Studies on Interaction of Polysaccharide-Templated SilverNanoparticles with Bovine Serum Albumin

Jing-Kun Yan • Pan-Fu Cai • Xiao-Qiang Cao •

Ting-Ting Fan • Hai-Le Ma

Received: 26 January 2013 / Accepted: 25 August 2013 / Published online: 4 September 2013

� Springer Science+Business Media New York 2013

Abstract In the present work, the interaction between

bovine serum albumin (BSA) and as-synthesized silver

nanoparticles (Oc-AgNPs) by using 4-acetamido-TEMPO-

oxidized curdlan (Oc), as a reducing and stabilizing agent, was

studied through fluorescence quenching method, ultraviolet

visible spectrum (UV–Vis), and circular dichroism measure-

ment. The results presented clearly indicate that the intrinsic

fluorescence of BSA molecule was effectively quenched after

the interaction with Oc-AgNPs by a static mechanism, which

is further confirmed by UV–Vis analysis. The apparent

binding constant (K), number of binding sites (n), and disso-

ciation equilibrium constant (KD) were calculated to be

7.5 9 105 M-1, 1.03, and 3.0 ± 0.6 lM, respectively. Fur-

thermore, a conformational change of BSA was also observed

when the Oc-AgNPs–BSA interactant formed.

Keywords Curdlan � 4-Acetamido-TEMPO � Silver

nanoparticles � BSA � Interaction

1 Introduction

It is well-known that albumins play an important role in

carrying drugs as well as endogenous and exogenous sub-

stances [1]. Serum albumins are the most abundant proteins

in plasma [2]. As the major soluble protein constituents of

the circulatory system, they have many physiological

functions [3]. Among the serum albumins, bovine serum

albumin (BSA) has a wide range of physiological functions

involving binding, transport and delivery of fatty acids,

porphyrins, bilirubin and steroids, etc. In general, it con-

tains 582 amino acid residues, and two tryptophans at

positions 134 and 212 as well as tyrosine and phenylala-

nine [4]. In view of water-soluble nature and well-char-

acterised structure of BSA, it has been often utilized as

protein model system for the study of interactions [5–7].

Recently, silver nanoparticles (AgNPs) have become the

focus of intensive research owing to their wide range of

applications in areas such as catalysis, optics, antimicro-

bials, and the biomaterial production [8, 9]. One kind of

important applications of AgNPs is used as a biosensor in

chemical and biochemical field for detection of proteins,

DNA sequencing, clinical diagnostics, etc. due to the sur-

face plasmon resonance (SPR) and large effective scatter-

ing cross section of individual silver nanoparticles. Among

them, there are some reports on interaction of BSA and

silver nanoparticles [10–12]. Furthermore, the AgNPs

could adsorb BSA leading to the formation of a protein

‘‘corona’’, and the stabilizing agents such as citric acid and

polyvinylpyrrolidone (PVP) in the preparation of AgNPs

play an important role in the corona formation [13].

Recently, studies on polysaccharides-conjugated Ag

nanoparticles have attracted intense attention for high-yield

and ‘‘green’’ production under relatively mild conditions

[14–17]. However, to the best of our knowledge, the

J.-K. Yan � P.-F. Cai � X.-Q. Cao � T.-T. Fan � H.-L. Ma

School of Food & Biological Engineering, Jiangsu University,

Zhenjiang 212013, China

J.-K. Yan (&) � H.-L. Ma

Physical Processing of Agricultural Products Key Lab of Jiangsu

Province, Zhenjiang 212013, Jiangsu, China

e-mail: jkyan_27@163.com

J.-K. Yan

Guangdong Province Key Laboratory for Green Processing of

Natural Products and Product Safety, South China University of

Technology, Guangzhou 510640, Guangdong, China

123

J Inorg Organomet Polym (2013) 23:1383–1388

DOI 10.1007/s10904-013-9940-8

interaction between BSA and polysaccharide-templated

silver nanoparticles has not yet been reported.

Herein, the major objective of the present study was to

investigate the interaction between BSA and polysaccha-

ride-templated silver nanoparticles by various spectro-

scopic measurements, such as fluorescence spectroscopy,

ultraviolet visible spectrum (UV–Vis), and circular

dichroism (CD). This study is also expected to provide

important insight into the interactions of the physiologi-

cally important protein BSA with silver nanoparticles.

2 Experimental Section

2.1 Materials

Commercial curdlan (DPw 6790; MW 1.1 9 106 kDa) was

obtained from Wako Pure Chemical Corporation (Osaka,

Japan). 4-acetamido-TEMPO-oxidized curdlan (Oc) was

prepared by the reported method [18]. The carboxylate

content and molecular weight (MW) of Oc was 4.87 mmol/g

and 5.7 9 105 kDa, determined by electric conductimetry

titration and size-exclusion chromatography with multi-

angle laser-light scattering (SEC–MALLS), respectively

[19]. Silver nitrate (AgNO3) and bovine serum albumin

(BSA, MW 6.6 9 104 kDa) were purchased from Sigma-

Aldrich Chemical Corporation (St. Louis, MO, USA). Other

chemicals and solvents were of laboratory grade, and used

without further purification.

2.2 Green Synthesis of Silver Nanoparticles

The silver nanoparticles was synthesized by directly

hybriding 1 mL of 10 mM AgNO3 solution in 1 mL of

1 mg/mL Oc aqueous solution at 100 �C for 120 min under

continuous stirring in the dark. The suspension was then

filtered through a filter (0.22 lm, Millipore) and centrifu-

gated at 30,000 rpm for 30 min, followed by redispersion

in acetone and water for removing excess Oc and Ag?. The

silver nanoparticles were then redispersed in water, and

dialyzed (MWCO, 14 kDa) against distilled water for 48 h.

The final solution was freeze-dried to obtained the

polysaccharide-templated silver nanoparticles, coded as

Oc-AgNPs.

2.3 Characterization of Silver Nanoparticles

The UV–Vis spectrum of Oc-AgNPs was recorded from

300 to 600 nm at room temperature using a Varian cary

100 spectrophotometer (Varian Co., USA), and the solution

containing only Oc was used as the blank. Transmission

electron microscopy (TEM, JEM-2100, JEOL Ltd., Japan,

200 kV) was used to characterize the size and morphology

of the as-prepared Oc-AgNPs. The tested sample was

prepared by placing a drop of sample solution (0.5 mg/mL)

on a 300 mesh carbon coated copper grids and then dried at

room temperature for 30 min. A wide-angle XRD instru-

ment (D8-Advance, Bruker Co., Germany) was used to

characterize the critical structure of the AgNPs. XRD

patterns with Cu Ka radiation (k = 0.15406 nm) at 40 kV

and 40 mA were recorded in the region of 2h from 30� to

80� with a step speed of 4�/min. Energy-dispersive X-ray

spectroscopy (EDX, Inca Energy-350, Oxford Co., UK)

attached to scan electron microscopy (SEM, JSM-7100F,

JEOL Ltd., Japan) was used to determine the elemental

composition of the nanoparticles.

2.4 Preparation of Oc-AgNPs and BSA Interactant

To prepare the interactant, the BSA solution (1.0 9 10-5

mol/L) was added to a different concentrations Oc-AgNPs

aqueous solutions, followed by incubation at 25 �C for

24 h. After incubation, the mixtures were stored at 4 �C

before tested.

2.5 Characterization of the Interactant

The UV–Vis spectra of free BSA and Oc-AgNPs-BSA

interactants were recorded in a Varian Cary 100 spectro-

photometer (Varian Co., USA) from 250–500 nm at room

temperature. The absorbance of free BSA and the inter-

actants were recorded at 280 nm. Fluorescence spectra and

fluorescence intensities of free BSA and Oc-AgNPs–BSA

interactants were recorded on a Fluoromax-4 fluorescence

spectrophotometer (HORIBA Jobin–Yvon, France), and

the emission spectra were recorded in the range of 290–

500 nm at an integration time of 1.0 s. The excitation and

emission slit width were both 5.0 nm. The CD spectra of

free BSA and Oc-AgNPs–BSA interactant were recorded

in a range of 200–250 nm on a circular dichroism spec-

trometer (JASCO J-815, Japan) at 25 �C. A relative a-helix

content of BSA molecule was determined as follows [20]:

% a-Helix = � h½ �208 � 4000� �

= 33000 � 4000ð Þ ð1Þ

where [h]208 is the mean residue ellipticity in deg cm2

dmol-1 at 208 nm.

3 Results and Discussion

3.1 Characterization of Oc-AgNPs

Oc was a water-soluble polysaccharide containing car-

boxylate (COO-) groups in C-6 position of curdlan mol-

ecule. The large number of hydroxyl and carboxylic groups

on this biopolymer facilitated the complexation of silver

1384 J Inorg Organomet Polym (2013) 23:1383–1388

123

ions. Thus, in the present work, Oc was used as both

reducing and stabilizing agent in preparation of silver

nanoparticles. Figure 1a shows the UV–Vis absorption

spectrum of Oc-AgNPs solution. There was a maximum

absorption peak at *410 nm appeared, which corre-

sponded to the SPR [21], indicating that the formation of

AgNPs. The shape of the plasmon band is symmetrical and

narrow, suggesting that AgNPs are spherical and mono-

disperse [22]. In addition, there are no peaks located

around 335 and 560 nm, indicating the complete absence

of nanoparticle aggregation [23, 24]. Meanwhile, the color

of the Oc-AgNO3 solution changed from colorless to

golden yellow, further indicating the formation of the

AgNPs. Figure 1b shows the typical transmission electron

microscopy (TEM) image of the Oc-AgNPs. The TEM

observation demonstrated that the particles are spherical in

shape and reasonably monodisperse. Nearly all the nano-

particles were well separated and no agglomeration was

noticed, and the mean diameter of the Oc-AgNPs was

calculated to be *15 nm. Figure 1c shows the XRD

spectrum of the Oc-AgNPs. The peaks at approximately

38.3�, 44.4�, 65.0�, 77.8� and 82.1� were attributed to the

(111), (200), (220), (311) and (222) planes of the cubic

structure of Ag [25]. No peaks related to other crystalline

phases were found. Among them, the diffraction peak at

38� was the only highly intense peak. Moreover, the ratio

between the (220) and (111) peaks was much lower than

the standard value (0.1 versus 0.4). These results further

demonstrated that the preferred orientation for the calcined

AgNPs was assigned to the (111) lattice plane. Figure 1d

shows the energy dispersive X-ray analysis (EDX) of the

silver nanoparticles. In the EDX spectrum, there was a

strong and typical optical absorption peak at about 3 keV

was attributed to the SPR of metallic silver nanocrystals

[26], indicating that the silver nanoparticles was formed in

the reaction medium.

3.2 Fluorescence Quenching and Stern–Volmer

Analysis

In this study, the interaction between Oc-AgNPs and BSA

was investigated by using fluorescence quenching method

30 40 50 60 70 80 90

200

400

600

800

(222)

C

Inte

nsity

2Theta (degree)

(111)

(200)

(220) (311)

B

100nm

0 1000 2000 3000 4000 5000 6000

0

200

400

600

800

1,000

Ag Ag

D

C

O

Na Ag

cps/

eV

eV

Ag

A

300 350 400 450 500 550 6000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

AB

SWavelength (nm)

Fig. 1 a The UV–Vis

absorption spectrum of the Oc-

AgNPs, and the inset is the

photo of the Oc-AgNPs;

b typical TEM image of the Oc-

AgNPs; c XRD pattern; d EDX

spectrum from SEM

300 350 400 450 500

0.0

2.0x106

4.0x106

6.0x106

8.0x106

Inte

nsity

(a.

u.)

Wavelength (nm)

a

e

Fig. 2 The fluorescence spectra of BSA (1.0 9 10-5 M) in the

presence of Oc-AgNPs at different concentrations (a. 0, b. 0.4, c. 0.6,

d. 0.8, e. 1.0 9 10-7 M)

J Inorg Organomet Polym (2013) 23:1383–1388 1385

123

[27]. Figure 2 shows the fluorescence spectra of BSA in the

presence of different concentrations of Oc-AgNPs. It can

be observed that free BSA and Oc-AgNPs–BSA interac-

tants exhibit maximum peaks at around 347 nm which

corresponding to the presence of tyrosine, tryptophan and

phenylalanine residues in BSA molecule. Moreover, the

fluorescence emission intensity at 347 nm decreased with

the increase of Oc-AgNPs concentration, indicating that the

intrinsic fluorescence of BSA molecule was quenched by

interacting with Oc-AgNPs.

Fluorescence quenching has been widely utilized for

revealing the accessibility of fluorophores in the protein

matrix to quenchers, and it is usually described by the

Stern–Volmer expression [28]:

F0=F ¼ 1þ Ksv Q½ � ¼ 1þ Kqs0 Q½ � ð2Þ

where F0 and F are the fluorescence intensities of BSA in

the absence and presence of quencher, Ksv is Stern–Volmer

constant, Kq is the biomolecular quenching rate constant,

[Q] is the concentration of quencher, and s0 is the average

lifetime of BSA, 10-8 s [29]. Figure 3a shows a linear plot

between F0/F against [Oc-AgNPs] according to Eq. (2).

From the slope, we could calculated the quenching rate

constant (Kq) as 5.0 9 1013 M-1 s-1, which was higher

than that of the maximum collisional quenching constant

(Kq) of various kinds of quenchers to biopolymers

(2.0 9 1010 M-1 s-1) [29]. This showed that the quench-

ing is not dynamic in nature.

For static quenching, the relationship between the fluo-

rescence intensity and the binding constant (K) can be

deduced from the following relation [10]:

log F0 � Fð Þ=F½ � ¼ n log Q½ � þ log K ð3Þ

where K is the binding constant of Oc-AgNPs with BSA,

[Q] is the concentration of Oc-AgNPs, n is the number

of binding sites. Figure 3b showed that the plot of

log[(F0-F)/F] versus log[Oc-AgNPs] according to the

Eq.(3). From the intercept and slope, we obtained the

binding constant ‘‘K’’ as 7.5 9 105 M-1 and binding sites

‘‘n’’ (1.03) for Oc-AgNPs with BSA. The value of ‘‘n’’

obtained from this study indicates that there is one avail-

able surface site between BSA molecule and Oc-AgNPs. It

seems likely that the presence of a hydroxyl group in the

tyrosine residues in the BSA moiety affects the interaction

of BSA with Oc-AgNPs, similar to AgTiO2 as reported by

Kathiravan et al. [10].

3.3 UV–Vis Spectral Study

Figure 4a shows the absorption spectra of BSA in the

presence and absence of Oc-AgNPs solution at different

concentrations. It can be observed that upon increasing the

concentration of Oc-AgNPs the absorption peak at around

280 nm is caused by the p?p* transition of aromatic

amino acid residues of BSA increase, indicating that the

BSA molecule has absorbed on the surface of Oc-AgNPs.

Similar observations have been stated in previous reports

[10, 30]. In addition, regardless of the concentration of Oc-

AgNPs, the absorption peak at *410 nm which was

characteristic for the formation of Ag0 nanoparticles

decreased markedly, further implying that the interaction

of BSA and Oc-AgNPs had been underwent.

In order to quantify the inherent strengths of the BSA/

AgNPs interactions, and the dissociation equilibrium con-

stant KD can be deduced by Eq. (4)

BSAþ Oc-AgNPs � BSA � � �Oc-AgNPs

KD ¼ BSA½ � � Oc-AgNPs½ �= BSA � � �Oc-AgNPs½ �: ð4Þ

The change in intensity of the absorption peak (280 nm)

as a result of the formation of the surface complex were

utilized to obtain KD. According to former report [31], the

KD can be further expressed as Eq. (5):

1= Aobs � A0ð Þ ¼ 1= Ac � A0ð Þ þ KD

� 1= Ac � A0ð Þ Oc-AgNPs½ � ð5Þ

where Aobs is the observed absorbance of the solution

containing different concentrations of Oc-AgNPs at

280 nm; A0 and Ac are the absorbance of free BSA and Oc-

AgNPs–BSA interactants at 280 nm, respectively.

0 2 4 6 8 10

1.0

1.1

1.2

1.3

1.4

1.5

1.6

F0/

F[AgNPs](10-7M)

A

-4.9 -5.0 -5.1 -5.2 -5.3 -5.4 -5.5

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

log[

(F0-

F)/F

]

Log [Oc-AgNPs]

BFig. 3 a Stern–Volmer plot of

F0/F versus [Oc-AgNPs], b plot

of log [(F0 - F)/F] versus log

[Oc-AgNPs]. The concentration

of BSA was 1.0 9 10-5 M

1386 J Inorg Organomet Polym (2013) 23:1383–1388

123

Figure 4b shows the linear relationship between KD�1/

(Aobs - A0) versus reciprocal concentration of Oc-AgNPs

with a slope equal to 1/(Ac-A0) and an intercept equal to

1/(Ac - A0). The value of KD derived from this plot is

3.0 ± 0.6 lM. Generally, the KD value found herein cor-

respond with expectations from literature findings on

comparable protein/NPs system. For example, Treuel et al.

[12] reported a high affinity of BSA to citrate-coated Ag-

NPs system with the lower KD value (5.0 9 10-3 lM), but

the KD value for the BSA/PVP-coated AgNPs system was

1.3 lM. In addition, Rocker et al. [32] investigated the

interactions of human serum albumin (HSA) with polymer-

coated NPs reporting a value of KD * 3.8 ± 1.5 lM from

kinetic studies. These results revealed that the citrate-

coated NPs had a stronger interaction with proteins than

that of polymer-coated NPs. This may be due perhaps to

the different mechanism in the interaction processes.

3.4 CD Spectra Analysis

In general, circular dichroism (CD) spectroscopy is often

applied to estimate the conformation of proteins [20, 33].

In the present work, The CD was utilized for investigating

the conformational change of BSA before and after inter-

action with Oc-AgNPs. In the Oc-AgNPs itself, no CD was

observed under the condition employed. Figure 5 shows

the CD spectra of BSA in the presence of various con-

centrations of Oc-AgNPs in the near ultraviolet. It can be

observed that the main features of free BSA spectrum

occurred at 208 and 222 nm were in line with that reported

before [34, 35]. When the interaction happened, the char-

acteristic peaks at 208 and 222 nm were both decreased

with increasing the concentration of Oc-AgNPs. The heli-

cal content of free BSA is 62.80 %. After the interaction,

however, it decreased from 45.07 to 35.94 % with the

increase of the concentration of Oc-AgNPs. Thus, the data

obtained from CD demonstrated that the higher order

structure of BSA molecule changed upon interaction

with Oc-AgNPs. Similarly, Wang et al. [36] investigated

the interaction between BSA and the self-aggregated

nanoparticles of cholesterol-modified O-carboxymethyl

chitosan (CCMC) by CD measurement, and concluded that

the higher order structure of BSA changed upon interaction

with CCMC self-aggregated nanoparticles.

4 Conclusions

In summary, the interaction between BSA and polysac-

charide-templated silver nanoparticles was investigated

with fluorescence spectrum, UV–Vis, and CD measure-

ments. These results revealed that BSA molecule was able

to absorb on the surface of Oc-AgNPs to form the inter-

actant by static quenching, and values of the binding rate

constant (K) and the dissociation equilibrium constant (KD)

was 7.5 9 105 M-1 and 3.0 ± 0.6 lM, respectively. The

higher order structure of BSA changed when the interac-

tion underwent. In this sense, Oc-AgNPs was hope to be a

novel carrier for the proteins, and the further investigations

are in progress now.

Acknowledgments This work was supported financially by the

China’s Postdoctoral Science Fund Projects (2013M531292), the

Open Project Program of Guangdong Province Key Laboratory for

250 300 350 400 450 5000.0

0.1

0.2

0.3

0.4

AB

SWavelength(nm)

a

f

280

410

A

1 2 3 4 5

8

10

12

14

16

18

20

1/(A

-A0)

1/[Oc-AgNPs] (µM-1)

BFig. 4 A Absorption spetrum

of BSA in the presence of Oc-

AgNPs: a. 1.0, b. 0.8, c. 0.6,

d. 0.4, e. 0.2, and f.

0 9 10-6 M; B the linear

dependence of 1/(A-A0) on the

reciprocal concentration of Oc-

AgNPs

200 210 220 230 240 250-25

-20

-15

-10

-5

0

5

(103 d

eg.c

m2 d

mol

-1)

Wavenumber (nm-1)

a

d

Fig. 5 CD spectra of BSA in the presence of Oc-AgNPs: a. 0, b. 1.0,

c. 2.5, d. 5.0 9 10-6 M

J Inorg Organomet Polym (2013) 23:1383–1388 1387

123

Green Processing of Natural Products and Product Safety (201201)

the Priority Academic Program Development (PAPD) of Jiangsu

Higher Education Institutions, School Foundation Financing Project

of Jiangsu University (105DG129), the Open Foundation of Jiangsu

Province Key Laboratory of Physical Processing on Agricultural

Products (JAPP2010-7), Jiangsu UIniversity Undergraduate Innova-

tion Project (2012097) and Jiangsu University Students’ Scientific

Research Project (11A343).

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