for physical and chemical adsorptions
on the surface and thus weakened the BSA adsorption. Adsorption of SAv displayed similar polymer-
surface-status dependence, i.e., more adsorption on vacuum-dried surface, less adsorption on hydrated
surface and the least adsorption on blocked surface. Compared with BSA, SAv showed more enhanced
adsorptions on P(LA-co-DHC/biotin) surface because of the specic interaction of biotin moieties in the
polymer with SAv molecules, especially on the blocked surface. The above semi-quantied results further
indicate that the FT-SPR system is suitable for investigating interactions between polymer surface and
bio-molecules.
2010 Elsevier B.V. All rights reserved.
1. Introduction
Biomaterials have found wide applications in disease diagnosis
and therapy, such as biosensors, surgical implants, tissue-
engineering scaffolds, drug-delivery devices (DDDs), etc. In these
applications, biomaterials are in contact with biomolecules, cells,
tissues or organs, and interactions of polymer surfaces with these
biomolecules, cells, tissues or organs play an important role
in determining biomedical functions of these materials. Protein
adsorption is known as the rst event following the implanta-
tion of biomaterials and it determines many subsequent events.
In some cases, protein adhesion can be a necessary process for a
biomaterial to perform its function, for example, when the bone
repair materials are implanted into human body, desired protein
adsorption on the materials will improve the cell adhesion and
growth. For this purpose, incorporation of RGD peptides on the
materials was reported to promote adherence, spread and prolif-
eration of the desired cells on the implant surface [1]. However,

Corresponding author. Tel.: +86 431 85262775; fax: +86 431 85262775.
E-mail address: xbjing@ciac.jl.cn (X. Jing).
in most cases, non-specic protein adsorption leads to undesir-
able events, including thrombus formation, foreign body reaction,
bacterial infection, and other responses [2]. For instance, when a
macromolecule was usedas a drug carrier andwas sent into human
body, too much protein adsorption may enlarge the particle size,
thus prevent the cell uptake, or even cause thrombus. In the inter-
vention diagnosis and therapy, if the device surface is stained by
undesired proteins, the sensitivity of the detector will degrade, or
even leads to adverse effects [3]. Most biosensors are based upon
specic interactions between antibody and antigen or between lig-
and and receptor [46], and if the sensor is stained by proteins
adsorbed non-specically, it will surely do damage to the accuracy
and sensitivity of the detection [7]. Similarly, undesired protein
adsorption also does great harmto in vitro biosensors, blood trans-
fusion vessels [8], marine coatings [9], and so on, in addition to the
in vivo applications above.
Therefore, lots of effort have been made to enhance or to reduce
protein adsorption on biomaterial surfaces, e.g., by adjusting the
hydrophilicity [10,11], charge [12,13] and microphase separation
[14] of the surface. PEGylation is one of the most popular strate-
gies oftenusedto modify the proteins [1518], materials [14,19,20]
as well as the surface of the biosensor platforms [7]. But most of
0927-7765/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2010.11.020
J. Wei et al. / Colloids and Surfaces B: Biointerfaces 83 (2011) 220228 221
these modications are empirical or qualitative, because quanti-
tative measurement of proteins adsorbed on material surface is
difcult.
For quantitative determination of protein adsorption or bind-
ing on polymer surfaces, several techniques have been developed
in recent years, such as quartz crystal microbalance (QCM)
[2123], surface plasmon resonance (SPR) [2426], enzyme-linked
immunoassay (ELISA) [23,27], etc. Among them, ELISA is based on
the specic interaction of some antigen/antibody pairs and often
needs special biological reagents and uorescent labeling, QCM
and SPR are based on the physical responses of the adsorbed layer
(weight, capacity or resonance angle) and thus are suitable for
varies of adsorptions or bindings. Recently, a new SPR technique,
FT-SPR, is developed. It is also based on the SPR effect in a thin
gold lm, but the resonance frequency is detected by Fourier trans-
form infrared (FT-IR) system working in the near infrared region.
Because of the high accuracy of frequency measurement of FT-IR
spectrometer, FT-SPR is becoming a powerful tool of investigat-
ing surface adsorption and other interactions [2831], especially
protein/protein, protein/DNA, protein/RNA, and DNA/DNA inter-
actions. But interactions between proteins and various polymer
surfaces are seldom reported to our knowledge [32,33].
Therefore, in this paper, a biotin-bearing copolymer is coated
on an FT-SPR chip to form extra-thin lms. Because of the spe-
cic biotinavidin interaction, these lms may display specic
adsorption of streptavidin as well as non-specic adsorption of
other proteins. Different lm surfaces are constructed by treat-
ing the lms with vacuum drying, hydration and blocking so that
relative levels of the specic and non-specic adsorptions are
achieved. By means of FT-SPR measurements, these relative levels
of protein adsorption are differentiated. In this way, factors deter-
mining the specic and/or non-specic proteins are investigated
and strategies for suppression of non-specic protein adsorption
and enhancement of specic protein adsorption are suggested. To
our knowledge, this is the rst report on the systematic study of
both specic and non-specic protein adsorption on polymer lms
by means of FT-SPR technology.
2. Material and methods
2.1. Materials
l-Lactide (LA) was prepared from lactic acid in our own lab-
oratory and recrystallized from ethyl acetate three times before
use. Diethyl zinc (ZnEt
2
) was kindly supplied by Prof. Xianhong
Wang in Changchun Institute of Applied Chemistry. Pentaerythri-
tol was purchased from Aldrich. Palladium hydroxide on activated
charcoal [Pd(OH)
2
/C] was obtained from Shanxi Kaidai Chemi-
cal Industry Corp. in China and used without further purication.
1,3-Dicyclohexylcarbodiimide (DCC) was supplied by Chengdu
Tenglong Corporation in China and 4-dimethylaminopyridine
(DMAP, 99%) was purchased from Acros. Biotin and bovine serum
albumin (BSA) was purchased from SigmaAldrich and strepta-
vidin (SAv) was from Promega, USA. Toluene and tetrahydrofuran
(THF) were dried over sodium/benzophenone under a nitrogen
atmosphere prior to use. N,N-dimethylformamide (DMF) was dried
over CaH
2
and then distilled under reduced pressure before use.
Triply-distilledwater (TDW) was usedfor all aqueous solutions and
rinsing. Other reagents were commercially available andwere used
as received.
2.2. Measurements
Nuclear magnetic resonance (NMR) spectra were recorded on
a Bruker AV 300MHz instrument in CDCl
3
or DMSO-d
6
at 25

C.
Chemical shifts were given in parts per million with respect to
tetramethylsilaneas aninternal reference. FT-IRspectraweretaken
on a Nicolet 6700 instrument.
2.3. Copolymer synthesis and characterization
2.3.1. Preparation of P(LA-co-DHC)
Typical procedure for the synthesis of carbonate monomer
9-phenyl-2,4,8,10-tetraoxaspiro[5,5]undecan-3-one (PTO) and
copolymer P(LA-co-DHC) has been reported by our group [34].
Briey, as shown in Scheme 1a, pentaerythritol and benzaldehyde
were reacted in an aqueous solution containing 10% hydrochloric
acid (HCl) to form benzalpentaerythritol. After re-crystallization,
benzalpentaerythritol was reacted with ethyl chloroformate under
the catalysis of triethylamine. The product was re-crystallized
from THF/diethyl ether twice to get the carbonate monomer
PTO.
PTO, LAandZnEt
2
were addedintoa driedvessel andthe copoly-
merization was carried on at 130

C for 2h. After purication of the

copolymer, the protective groups were removed by hydrogenation
to get the copolymer P(LA-co-DHC) with double hydroxyl groups
on each carbonate unit (Scheme 1b). The structure of copolymer
P(LA-co-DHC) was conrmed by
1
H NMR.
2.3.2. Biotinylation of P(LA-co-DHC)
Biotinylation of P(LA-co-DHC) was carried out by esterica-
tion with biotin (Scheme 1c). Biotin was dissolved in 10mL DMF
at 70

C, then the solution was cooled in an ice/water bath. The

copolymer was added into the solution, followed by addition of
DCC and DMAP as the condensation agent and catalyst, respec-
tively. The mixture was stirredfor 24hat 0

C. The dicyclohexylurea
(DCU) formed was ltered out and the ltrate was concentrated
in vacuum. The concentrated product was precipitated into a large
amount of diethyl ether, ltered, andwashedwithisopropanol sev-
eral times. Thewhiteprecipitates weredriedunder vacuumat room
temperature.
2.4. Preparation and treatments of P(LA-co-DHC/biotin) lms
The following procedure was adopted to prepare the vacuum
dried copolymer lms: (1) 25mg of P(LA-co-DHC/biotin) was dis-
solved in 5mL of CHCl
3
completely; (2) the gold-lm of an SPR
chip (GWC Technologies Inc.) was dipped into the copolymer solu-
tion (the other side without gold was not allowed to contact with
the polymer solution) and the extra liquid was removed by a lter
paper toget a thinlmof thecopolymer; (3) thelmontheSPRchip
was dried rstly under ambient condition for 2h and then under
vacuumfor 24hto remove the solvent completely. Three suchsam-
ples were prepared. One of them was kept dry and was denoted as
vacuum-driedlm. The other twowere further treatedtoprepare
the hydrated and blocked lms, respectively, and all the experi-
ments were carried out in triplicate.
After the above procedure, a vacuum-dried chip was immersed
in TDW of 70

C for 1h and then 4

C for 11h to get a hydrated lm

for SPR measurements.
To prepare the blocked lm, another vacuum-dried chip was
soaked in a blocking reagent solution at 4

C for 12h, which con-

sisted of 0.5% poly(vinylpyrrolidone) (PVP) and 0.02% gelatin in
TrisHCl buffered saline (0.05M, pH 8.4) [35], and then washed
with TDW to remove the extra blocking reagent.
The lms were vacuum-dried before the atomforce microscopy
(AFM), X-rayphotoelectronspectrometer (XPS) andthickness mea-
surements to fulll the requirements of these analyses, while
they were immersed into TDW at room temperature for one hour
before confocal laser scanning microscopy (CLSM) and FT-SPR
222 J. Wei et al. / Colloids and Surfaces B: Biointerfaces 83 (2011) 220228
+
O
O
H
3
C
CH
3
O
O
Et
2
Zn O
O
O O
O O
Ph
O
m
n
H
2
, Pd (OH)
2
/C
O
O
O O
OH OH
O
m
n
2 h 130C
(b) PTO
P(LA-co-DHP) P(LA-co-DHC)
50C 48 h
O
H
OH OH
OH OH
+
OH OH
O O
Ph
Cl O
C
2
H
5
O
HCl, H
2
O
20C
5 h
O O
O O
Ph
O
TEA/THF, 0C
2 h
(a)
PTO
O
O
O O
O
m n
N
N
H
H
O
S
HO
O
+
DCC/DMAP
THF, 0C 24 h
(c)
N
N
H
H
O
S
O
O
N
N
H
H
O
S
O
O
P(LA-co-DHC)
P(LA-co-DHC/biotin)
Scheme 1. Preparation procedure of P(LA-co-DHC/biotin). (a) Synthesis of monomer, 9-phenyl-2,4,8,10-tetraoxaspiro[5,5]undecan-3-one (PTO); (b) polymerization and
deprotection of copolymer P(LA-co-DHC); (c) biotinylation of copolymer P(LA-co-DHC).
measurements to achieve equilibrated water absorption of the
lms.
2.5. Characterization of the copolymer lms
Atom force microscopy (AFM) measurements were performed
for the smoothness of the copolymer lm on the SPR chip with SPI
3800/SPA 300HV (Seiko Instrument Inc.) in tapping mode at room
temperature in air. The tip was of OMCL-ACTS-W type.
The lm thickness was determined using a Dektak 6M sur-
face proler (Veeco Instruments Inc., USA) by purposely scratching
the polymer-coated area and by subsequent prole analysis in the
vicinity of the scratches.
The surface elemental compositionof the polymer lmwas ana-
lyzed on an Escalab-MKII X-ray photoelectron spectrometer (VG
Scientic Ltd., UK) using Mg K radiation (1253.6eV) as the X-ray
source for excitation. The typical operating pressure in the analyt-
ical chamber was in the range of 10
9
to 10
10
Torr. The binding
energy of the experimental spectra was calibrated on the basis of
the most intense peak of C
1s
at 284.5eV.
The contact angle of the lms were measured by a DSA-10 drop
shape analyzer (Germany) equipped with a charge-coupled device
(CCD) camera. The water droplets hada xedvolume of 2L. When
the water droplet attached the lm surface, the picture was cap-
tured by the CCD camera and the contact angle was calculated by
the DSA software.
2.6. Confocal laser scanning microscopy (CLSM) measurements
2.6.1. FITC labeling of BSA
200mg of BSA (0.003mmol) was dissolved in 1.5mL of TDW
and FITC (uorescein isothiocyanate, 5mg, 0.013mmol) solution
in 10mL carbonate buffer (1000mL buffer consisted of NaHCO
3
7.56g, Na
2
CO
3
1.06g and NaCl 7.36g, pH 9.0) was added into the
BSA solution dropwise under stirring. The reaction lasted for 2h in
darkness. After the reaction, the solution was dialyzed against PBS
(phosphate buffered saline, pH 7.4) for 3 days, followed by freeze-
drying.
2.6.2. CLSM measurement
For CLSM measurement, the copolymer was spin-coated onto
glass chips and the coatings were treated in the same manner as
for those coated on the SPR chips. The treated glass chips were
immersed in BSA-FITC solution (1mg/mL in PBS, pH 7.4) for 5min,
then washed with TDW for 5 times and dried at room temper-
ature. Finally, each chip was covered with a cover glass with a
thin layer of glycerin in between. CLSM images were collected
with a Leica TCS SP2 CLSM (Leica Microsystems Heidelberg GmbH,
Germany) equipped with an 20 dry objective (NA=0.7) using
digital zooms of 132 attached to a Leica DM IRE2 inverted
microscope. Confocal optical sections were collected in the image-
scan xyz mode. The samples were excited by a 543nm He/Ne
laser.
J. Wei et al. / Colloids and Surfaces B: Biointerfaces 83 (2011) 220228 223
Fig. 1. (A) Schematic illustration of the SPR sensor; (B) a typical reection curve with a resonance peak; (C) a typical adsorption curve determined by FT-SPR.
2.7. Fourier-transform surface plasmon resonance (FT-SPR)
detection
FT-SPR measurements were carried out using a SPR-100 (GWC
Instruments, WI, USA) module coupled with a Nicolet 6700 FT-
IR spectrometer (Thermo-Electron, Madison, WI, USA) working
in the near infrared (NIR) region which was equipped with a
tungsten halogen near-infrared light source, a CaF
2
beam split-
ter and an InGaAs detector. The SPR-100 module consists of a
sample cell and a set of light path accessory. Structure of the
sample cell is shown in Fig. 1A. The NIR light beam enters the
glass prism at a xed incidence angle, reaches and is reected
by the gold lm on the SPR chip, and nally detected by the FT-
IR spectrometer. When a surface plasmon resonance takes place,
a minimum light intensity of the reected beam is obtained at
a certain NIR frequency (Fig. 1B) due to the resonance absorp-
tion, and this frequency is called resonance frequency. Unlike the
other SPR devices which measure the intensity of the reected
beam at a xed frequency as a function of incidence angle to
obtain the resonance angle, FT-SPR measures the intensity of the
reected beam as a function of incident beam frequency at a given
incidence angle. Because of the high accuracy and sensitivity of
Fourier-transform infrared spectroscopy, FT-SPR is more sensitive
than other techniques based on direct measurement of resonance
angle.
According to the principle of FT-SPR, the intrinsic resonance fre-
quency is determined by the thickness and refractive index of the
gold lm of the SPR chip. When the gold lm is in contact with a
layer of solidor liquid, the resonance frequency will shift toa higher
value (Fig. 1C) because of the involvement of this layer in the sur-
face plasmon resonance, and the frequency shift (termed as SPR
response) is dependent on the refractive index and thickness of
the layer. Therefore, the SPR chip serves as a sensor to the chemical
composition, density and/or thickness of the layer in contact with
the gold surface.
In the FT-SPR module, the gold lm on the SF-10 glass chip
is 45nm in thickness and is a square of 18mm18mm. In the
center of the square, a circular sample cell of 10mm in diam-
eter is formed by a rubber O-ring. The capacity of the ow
cell was 60L. The other side of the chip is brought into opti-
cal contact with the SF-10 equilateral prism using a refractive
index matching uid (Cargille Laboratories, USA). Circulation was
carried out by a Masterex L/S (Cole-Parmer, USA) peristaltic
pump at a circulating rate of 0.18mL/min. FT-SPR data were
collected using the Omnic software version 7.0 (Thermo Elec-
tron), at the resolution of 32cm
1
over the range from 6000 to
12,000cm
1
.
In the present study, the P(LA-co-DHC/biotin) lm on the SPR
chip was the rst layer. A protein solution was pumped through
the outer surface of polymer coating. When protein molecules
were adsorbed onto the P(LA-co-DHC/biotin) coating, the reso-
nance frequency of the whole systemwould change. The frequency
shift would correspond to the proteins adsorbed on the polymer
surface.
Experimentally, PBS was rst circulated through the ow cell
and the resonance frequency was adjusted to ca. 9000cm
1
. This
frequency was considered as the baseline of adsorption. Then, BSA
or SAv solution in PBS buffer (pH 7.4) of 0.2mg/mL was allowed
to ow through the cell, and its resonance frequency was plot-
ted against time to demonstrate the protein adsorption process.
After the resonance frequency leveled off, PBS was circulated again
through the cell to rinse the adsorbate layer on the SPR chip. The
resonance frequency decreased correspondingly. The difference of
the nal resonance frequency fromthe initial baseline was consid-
ered to reect the amount of the protein adsorbed on the polymer
lm.
224 J. Wei et al. / Colloids and Surfaces B: Biointerfaces 83 (2011) 220228
Fig. 2. AFM images and roughness of P(LA-co-DHC/biotin) coated SPR chip (A and B) and bare SPR chip (C and D).
3. Results and discussion
3.1. P(LA-co-DHC/biotin) copolymer
P(LA-co-DHP) copolymer was prepared by ring-opening copoly-
merization of l-lactide (LA) and cyclic carbonate PTO at 130

C
under the catalysis of diethyl zinc as shown in Scheme 1b. After
removal of the protective groups, biotin was attached to the pen-
dant OHgroups of the copolymer withthe helpof DCCandDMAP as
showninScheme 1c. The structure of eachsynthesizedproduct was
conrmed by
1
H NMR spectrum (for details, see Fig. S1 in the sup-
plementary information).
3.2. P(LA-co-DHC/biotin) lm on the SPR chips
The main concern of the present study is to reduce non-specic
adsorption and to enhance specic adsorption of proteins. There-
fore, preparation of appropriate samples is critical and essential.
P(LA-co-DHC/biotin) was used as the starting material. Its main
constituent is poly(l-lactide), awell-knownbiodegradablepolymer
already approved by FDA, USA as biomedical materials. Reduc-
tion of non-specic protein adsorption on it is of signicance for
its medical applications. Incorporation of DHC units is for attach-
ment of biotin, that is expected to result in specic adsorption of
stretavidin. Therefore, non-specic andspecic adsorptions coexist
on the P(LA-co-DHC/biotin) lms. The levels of these adsorptions
are adjusted by the surface treatments, i.e., simple vacuum-drying,
hydrationandblocking. It is reportedthat hydrophilic lms areusu-
allyprotein-resistant comparedtohydrophobic ones. Inthepresent
study, the lms were treated in 70

C water for 1h. In this way,

the biotin moieties may migrate to the outer surface of the lm
due to the molecular motion above Tg (Tg of P(LA-co-DHC) is 50

C
[34]). Free water molecules may be adsorbed onto the lm surface
via H-bond formation between water and CO and NH groups in
the copolymer. Therefore hydrophilicity would be improved after
this treatment. In the literature, inert polymers such as PVP, BSA,
gelatin, lactoprotein, etc. areusedtosuppress proteinadsorptionon
material surface[3538]. Surface-blockingtreatment inthepresent
study, on the one hand, introduces a water soluble coating onto the
lm so as to enhance its hydrophilicity and protein resistance, and
on the other hand, all or most active sites for non-specic adsorp-
tions are saturated by surface blockers but biotin moieties are left
alone for specic adsorption of streptavidin. In short, the three
kinds of P(LA-co-DHC/biotin) lms should have different levels of
hydrophilicity, non-specic andspecic adsorptions. The measure-
ments in the next sections will support this expectation.
3.2.1. Surface morphology and lm thickness
The FT-SPR measurements are performed in a ow cell with
a specied apparent area dened by the O-ring diameter (total
amount of adsorption) and by the NIR beam diameter (FT-IR sig-
nal). Obviously, surface morphology and roughness will determine
the real surface area and thus inuence protein adsorption. There-
fore, the surface morphologies of the polymer-coated and bare SPR
chips were examined by AFM and the images were given in Fig. 2.
From these images, mean surface roughness (Ra) was calculated
J. Wei et al. / Colloids and Surfaces B: Biointerfaces 83 (2011) 220228 225
403 402 401 400 399 398 397
1000
1500
2000
2500
3000
3500
4000
C
o
u
n
t
s

/

s
Binding Energy (eV)
Dried surface
Hytrated surface
Blocked surface
N1s
167 166 165 164 163 162 161
100
200
300
400
500
600
700
C
o
u
n
t
s

/

s
Binding Energy (eV)
Dried surface
Hytrated surface
Blocked surface
S2p
536 534 532 530
0
3000
6000
9000
12000
15000
18000
C
o
u
n
t
s

/

s
Binding Energy (eV)
Dried surface
Hytrated surface
Blocked surface
O1s
292 290 288 286 284 282
0
2000
4000
6000
8000
10000
12000
C
o
u
n
t
s

/

s
Binding Energy (eV)
Dried surface
Hytrated surface
Blocked surface
C1s
Fig. 3. XPS proles of elements C, O, N and S on the surfaces of the dried, hydrated and blocked P(LA-co-DHC/biotin) lms.
using SPIMAN software. It could be seen that Ra values of the vac-
uumdried lm(1.95nm), hydrated lm(1.85nm) and blocked lm
(2.31nm) were close to that of bare gold chip (1.66nm). They are
smooth enough for FT-SPR analysis. Because an additional layer of
blocking coating is formed on the polymer lm, the higher Ra value
(2.31nm) is understandable. Correspondingly, higher adsorption
should be observed on the blocked lm. In fact, the opposite is
observed as shown in Sections 3.3 and 3.4. Therefore, contribution
of the surface roughness to the adsorption is not a determining
factor compared to others and will be ignored in the following
discussion.
It was reported that there is a thickness limitation in SPR mea-
surement. For solid lm, this thickness limit is about 300nm [39].
In this study, the vacuum-dried lms on SPR chips were about
658nm (n=6, three chips, two points on each) in thickness
according to the surface prole measurement, suggesting that the
lm thickness was suitable for SPR measurements. Practical mea-
surements showed that hydration and blocking treatments did not
cause signicant change in lm thickness. Therefore, there was
enough room for protein adsorption.
3.2.2. XPS analysis
The surface element compositions after different treatments
wereexaminedbyXPS. Theresults arecollectedinFig. 3andTable1.
Because the XPS chamber was in high vacuum, the XPS samples
lost all or most of the water molecules adsorbed on their surfaces
and the detected data reected chemical composition on the lm
surface of the lms without the interference from the adsorbed
water.
It is seen that the elemental composition was dependent on
surface treatments. As shown in Scheme 1, sulfur and nitrogen ele-
ments only existedinthe biotinmoieties inP(LA-co-DHC/biotin). In
thevacuum-driedlm, thedeterminedcontents of sulfur andnitro-
gen stood to some extent for the average content of biotin group
in the polymer. These two contents increased whereas oxygen
content decreased after hydration treatment (Table 1), conrming
migration of the biotin moieties to lmsurface. Polar structure and
possibility of H-bond formation of the biotin moieties as well as
mobility of the molecular chains above Tg were responsible for this
migration. After blocking treatment, sulfur content on the surface
decreased while nitrogen content increased because the blocking
layer composed of PVP and gelatin contained abundant nitrogen
and much less sulfur. This blocking agent layer is responsible for
suppression of the non-specic adsorption of proteins as demon-
strated later in Section 3.2.3.
3.2.3. Contact angle of the lm surfaces
To measure the hydrophilicity of the P(LA-co-DHC/biotin) lms
after different treatments, contact angle measurements were
performed by DSA-10 drop shape analyzer on these lms. As
shown in Fig. 4, the vacuum-dried lm had highest contact
angles ( =95.210.2

), the blocked lm gave the lowest con-

tact angle ( =61.22.8

), and the hydrated lm was in between

Table 1
Surface element contents of the lms based on XPS analysis.
Samples Surface element contents (mol%)
C O N S
Vacuum-dried lm 63.33 35.65 0.80 0.22
Hydrated lm 67.74 27.68 3.52 1.06
Blocked lm 65.31 30.69 3.75 0.25
Theoretical ratios of the
copolymer (calculated
from
1
H NMR)
60.18 38.41 0.94 0.47
226 J. Wei et al. / Colloids and Surfaces B: Biointerfaces 83 (2011) 220228
Fig. 4. Contact angle analysis on P(LA-co-DHC/biotin) lms. The data were collected at three points on each chip and the experiment was carried out in duplicate. (Data were
given in meanSD, n=6.)
100 80 60 40 20 0
-10
0
10
20
30
40
50
60
70
80
PBS rinse
Blocked film
Hydrated film
Dried film
Streptavidin (0.2mg/ml PBS)
S
P
R

R
e
s
p
o
n
s
e

i
n

W
a
v
e

N
u
m
b
e
r

(
c
m
-
1
)
B
50 40 30 20 10 0
0
10
20
30
40
50
60
70
80
S
P
R

R
e
s
p
o
n
s
e

i
n

W
a
v
e

N
u
m
b
e
r

(
c
m
-
1
)
A
BSA (0.2mg/ml PBS)
Time (min) Time (min)
PBS rinse
Dried film
Hydrated film
Blocked film
Fig. 5. FT-SPR analyses on P(LA-co-DHC/biotin) lm surface for non-specic absorption of BSA (0.2mg/mL) (A) and specic interaction of streptavidin (0.2mg/mL) (B).
( =73.33.7

). This order of contact angles reects the extents of

hydrophilicity of these lms, indicating our successful modication
to the lm surfaces.
3.3. Non-specic adsorption of BSA
It is well-known that BSA is an inert protein. It does not speci-
callyinteract withthe copolymers synthesizedinthe present study.
Therefore, it was used as a model protein for non-specic adsorp-
tion. FT-SPR experiments were carried out for this purpose. In the
experiment, BSA solution (0.2mg/mL in PBS buffer, pH 7.4) was
pumpedinto the owcell to contact the lmsurface andthe FT-SPR
response was plotted as a function of adsorption time. As shown
in Fig. 5A, with BSA adsorption, the resonance moved to a higher
wavenumber. After the equilibrium as indicated by the leveling of
the curve was established, PBS was pumped into the ow cell, the
resonance frequency moved down, indicating the washing-off of
the un-adsorbed BSA on the surface, and another equilibrium was
established, corresponding to the adsorption of BSA on the poly-
mer surface. As shown in Fig. 5A, the vacuum-dried polymer lm
had the most serious non-specic adsorption, probably due to its
hydrophobic nature. After hydration, some of the biotin moieties
migrated from the inner to the surface of the lm and the sur-
face hydrophilicity was enhanced, so the adsorption decreased by
a factor of two. The blocked lm displayed the lowest non-specic
adsorption because the blocking process not only caused the same
500 1000 1500 2000 2500 3000 3500 4000
b
c
Wavenumber (cm
-1
)
a
Fig. 6. FT-IR spectra of P(LA-co-DHC/biotin) copolymer lms on the SPR chip before
(a) and after (b) BSA adsorption. The lms on the gold chip were detected by an 80

grazing angle accessory and the grazing reectionabsorption spectra were trans-
ferred to transmittance. For comparison, transmittance spectrum of BSA powders
was recorded using a KBr pellet and shown as spectrum c.
J. Wei et al. / Colloids and Surfaces B: Biointerfaces 83 (2011) 220228 227
Fig. 7. CLSM images of FITC-labeled BSA absorbed on the P(LA-co-DHC/biotin) lms non-specically. The same laser intensity was used for excitation of the samples. (A)
Vacuum-dried lm; (B) hydrated lm; (C) blocked lm (bar =10m.)
Table 2
SPR responses (cm
1
) of the protein adsorptions on P(LA-co-DHC/biotin) lms.
Samples SPR response to SAv SPR response to BSA SPR response to SAv binding Non-specic/specic ratio
R
SAv
R
BSA
R=R
SAv
R
BSA
R
BSA
/R
Vacuum-dried lm 65 54 11 4.9
Hydrated lm 48 28 20 1.4
Blocked lm 31 7 24 0.29
effect of hydration, but also blocked the active sites of physical or
chemical adsorption of BSA.
The above observation was conrmed by FT-IR and CLSM mea-
surements. The P(LA-co-DHC/biotin)-coatedSPRchipwas analyzed
by grazing reectionabsorption infrared spectroscopy. As shown
in Fig. 6, after BSA adsorption, characteristic absorption bands over
37002700cm
1
(H-bonded NH, see spectrum c) and those over
17001500cm
1
(amide I and amide II) of BSA was observed in its
FT-IRspectrum(spectrumb vs. a), indicating that a layer of BSAwas
left on the polymer surface after PBS washing. For CLSM measure-
ment, a layer of P(LA-co-DHC/biotin) was coated on a glass slide
and FITC-labeled BSA was adsorbed on the polymer surface and
treated as the P(LA-co-DHC/biotin)-coated SPR-chip. Fig. 7 showed
the CLSM images obtained. Under the same excitation condition,
the vacuum-driedlmdisplayedthe most intense uorescence and
the blocked lm displayed the weakest, corresponding to the BSA
thicknesses on the polymer surface.
3.4. Specic adsorption of streptavidin
It is well known that there is very specic interaction between
biotin and avidin. Their coordination constant, 10
15
per mole, is
one of the highest for naturally occurring ligand-receptor pairs.
Therefore, streptavidin (SAv) was chosen as a model protein to
demonstrate the specic adsorption on P(LA-co-DHC/biotin) sur-
face. When a streptavidin solution (0.2mg/mL in PBS buffer, pH
7.4) was allowed to pass the polymer surface, the FT-SPR system
detected minimumfrequency shift as shown in Fig. 5B. The adsorp-
tion and rinsing process looked similar to that of BSA (Fig. 5A).
Among the three samples used, the amount of the adsorbed SAv
was the most on the vacuum-dried lm and the least on the
blocked lm. The frequency shifts were 65, 48 and 31cm
1
(R
SAv
,
Table 2), respectively. It is interesting to notice that the three FT-
SPR responses to SAv were more than those to BSA (R
BSA
=54,
28, and 7cm
1
, respectively). This difference may be attributed
to specic adsorption of SAv, i.e., coordination of SAv with biotin
on the lm surface. This is because SAv is a protein too, it may
be adsorbed on the lm non-specically as BSA is. Therefore, the
observed SPR responses to SAv (R
SAv
) can be divided into two parts
due to non-specic adsorption and specic binding of SAv, respec-
tively. By assuming that the SPR responses to SAv adsorption are
equal to those to SBA, R=R
SAv
R
BSA
may be considered as the
SPR responses to SAv binding. As listed in Table 2, R values for
the three samples are 11, 20, and 24cm
1
, respectively, in oppo-
site order to non-specic adsorption. Because biotin is responsible
for the specic binding of SAv, these R values surely reect the
amounts of biotin moieties exposed on the lm surface. The water
treatment at 70

C for 1h leads to almost twofold increase of SAv

binding. It conrms successful migration of the biotin moieties
to the lm surface. Blocking with PVP and gelatin results in an
SPR response of 24cm
1
, even more than that to hydration. This
implies that the blocking treatment does not block specic bind-
ing between biotin and SAv although it suppresses the non-specic
adsorptions of SAv as well as BSA signicantly.
4. Conclusion
A biotin bearing copolymer was prepared and was coated onto
a FT-SPR chip to construct a model system to study the specic
adsorption of streptavidin and non-specic adsorption of BSA on
the polymer surface. To compare the factors inuencing the sur-
face adsorption, the polymer lms were vacuum-dried, hydrated
at 70

C, and treated with a blocking agent solution consisting of

0.5% PVP and 0.02% gelatin in TrisHCl buffered saline (0.05M,
pH 8.4). These polymer lms were characterized by AFM, XPS
and contact angle measurement. The BSA adsorption process and
quantity were monitored by CLSM and FT-SPR. Results showed
that the vacuum-dried lm had the most BSA adsorption; hydra-
tion treatment led to migration of the biotin moieties from inner
lm to surface and thus resulted in less BSA adsorption; blocking
layer on the polymer surface saturated the active sites for phys-
ical and chemical adsorptions on the surface and thus weakened
the BSA adsorption. Adsorption of streptavidin displayed simi-
lar polymer-surface-status dependence, i.e., more adsorption on
vacuum-dried surface, less adsorption on hydrated surface and
least adsorption on blocked surface. Compared with BSA, SAv
showed more adsorptions on P(LA-co-DHC/biotin) surface because
of the specic binding of the biotin moieties in the polymer with
the SAv molecules. Compared to vacuum-dried sample, hydrated
and blocked samples showed improved specic binding of biotin
228 J. Wei et al. / Colloids and Surfaces B: Biointerfaces 83 (2011) 220228
with SAv because the biotin moieties migrated to the surface dur-
ing the sample treatments. In short, surface hydration and blocking
are simple and effective means of post-treatment of biomedical
polymer materials for both suppression of non-specic protein
adsorption and enhancement of specic interaction of bioactive
proteins like streptavidin. The above semi-quantied results also
indicate that the FT-SPR system is suitable for investigating inter-
actions between polymer surface and bio-molecules. The sample
preparation is simple. Fluorescent or immunological labeling is not
needed. The detection is sensitive and semi-quantitative.
Acknowledgements
Financial support was provided by the National Natural Science
Foundation of China (Project No. 20674084, 50733003, A3 Fore-
sight Program No. 20621140369), by 100 Talents Program of the
Chinese Academy of Sciences (No. KGCX2-YW-802), and by the
Ministry of Science and Technology of China (973 Project, No.
2009CB930102; 863 Project, No. 2007AA03Z535).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.colsurfb.2010.11.020.
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