Chitosan as an active support for assembly of metal nanoparticles and application of the resultant bioconjugates in catalysis

Article abstract of DOI:10.1016/j.carres.2009.10.008

Metal nanoparticle-chitosan (NPs-chitosan) bioconjugates were formed by exposure of chitosan to an aqueous solution of metal salts under thermal treatment. The metal nanoparticles that are formed strongly bound to chitosan, which encouraged us to investigate their catalytic performance. It was demonstrated that the metal NPs-chitosan bioconjugates functioned as effective catalysts for the reduction of 4-nitrophenol in the presence of NaBH₄, which was monitored by means of spectrophotometry as a function of reaction time. The silver NPs-chitosan bioconjugates exhibited excellent catalytic activity and were reusable for up to seven cycles. In contrast, the gold NPs-chitosan catalyst displayed poor catalytic activity, even in the second cycle. A highlight of our approach is that chitosan simultaneously acts as an active support for the synthesis and assembly of nanoparticles, and the resultant bioconjugates bear the advantage of easy separation from the reaction medium.

Full text of DOI:10.1016/j.carres.2009.10.008

Carbohydrate Research 345 (2010) 74–81
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Chitosan as an active support for assembly of metal nanoparticles
and application of the resultant bioconjugates in catalysis
a,
Dongwei Wei a, , , Yongzhong Ye , Xueping Jia b, Chao Yuan a, Weiping Qian c
*
a College of Life Science, Henan Agricultural University, Zhengzhou 450002, PR China
b School of Chemistry and Chemical Engineering, Nantong University, Nantong 226007, PR China
c State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2009
Received in revised form 11 October 2009
Accepted 13 October 2009
Available online 22 November 2009
Metal nanoparticle–chitosan (NPs–chitosan) bioconjugates were formed by exposure of chitosan to an
aqueous solution of metal salts under thermal treatment. The metal nanoparticles that are formed
strongly bound to chitosan, which encouraged us to investigate their catalytic performance. It was
demonstrated that the metal NPs–chitosan bioconjugates functioned as effective catalysts for the reduc-
tion of 4-nitrophenol in the presence of NaBH4, which was monitored by means of spectrophotometry as
a function of reaction time. The silver NPs–chitosan bioconjugates exhibited excellent catalytic activity
and were reusable for up to seven cycles. In contrast, the gold NPs–chitosan catalyst displayed poor cat-
alytic activity, even in the second cycle. A highlight of our approach is that chitosan simultaneously acts
as an active support for the synthesis and assembly of nanoparticles, and the resultant bioconjugates bear
the advantage of easy separation from the reaction medium.
Keywords:
Chitosan
Metal nanoparticles
Bioconjugates
Catalysis
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
in solvents of varying polarities, they demonstrated that exposure
of the gold nanoparticle–spider-silk bioconjugates to vapors of
Much attention has been focused on the preparation of metal
nanoparticles (especially gold and silver nanoparticles) because
of their unusual properties compared to bulk metals and their
important applications as antimicrobial agents, in catalysis, in op-
tics, and as sensors.1–3 In particular, the preparation of metal nano-
particles immobilized on various matrices has attracted intensive
research interest, because excellent synergy and bifunctional ef-
fects are expected.4–9 For instance, through spontaneous reduction
methanol and chloroform led to changes in electrical transport
through the nanoparticles, thus opening the possibility of develop-
ing a vapor sensor.9
There have been a large number of reports on the preparation of
metal nanoparticles on matrix materials through various strategies
such as thermal, electrochemical, photochemical, and sonochemi-
cal reduction.10–14 Although the preparation methods for metal
nanoparticles are varied, the utilization of biomolecules for the
synthesis of metal nanoparticles via the thermal reduction method
is desirable for a mild and ecofriendly process.15–18 Chitosan, a
polysaccharide biopolymer formed by the deacetylation of
naturally occurring chitin, displays unique polycationic, chelating,
and film-forming properties due to the presence of active amino
and hydroxyl functional groups.19 With the development of envi-
ronmentally friendly industries, chitosan and its derivatives, which
combine nontoxicity, biocompatibility, biodegradability, and bio-
activity with desirable physical and mechanical properties, are
becoming increasingly important.20–25 In our recent work, we re-
ported the preparation of gold nanoparticles by thermal and pho-
tochemical reduction methods in chitosan–acetic acid aqueous
ꢀ
of AuCl4 ions by bis(2-(4-aminophenoxy)ethyl) ether at the li-
quid–liquid interface, gold nanoparticles were immobilized on this
polymeric matrix, and thereafter were used as a support for pep-
sin.7 Recognizing the film-forming properties of chitosan, gold
nanoparticles were immobilized in it, and the resultant gold–chito-
san films were employed in trace analysis as surface-enhanced
substrates (SERs) for Raman spectroscopy.8 Silver nanoparticles
were spontaneously incorporated into poly(vinyl alcohol) film by
thermal treatment, and the resultant materials were found to be
promising candidates for optical limiting applications.3 The Sastry
group reported the formation of gold nanoparticle–spider-silk
bioconjugates by reaction of spider silk with aqueous chloroauric
acid. In view of the contraction/expansion behavior of the fibers
solution.13,26,27 Therein, we demonstrated that AuCl4 ions could
ꢀ
be reduced to zero-valent gold nanoparticles by chitosan itself
without any additional reductant in thermal reduction.26,27 In our
current work, we present a further study on the preparation of
metal nanoparticle–chitosan (NPs–chitosan) bioconjugates by
exposure of chitosan to an aqueous solution of metal salts under
* Corresponding author. Tel.: +86 371 63555790; fax: +86 371 63554528.
E-mail addresses: weidongwei@henau.edu.cn, weidongwei2008@gmail.com (D.
Wei).
These two authors contributed equally to this work.
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.10.008

D. Wei et al. / Carbohydrate Research 345 (2010) 74–81
75
thermal conditions. Significantly, the silver nanoparticles thus pre-
pared were found to function as an effective catalyst for the reduc-
tion of 4-nitrophenol to 4-aminophenol, and they exhibited
excellent catalytic activity and reusability for up to seven cycles.
Nitrophenols and their derivatives, which result from the produc-
tion processes of pesticides, herbicides, insecticides, and synthetic
dyes, are some of the most refractory pollutants that can occur in
industrial wastewaters. Now we are aware of the fact that water
pollution by phenol and phenolic compounds is of great public
concern.20,28 Therefore, this study also becomes much more fasci-
nating from the point of pollution abatement. Besides, as there is
a great demand for aromatic amino compounds in industry, the
reaction becomes academically as well as technologically impor-
tant. Also, it is worth noting that our current strategy for the syn-
thesis of metal NPs–chitosan bioconjugates has some significant
features: (1) nanoparticles are formed under mild and ecofriendly
conditions in which chitosan simultaneously acts as both a reduc-
tant and scaffold for nanoparticles. (2) The resultant materials have
the advantage of easy separation from the reaction medium, which
present the possibility of their functioning as reusable catalysts. (3)
Chitosan is nontoxic and biocompatible. Thus the metal NPs–chito-
san bioconjugates formed are also very promising candidates for
pharmaceutical and biomedical applications.
as catalysts for the reduction of 4-nitrophenol. The parallel sam-
ples after centrifugation and redispersion were characterized by
TEM and FTIR. All the reactions were carried out in aqueous solu-
tion at pH 5.9. To investigate the pH effect on the formation of me-
tal NPs–chitosan bioconjugates, 1 M NaOH solution was used to
adjust the pH of the medium.
2.4. Catalytic properties of metal NPs–chitosan bioconjugates
for nitrophenol reduction
The reduction of 4-nitrophenol by NaBH4 was studied as a mod-
el reaction to probe the catalytic activity of metal (gold or silver)
NPs–chitosan bioconjugates in a heterogeneous system for 4-nitro-
phenol reduction. The catalytic reaction was carried out in a stan-
dard quartz cell with a 1-cm path length and ꢂ3.2 mL volume. The
procedures were as follows: 1 mL of 15 mM NaBH4 was added to
2 mL of triply distilled water containing 2.0 mM 4-nitrophenol
(120 lL) in a quartz cell. The color changed from light yellow to
yellow-green. Immediately after the addition of metal NPs–chito-
san bioconjugates, the absorption spectra were recorded in 1-min
intervals in the range of 200–600 nm at room temperature (25 °C).
3. Results and discussion
3.1. Formation and characterization of metal NPs–chitosan
bioconjugates
2. Experimental
2.1. Reagents
Figure 1 shows the representative photographs of metal NPs–
chitosan bioconjugates formed by exposure of chitosan flakes to
aqueous solution of metal salts under thermal treatment at desig-
nated parameters. It is clear from Figure 1 that when chitosan
flakes are exposed to hydrochloroauric acid, the colorless chitosan
(Fig. 1a) turns red (Fig. 1b–d). A characteristic surface plasmon res-
onance (SPR) band for gold nanoparticles suggests the formation of
gold nanoparticles.26 It is also apparent that there is a progressive
increase in the red color with an increase of hydrochloroauric acid
concentration, which is attributed to the increased content of gold
nanoparticles. Furthermore, when chitosan flakes are exposed to
silver nitrate, the silver NPs–chitosan samples formed exhibited a
yellowish-brown or brown color, and a characteristic SPR band
for silver nanoparticles (Fig. 1e–g).27 Also, the color of the silver
NPs–chitosan samples changed from yellow to brown with an in-
crease in silver nitrate concentration, which is attributed to the in-
creased amount of silver nanoparticles. Moreover, it is apparent
that the color of the samples formed is different before and after
the adjustment of the pH of the medium (compared Fig. 1e–g with
Fig. 1h–j). It is known that the SPR band for nanoparticles is sensi-
tive to the size, shape, and spatial distribution of the particles.16
Hence, the difference in the color of the silver samples suggest
the pH of the medium has an effect on the interaction of chitosan
with silver nitrate, and accordingly, the characteristics of silver
nanoparticles, since the color of metal nanoparticles is related to
the SPR excitation.
Figure 2a shows the optical spectra of the metal NPs–chitosan
bioconjugates by exposure of chitosan flaks to triply distilled
water, hydrochloroauric acid, and silver nitrate, respectively, at
95 °C. It is evident from Figure 2a that the sample from the expo-
sure of chitosan flakes to triply distilled water exhibits no absorp-
tion band, and the samples from the exposure to hydrochloroauric
acid or silver nitrate show their corresponding characteristic SPR
bands, further demonstrating that the color of NPs–chitosan sam-
ples originate from the metal nanoparticles that are formed in
the chitosan matrix.26 Figure 2b compares the optical spectra of
the samples formed in the presence and absence of chitosan. It is
clear from Figure 2b that the samples formed in the presence of
chitosan show the characteristic SPR band for metal nanoparticles
Chitosan flakes, from crab shells (Practical grade >85% deacety-
lated; Brookfield viscosity >200,000 cps) were purchased from Sig-
ma–Aldrich. Sodium borohydride (NaBH4), silver nitrate (AgNO3)
hydrogen tetrachloroaurate hydrate (HAuCl4ꢁ3H2O), and 4-nitro-
phenol (C6H5NO3) were of analytical grade. All compounds were
used as received. All solutions were prepared with triply distilled
water.
2.2. Instruments
The optical properties of the products were measured by a Shi-
madzu UV–vis–NIR spectrophotometer (UV-3150). Transmission
electron microscopy (TEM) was carried out on a JEM-2000EX
microscope at an accelerating voltage of 120.0 kV. Fourier-trans-
form infrared spectroscopy (FTIR) was recorded on a Nicolet Nexus
870 FTIR instrument.
2.3. Formation of metal (gold or silver) nanoparticle–chitosan
bioconjugates
The synthesis of metal (gold or silver) NPs–chitosan bioconju-
gates was carried out by exposure of chitosan flakes to an aqueous
solution of metal salts (HAuCl4ꢁ3H2O or AgNO3). In a typical proce-
dure, 30 mg of chitosan flakes were added to a test tube with 1 mL
of the designated concentration of metal salts solution and 6 mL of
the triply distilled water at room temperature. The nanoparticle
conjugate settled at the bottom of the tube after storage for
10 min, even without centrifugation. After the supernatant solu-
tion became colorless (about 30 min), the test tubes containing
the above mixtures were heated to the selected temperature (be-
tween 45 and 95 °C) and left standing for about 12 h. Then, the
resultant mixture was cooled and mixed for homogenization for
UV–vis spectroscopy measurements. To confirm the binding of me-
tal nanoparticles onto chitosan for subsequent catalytic reactions,
the resultant mixtures containing metal NPs–chitosan bioconju-
gates were centrifuged and redispersed in triply distilled water at
least four times. Thereafter, the bioconjugates were dried and used

76
D. Wei et al. / Carbohydrate Research 345 (2010) 74–81
Figure 1. Formation of metal NPs–chitosan bioconjugates by exposure of 30 mg of chitosan flakes at 95 °C to (a) triply distilled water, (b) 0.2, (c) 0.5, (d) 1.0 mM HAuCl4, (e)
1.0, (f) 3.0, (g) 6.0 mM AgNO3, (h) 6.0, (i) 6.0, and (j) 12.0 mM of AgNO3. Therein, (a–g) pH 5.9, (h) pH 7.0, and (i and j) pH 9.0.
1.8
1.5
1.2
0.9
0.6
0.3
0.0
nevertheless, show no absorption band in the UV–vis region. Also,
no color change in the medium solution was observed during the
reaction process when chitosan is absent, suggesting the reduction
of metal salts did not occur. Therefore, it is concluded that chitosan
acts as a reductant for the formation of metal nanoparticles, and
when chitosan is absent, the reduction of metal salts cannot be
realized. Moreover, we also studied system with exposure of chito-
san to metal salt solution at pH 5.9, 7.0, and 9.0, respectively, as
shown in Figure 2c. It is apparent from Figure 2c that at pH 5.9 (tri-
ply distilled water), no absorption band is observed, even from
exposure of chitosan to silver nitrate for 12 h at 45 °C. Also no color
change in solution was observed. However, the solution color
quickly changed from colorless to bright yellow when the pH of
the medium was adjusted to 7.0 or 9.0 by 1 M sodium hydroxide
solution, and concomitantly the characteristic absorption band of
silver nanoparticles appeared. The above experimental results
demonstrate that the pH of the medium affects the reduction of sil-
ver nitrate salts by chitosan. The reason may be that the redox po-
tential of the silver nitrate salts is related to the pH of the medium:
the pH elevation favors the hydrolyzation (hydrolysis) of silver
ions, and, therefore, the formation of Ag2O, which advantageously
accelerates the reduction of silver nitrate by chitosan. In contrast,
the pH elevation disfavored the hydrolyzation (hydrolysis) of
hydrochloroauric acid salts by the increase in the number of hydro-
xyl radicals and, accordingly, slowed down the reduction of hydro-
chloroauric acid salts by chitosan. As a result, the reduction of
hydrochloroauric acid salts by chitosan did not occur even when
incubated for 12 h at 45 °C at pH 5.9, 7.0, and 9.0. A detailed discus-
sion of the mechanism is presented below.
TEMimages and the electron diffraction pattern further confirmed
the formation of metal NPs–chitosan bioconjugates as demonstrated
in Figure 3. It is evident from Figure 3 that metal nanoparticles were
formed in the chitosan matrix, whereas there are some differences
in nanoparticle size and size distribution under the different reaction
parameters (compare Fig. 3a–c and Fig. 3b–d, respectively). Further-
more, itisclearthatwhenthepHofthemediumwasadjusted, thesize
distribution of silver nanoparticles was altered, which is in accord
with the corresponding illustration in Figure 1e–j. The electron dif-
fraction patterncollected during TEM images furtherrevealedthe for-
mation of gold (Fig. 3c) or silver (Fig. 3f) nanoparticles in the chitosan
matrix.27
a
b
c
1.0 mM HAuCl4
6.0 mM AgNO3
only chitosan
300
400
500
600
700
800
Wavelength (nm)
1.5
1.2
0.9
0.6
0.3
0.0
1.0 mM HAuCl4
6.0 mM AgNO3
only HAuCl4
only AgNO3
300
400
500
600
700
800
Wavelength (nm)
2.0
1.6
1.2
0.8
0.4
0.0
pH 5.9
pH 7.0
pH 9.0
The formation of metal NPs–chitosan bioconjugates was further
confirmed by FTIR measurements. Figure 4 shows the FTIR spectra
of the chitosan and NPs–chitosan samples. It is clear from Figure 4
that, although there is the possibility of overlapping between the
N–H and the O–H stretching vibrations, the strong broad band at
3300–3500 cmꢀ1 is characteristic of the N–H stretching vibra-
tion.29 The gradual blue shift of transmittance in this band region
with the increased silver concentration indicates that the N–H
vibration was affected by the attachment of silver. The attachment
of silver to nitrogen atoms in the chitosan molecules reduced the
vibration intensity of the N–H bond due to the increase in molec-
ular weight with silver binding. Our results are consistent with
that of Panigrahi et al. who suggested that the N–H stretching at
3447 cmꢀ1, which is attributed to the C–NH3 on pure resin parti-
cles, shows a blue shift after immobilization of the gold particles
onto the resin beads because of the new Au–N bond formation.28
300
400
500
600
700
800
Wavelength (nm)
Figure 2. UV–vis spectra of samples obtained under different reactive parameters
(as indicated on each plate): (a) 95 °C, (b) 80 °C, (c) 45 °C and 6.0 mM AgNO3.
centered at about 530 nm (gold nanoparticles) or 420 nm (silver
nanoparticles).27 The samples formed in the absence of chitosan,

D. Wei et al. / Carbohydrate Research 345 (2010) 74–81
77
Figure 3. TEM images of metal NPs–chitosan bioconjugates by exposure of 30 mg of chitosan flakes to (a) 1.0 mM HAuCl4 at 95 °C, (b) 6.0 mM AgNO3 at 95 °C, (c) 1.0 mM
HAuCl4 at 80 °C, (d) 12.0 mM AgNO3 at 80 °C, (e) 6.0 mM AgNO3 at 45 °C, and (f) 6.0 mM AgNO3 at 45 °C. Therein, (a–d) pH 5.9, (e) pH 7.0, and (f) pH 9.0.
Moreover, it is noted that metal NPs–chitosan samples (Fig. 4e–h)
show a new band at about 1740 cmꢀ1 that corresponds to the car-
bonyl stretch vibrations in ketones, aldehydes and carboxylic acids.
The presence of the 1740 cmꢀ1 signal in the metal
NPs–chitosan samples indicates that the reduction of the silver
ions is coupled to the oxidation of the hydroxyl groups in the chito-
san molecules and/or their hydrolyzates,27 but this signal was
obviously not observed when the metal salt concentrations were
low enough. Hence, Figure 4 gives sound evidence that gold or sil-
ver nanoparticles are strongly bound to the chitosan matrix.
silver NPs–chitosan bioconjugates should be represented by the
following reactions.
Step A: The adsorption of silver ions onto chitosan.
R-NH2 þ Hþ ꢀ R-NHþ
ð1Þ
ð2Þ
ð3Þ
ð4Þ
3
R-NH2 þ Agþ ! R-NH2Agþ
R-NHþ3 þ Agþ ! R-NH2Agþ þ Hþ
R-NH2Agþ þ H2O ! AgOH þ R-NHþ
3
where R represents all other components except –NH2 in chitosan.
Step B: The formation of silver NPs–chitosan bioconjugates.
3.2. Proposed mechanism for the formation of metal NPs–
chitosan bioconjugates
2AgOH ꢀ 2Agþ þ 2OHꢀ ꢀ Ag O þ H2O
ð5Þ
ð6Þ
ð7Þ
Ag O þ R0CH2OH ! R0CHO þ22Ag þ 2H2O
2
Mills and co-workers30 and Liu and co-workers31 have investi-
gated the reduction of silver ions in alcoholic solutions. They dem-
onstrated that Ag+ ions could be reduced and form metal particles;
meanwhile, the alcohol molecules were transformed into their cor-
responding aldehydes and acids. According to the previous reports
and our experimental results, the mechanism for the formation of
Ag2O þ R0CHO ! R0COOꢀ þ 2Ag þ 2H2O
where R0 represents all other components except –CH2OH in
chitosan.
It is mentioned above that the amine groups in chitosan mol-
ecules have strong complexation abilities with metal ions, and,

78
D. Wei et al. / Carbohydrate Research 345 (2010) 74–81
3.3. Catalytic properties of metal NPs–chitosan bioconjugates
for 4-nitrophenol reduction
a
b
NO2
NH2
c
d
e
NaBH4
ð8Þ
metal nanoparticles/chitosan
OH
OH
f
Chitosan has been extensively used as a support for catalysis
during the past few decades.20,21,33 One of the important applica-
tions of metal nanoparticles is to activate/catalyze some reactions
that are otherwise unfeasible.28,34,35 To this end, the catalytic func-
tion of metal NPs–chitosan bioconjugates on the reduction of 4-
nitrophenol as a model reaction was investigated in the study. It
was observed that the UV absorption peak of 4-nitrophenol under-
went a red shift from 317 to 400 nm (due to the generation of 4-
nitrophenolate ions)36 immediately upon the addition of an aque-
ous solution of NaBH4. At the same time, there is a significant
change in solution color from light yellow to yellow-green. The
absorption peak at 400 nm remained unaltered for a long duration
when a metal NPs–chitosan was absent, demonstrating the reduc-
tion of 4-nitrophenolate ions could not be realized with only the
strong reducing agent NaBH4.36 Herein we first demonstrate the
reduction of 4-nitrophenol in the presence of silver–chitosan cata-
lyst. Figure 5 shows the UV–vis spectra for the reduction of 4-nitro-
phenol with the addition of silver–chitosan samples measured at
1-min intervals during the reaction. It is evident from Figure 5 that,
as the absorption band of 4-nitrophenolate ions centered at
400 nm decreases and disappears, and two new peaks at 300 nm
and 230 nm appear, which are attributed to the generation of 4-
aminophenol37 as represented in Eq. 8. Concomitantly, evolution
of small bubbles of hydrogen gas surrounding the catalyst particles
helps to stir the solution. As a result, the catalyst particles remain
well distributed in the reaction mixture during the progress of the
reaction and offer favorable conditions for a smooth reaction.
Interestingly, upon the addition of metal NPs–chitosan samples,
the reaction solution caused a fading and ultimate bleaching of
the yellow-green color derived from 4-nitrophenolate ions, sug-
gesting the occurrence of a reduction reaction. To exclude the pos-
sibility that the reduction reaction might be activated by chitosan
g
h
1600
2000
2400
2800
3200
3600
4000
-1
Wavenumber (cm )
Figure 4. FTIR spectra of the metal NPs–chitosan bioconjugates prepared by
exposure of 30 mg of chitosan flakes to (a) triply distilled water, (b) 1.0 mM HAuCl4,
(c) 3.0 mM, (d) 6.0 mM, (e) 12.0 mM, (f) 6.0 mM, (g) 6.0 mM, and (h) 12.0 mM
AgNO3. Therein, (a–e) pH 5.9, (f) pH 7.0, (g and h) pH 9.0.
therefore, chitosan has been investigated as an absorption mate-
rial for recovering heavy metal ions from industrial effluents.23,28
For the equations shown above, the two initial steps are the bind-
ing of silver ions to the amine groups in chitosan molecules due
to the sharing of lone electron pairs from the nitrogen atom in
chitosan with silver ion (Eq. 1) and a competitive adsorption of
Ag+ over H+ to the nitrogen atom (Eq. 2). In addition, the com-
plexes of R–NH2Ag+ are subject to the reaction in Eq. 3 due to
the greater binding force of Ag ions with the OHꢀ group from
water than with the nitrogen of the amino group. Thus, a two-
step process is considered for the formation of silver nanoparti-
cles in the chitosan matrix, viz., the formation of silver NPs–chito-
san bioconjugates: First, silver ions are diffused onto the surface
of chitosan molecules and interacted with OHꢀ to form Ag2O par-
ticles (Eq. 4). Second, the Ag2O particles are reduced by chitosan
and adsorpted on to chitosan. At the same time, the chitosan –OH
group are oxidized to their corresponding aldehydes and acids
(Eqs. 5 and 6).
2.0
1.6
1.2
0.8
0.4
0.0
The formation of gold NPs–chitosan bioconjugates also took
place in two steps, viz., the adsorption of gold ions onto chitosan
and the subsequent reduction of gold species by chitosan. Never-
theless, the pH of the medium has distinct influences on the reduc-
tion of two kinds of metal precursors. For the reduction of silver
salts, the reaction is shifted toward the formation of Ag2O (Eq. 5)
with the increasing pH of the medium, viz., the increasing hydrox-
ide ion concentration. Accordingly, the increased Ag2O concentra-
tion favors the reduction of the silver species by chitosan (Eqs. 6
and 7). Hence, the elevation of the pH of the medium accelerates
the reduction of silver nitrate with chitosan. On the other hand,
for the interaction of hydrochloroauric acid with chitosan, increas-
ing pH of the medium, viz., the increasing hydroxide ion concentra-
tion disfavors the hydrolyzation of AuCl4ꢀ, and, consequently, the
200
300
400
500
600
formation of Aun+ 32
thus slowing down the reduction of hydro-
,
Wavelength (nm)
chloroauric acid with chitosan. Therefore, the elevation of the pH
of the medium did not facilitate the reduction of hydrochloroauric
acid with chitosan.
Figure 5. UV–vis absorption spectra for reduction of 4-nitrophenol measured at 1-
min intervals by the silver NPs–chitosan catalyst for exposure of 30 mg of chitosan
flakes to 6.0 mM AgNO3 (95 °C) as catalyst in the third cycle.

D. Wei et al. / Carbohydrate Research 345 (2010) 74–81
79
instead of the silver NPs–chitosan samples, chitosan alone was
added into the aqueous solution of 4-nitrophenol and NaBH4 mix-
ture. It was found that no change in the color and position of the
absorption band at 400 nm of 4-nitrophenolate ions was observed,
which suggests that the reduction of 4-nitrophenol did not proceed
when silver NPs–chitosan samples were absent.36 Thus, the reduc-
tion of 4-nitrophenol by NaBH4 has been clearly demonstrated to
be activated by silver NPs–chitosan as catalyst. In our system,
the silver particles start the catalytic reduction by relaying elec-
trons from the donor BH4ꢀ to the acceptor 4-nitrophenol right after
their adsorption onto the catalyst particle surface. A detailed dis-
cussion is presented below.
In industrial applications, catalysis may be performed in homo-
geneous systems. However, in the case of expensive catalysts such
as those involving precious and strategic metals, it is important to
recover the metal catalysts at the end of the process.21,28,38 There-
fore, we prepared the above mixtures by centrifuging and redi-
spersing them in triply distilled water at least four times for
subsequent reuse experiments. As shown in Figure 6, the catalytic
activities for reuse are estimated by the translation time of 95%
4-nitrophenol. It is evident from Figure 6 that the silver NPs–chito-
san samples are not only very effective for the catalytic reduction
of aromatic nitro compounds, but also are recoverable and can be
recycled a number of times. Figure 6 shows the representative re-
sults of the silver NPs–chitosan samples as catalysts for seven suc-
cessive cycles. It is clear from Figure 6 that the translation time of
95% 4-nitrophenol is 5 min for the first cycle, and for the 3rd and
5th reuse cycles, the translation time is 7 min and 11 min, respec-
tively. Moreover, the translation time of 95% 4-nitrophenol is 2.6
times (13 min) of the initial time (5 min) during the 7rd reuse cy-
cle. Hence, above results clearly underline the fact that, although
the catalytic activity of the silver NPs–chitosan samples gradually
falls off during the recycles, the reduction of 4-nitrophenol by
NaBH4 could also be done fast for the 7rd reuse cycle compared
to the system in which a catalyst is absent, viz, silver NPs–chitosan
samples as the catalyst possess remarkable reuse characteristics.
As demonstrated in the Experimental (Section 2.3), the loss of sil-
ver nanoparticles during the reuse cycle is not observed or mea-
sured by UV–vis spectrometry. Thus, the decrease in catalyst
activity during the successive reuse cycles may be attributed to
the deactivation of active sites on the silver catalyst surface due
to the increasing absorbent.
The activity of gold NPs–chitosan samples as a catalyst for the
reduction of 4-nitrophenol by NaBH4 was also investigated. Similar
results were observed for the gold NPs–chitosan samples, and the
results are shown in Figure 7. It is clear from Figure 7 that upon the
addition of gold NPs–chitosan samples to the reaction system, the
peak height at 400 nm attributed to 4-nitrophenolate ions gradu-
ally decreases with time. With the gradual decrease in peak height
at 400 nm, a new peak appeared at 295 nm, indicating the forma-
tion of 4-aminophenol.36 At the same time, a fading and ultimate
bleaching of the yellow-green color in the aqueous solution and
evolution of small bubbles of hydrogen gas surrounding the cata-
lyst particles were observed when the gold NPs–chitosan sample
was added to the reaction system, indicating the reduction of 4-
nitrophenol and the formation of 4-aminophenol.37 In another
experiment, we found that the translation time from 4-nitrophenol
to 4-aminophenol with the addition of gold NPs–chitosan samples
prepared for 1 h is much shorter for the gold NPs–chitosan samples
prepared for 12 h, as is illustrated in Figure 7 (compare Fig. 7a and
b). That is to say, the catalytic activity of gold NPs–chitosan sam-
ples prepared for 1 h is much higher than that of the gold NPs–
chitosan samples prepared for 12 h. The distinctive difference in
the activity of these two particle samples prepared under different
reaction times may be that the gold nanoparticles in the chitosan
samples prepared for 1 h are still growing, and the samples are
1.6
a
1.2
0.8
0.4
0.0
100
80
200
300
400
500
600
Wavelength (nm)
1.6
1.2
0.8
0.4
0.0
b
60
1
3
5
7
40
20
0
200
300
400
500
600
0
2
4
6
8
10
12
14
Time (min)
Wavelength (nm)
Figure 6. Conversion-time plot for reduction of 95% 4-nitrophenol by the silver
NPs–chitosan catalyst for exposure of 30 mg of chitosan flakes to 6.0 mM AgNO3 at
95 °C as a function of time during the reuse cycles.
Figure 7. UV–vis absorption spectra for reduction of 4-nitrophenol measured at 1-
min intervals by the gold NPs–chitosan catalyst for exposure of 30 mg of chitosan
flakes to 1.0 mM gold precursors with chitosan (a) for 1 h and (b) for 12 h at 95 °C.

80
D. Wei et al. / Carbohydrate Research 345 (2010) 74–81
made up of smaller particles. Smaller particles are composed of a
higher fraction of coordinatively unsaturated surface atoms, which
increases the surface roughness and promotes the chemisorption
of the 4-nitrophenolate ions and thereby facilitates the reaction.
While gold nanoparticles prepared for 12 h are fully grown, the
surfaces of the larger gold particles are terminated primarily by
low-index, high-coordination surfaces related to the lower surface
roughness. The lower surface roughness is unfavorable for the
chemisorption of the nitrophenolate ions and thereby does not
facilitate the reaction. In fact, the UV–vis observation for the for-
mation of gold nanoparticles did reveal that the absorption peak
of gold NPs–chitosan samples prepared for 1 h was increasingly
strengthening, and that the absorption peak of samples prepared
for 12 h was steady, which also was confirmed by their color devel-
opment during the formation process of the gold NPs–chitosan
samples. In the recent past, it has been reported by the Pal group
that, for some redox reactions, the rate of catalysis involving the
growing metal nanoparticles was higher than that involving fully
grown nanoparticles,37,39 which is in accord with our results. Fur-
thermore, other experimental results revealed that the silver NPs–
chitosan samples as catalyst follow the same law for this reduction
reaction, that is, the catalytic activity involving the silver NPs–
chitosan samples prepared for 1 h was higher than that involving
silver NPs–chitosan samples prepared for 12 h. It is also worth-
while to mention that when the silver NPs–chitosan samples pre-
pared from high silver concentrations acted as a catalyst, the
reduction of 4-nitrophenol could be done quickly, and the transi-
tion from 4-nitrophenol to 4-aminophenol could not be measured
by a UV–vis spectrometer.
The reusable activity of gold NPs–chitosan samples as catalysts
was explored. After the first cycle, the gold NPs–chitosan samples
were centrifuged and redispersed in triply distilled water at least
four times for subsequent reuse. It was found the gold NPs–chito-
san samples thus prepared displayed poor catalytic activity even in
the second cycle. The reduction from 4-nitrophenol to 4-amino-
phenol could not be achieved even with gold NPs–chitosan sam-
ples that were prepared for 1 h as the catalyst. The reason may
be that the catalytic activity of a catalyst is related to the active
sites in its surface, and gold NPs–chitosan samples easily absorb
the reactant or product, thus making them poisoned with loss of
activity. Therefore, even smaller particles with a higher activity
of unsaturated surface atoms available for catalytic activity also
deactivate in the second reuse. Additionally, the loss of the cata-
lytic activity for the metal NPs–chitosan samples are also related
to the interactions between chitosan and the metal species, since
the diffusion of reactants onto the chitosan matrices is influenced
by the network and the charge of chitosan. Further investigations
are required to fully understand the exact reasons.
alytic reaction by metal nanoparticles.36–39 Pradhan et al. reported
that the delay time t0 for the reduction of aromatic nitro compounds
in an oxygen atmosphere is greater than in a nitrogen atmosphere
where it is smaller and negligible, in comparison to that for ambient
conditions.37 In the present case, the positive charge of the chitosan
matrix activates the negatively charged 4-nitrophenolate and boro-
hydride ions for adsorption and therefore facilitates the electron
ꢀ
transfer from BH4 (donor) ion to the nitrophenolate (acceptor)
ion through the metal surface. As soon as NaBH4 is added, the metal
particles start the catalytic reduction by relaying electrons from the
donor BH4ꢀ to the acceptor 4-aminophenol right after their adsorp-
tion onto the catalyst particle surface. Moreover, evolution of small
bubbles of hydrogen gas surrounding catalyst particles is essential
for stirring the solution. Accordingly, the catalyst particles remain
well distributed in the reaction mixture and offer favorable condi-
tions for the reaction to occur smoothly. As NaBH4 was present in
large excess, its consumption for the reduction of oxygen did not al-
ter its concentration noticeably. The induction period observed in
the initial stages of the reaction is different as the particle properties
are varied.
4. Conclusions
In this study, we have shown the feasibility of our route to form
metal NPs–chitosan bioconjugates by exposure of chitosan to an
aqueous solution of metal salts in which chitosan simultaneously
acts as a reductant and scaffold for the formation of nanoparticles.
Compared to the traditional chemical reduction approach, our
method for the formation of metal NPs–chitosan bioconjugates is
in agreement with the ‘green’ requirement nowadays. Also, the cat-
alytic function of the resultant bioconjugates to activate the reduc-
tion of aromatic nitro compounds in the presence of NaBH4 has
been clearly confirmed by both visual observation and UV–vis
spectra. It was demonstrated that silver NPs–chitosan bioconju-
gates exhibited excellent reuse characteristics over seven succes-
sive reaction cycles. However, the gold nanoparticles as prepared
are only effective for the first cycle and are deactivated in the sec-
ond cycle. It is also significant to explore the pharmaceutical and
biomedical applications of metal NPs–chitosan bioconjugates in
view of the unusual properties of metal nanoparticles and the bio-
compatible characteristics of chitosan.
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