10.1016-j.jelechem.2014.11.025

Accepted Manuscript

Carboxymethyl cellulose assisted immobilization of silver nanoparticles ontocelluse nanofibers for the detection of catechol

Jiapeng Fu, Dawei Li, Guohui Li, Fenglin Huang, Qufu Wei

PII: S1572-6657(14)00517-7DOI: http://dx.doi.org/10.1016/j.jelechem.2014.11.025Reference: JEAC 1902

To appear in: Journal of Electroanalytical Chemistry

Received Date: 19 August 2014Revised Date: 3 November 2014Accepted Date: 17 November 2014

Please cite this article as: J. Fu, D. Li, G. Li, F. Huang, Q. Wei, Carboxymethyl cellulose assisted immobilizationof silver nanoparticles onto celluse nanofibers for the detection of catechol, Journal of ElectroanalyticalChemistry (2014), doi: http://dx.doi.org/10.1016/j.jelechem.2014.11.025

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1

Carboxymethyl cellulose assisted immobilization of silver

nanoparticles onto celluse nanofibers for the detection of

catechol

Jiapeng Fu, Dawei Li, Guohui Li, Fenglin Huang, Qufu Wei*

Key Laboratory of Eco–Textiles, Ministry of Education, Jiangnan University, Wuxi, Jiangsu,

214122, China

E-Mails: [email protected] (J.F.); [email protected] (D.L); [email protected] (G.L);

[email protected] (F.H.)

*E-mail address of corresponding author: [email protected]; Tel.: +86-510-8591-3653;

Abstract: We report a facile approach to synthesize and immobilize silver nanoparticles (AgNPs)

onto carboxymethyl cellulose (CMC)-modified electrospun cellulose nanofibers and demonstrate

the potential application of as-prepared AgNPs-CMC/cellulose composite nanofibrous mats as

effective biosensor substrate materials. Cellulose nanofibers were prepared by the combination of

electrospinning with deacetylation. Then, CMC was adsorbed onto cellulose nanofibers to complex

silver ions through the chemical binding with the free carboxyl groups of CMC for subsequent

reductive formation of AgNPs. The AgNPs-CMC/cellulose nanofibers immobilized with laccase

(Lac) by electrostatic interactions were used as biosensor substrate materials for catechol detection.

The cyclic voltammetries revealed that the AgNPs-CMC/cellulose nanofibers was beneficial to the

immobilization of Lac and facilitated the direct electron transfer between Lac and electrode.

Lac/AgNPs-CMC/cellulose/glassy carbon electrode exhibited a detection limit of 1.64 µM (S/N =

3), and a wide linear range from 4.98 µM to 3.65 mM, as well as good repeatability, reproducibility,

stability, and selectivity. The CMC/cellulose nanofibrous mats have great potential applications as

substrate materials for different biosensors by immobilizing other different functional nanoparticles

or enzyme on them.

Keywords: electrospun; cellulose nanofibers; carboxymethyl cellulose; silver nanoparticles;

catechol detection; biosensors

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1. Introdution

In recent years, polymeric nanocomposites and polymeric nanomaterials have received a great

deal of attention in a wide variety of domains including catalysis [1], enzyme immobilization [2, 3],

biosensor [4, 5] and so on. Among these polymers, cellulose has been evaluated by Pelton as

“particularly protein and biomolecule friendly” [6]. Cellulose is one of the most abundant

renewable biopolymers on earth and it has desirable inherent characteristics such as hydrophilicity,

biocompatibility, low toxicity, disposability, and affordability. In addition, its derivatives, including

cellulose nitrate, cellulose acetate (CA) and carboxymethyl cellulose (CMC), exhibit the similar

properties. These properties make cellulose-based materials ideal support for enzyme

immobilization for various applications [7, 8]. Meanwhile, polymer-based electrospun nanofibrous

mats are considered as the most appropriate form of support for enzyme immobilization due to the

properties such as high specific surface area, inter-fiber porosity, and the low hindrance of mass

transfer, easy recoverability as well as potential applicability for continuous operations [9-11].

However, direct electrospinning of cellulose has been extremely limited because its high

crystallinity prevents its dissolution in common solvents [12]. Alternatively, cellulose nanofibers

have been fabricated via electrospinning of CA and subsequent deacetylation [13, 14].

Frequently, electrospun cellulose nanofibrous mats have been employed as the immobilization

matrix for direct covalent conjugation of enzymes [3, 15, 16]. However, the covalent coupling

methods could affect the stability of enzyme, leading to the large loss of enzyme activity. Its poor

repeatability and reuseability limited the practical applications in biosensing. In order to overcome

these problems, the adsorption of biomolecules to CMC which was based on cellulose supports was

investigated [17, 18]. CMC was chosen owing to its physical and chemical characteristics. CMC is

negatively charged in aqueous solutions due to carboxyl groups (pKa of about 4.5) and it can be

adsorbed irreversibly on cellulose in the presence of salt [17, 19, 20]. Such adsorptions could

influence the surface swelling and hydration of cellulosic fibers, which were expected to promote

functionality and stability of immobilized biomolecules [21].

Apart from the stability of the enzyme, the efficient and facile signal transduction for

improving sensitivity and elimination of interferences is the most critical issue in the application of

biosensors [22]. However, the poor electrical conductivity of polymer nanofibrous mats could not

meet the demand of biosensors in term of sensitivity. Metal nanoparticles have been integrated to

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polymer nanofibers to achieve the adequate sensitivity and stability because of their excellent

conductivity and biocompatibility [1, 5, 23-25]. Among various nanoparticles used, silver

nanoparticles (AgNPs) have revealed good conductivity and biocompatibility. Besides, silver

nanoparticles could facilitate efficient electron transfer and enhance the response current in

biosensors [26-28]. In this work, the CMC adsorbed onto the cellulose nanofibers was used as a

nanoreactor to complex Ag ion through the chemical binding with the carboxyl groups of CMC for

subsequence reductive formation and immobilization of AgNPs (see Scheme 1). The aim was to

combine the biocompatible and hydrophilic properties of CMC-modified cellulose mats with the

excellent conductivity of AgNPs. The potential application of the obtained AgNPs-CMC/cellulose

composite nanofibrous mat as a biosensor substrate material was demonstrated by immobilizing the

laccase (Lac) onto the mats for catechol detection. The fabricated

Lac/AgNPs-CMC/cellulose/glassy carbon electrode (GCE) for catechol detection exhibited an

excellent biosensing performance.

(Scheme. 1)

2. Materials and methods

2.1. Materials

Cellulose acetate (CA, 39.8 wt % of acetyl content, Mn = 30,000), acetone,

N,N-dimethylacetamide (DMAc), carboxymethyl cellulose (CMC), silver nitrate (AgNO3), sodium

borohydride (NaBH4), NaCl, CH3COOH, CH3COONa, guaiacol, vanillin, phenol and

3,5-dinitrosalicylic acid were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,

China). Catechol was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Laccase (Lac)

and Nafion were purchased from Sigma-Aldrich Chemical Co., Ltd. (St. Louis, MO, USA). All

reagents were used as received without further purification. All aqueous solutions were prepared

with deionized water.

2.2. Fabrication of CMC-modified cellulose nanofibers

The cellulose nanofibers were fabricated by electrospinning 15 wt % CA in a mixture solution

of acetone and DMAc (3:2/ v:v), followed by alkaline hydrolysis to convert CA nanofibers to

4

regenerated cellulose nanofibers. The electrospinning solution was loaded into a 20 mL syringe and

fed at 1 mL/h with a syringe pump. The applied voltage between the tip and collector was 18 kV.

The electrospun fibers were collected on a drum collector wrapped with aluminum foil located 16

cm from the needle tip. The electrospun CA nanofibers were deacetylated in 0.05 M NaOH ethanol

solution at ambient temperature for 12 h. After deacetylation, the fibers were rinsed with an excess

of water to remove residual NaOH and ethanol.

The fabricated cellulose nanofibrous mats were modified by adsorption of CMC from aqueous

solution. Before adsorption on cellulose nanofibers, CMC was dissolved in 50 mM NaOAc buffer at

pH 5.0 and 50 mM ionic strength adjusted with NaCl. The concentration of CMC in the solution

was 0.5 mg/mL. Then the fabricated cellulose nanofibrous mats were soaked in aqueous solution to

adsorb the CMC for 2 h. Thereafter, rinsing with polymer-free buffer solution was applied.

2.3. Synthesis of AgNP-immobilized composite nanofibers

The CMC-modified cellulose nanofibrous mats were then immersed into an aqueous solution

of AgNO3 (4 mM) for 2 h to allow silver ions to complex with available free carboxyl groups of

CMC through ionic exchange followed by three rinses with water. NaBH4 solution (20 mM) was

dropped gradually onto the nanofibrous mats complexed with silver ions until there were no bubbles

of hydrogen gas produced. Finally, the formed AgNPs-immobilized composite mats were rinsed

three times with deionized water, and vacuum-dried at room temperature for 12 h. The obtained

composite mats were stored in a desiccator for further use.

2.4. Preparation of biosensor for catechol detection

Prior to the preparation of the biosensor, the glassy carbon electrode (GCE) was polished with

0.05 µm alumina slurry on a polishing cloth, rinsed thoroughly with water and sonicated in water

for 5 min. The AgNPs-CMC/cellulose fibrous mat had a mass per sheet area of about 0.89 mg/cm2.

The AgNPs-CMC/cellulose fibrous mat was incubated with 3 mg/mL of laccase solution (10 mL) at

4oC overnight in a humidity chamber. The pH value of laccase solution was adjusted to 4 using 0.1

M acetate buffer solution. Then, the AgNPs-CMC/cellulose fibrous mat modified by laccase was

glued by Nafion aqueous solution (1 wt %) on the surface of the pretreated GCE and left to dry at

room temperature. This modified electrode is denoted as Lac/AgNPs-CMC/cellulose/GCE. For

comparison with Lac/AgNPs-CMC/cellulose/GCE, Lac/CMC/cellulose/GCE, Lac/GCE and GCE

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were also prepared with similar procedures as described above. All the modified electrodes were

stored at 4 oC in a refrigerator before use.

2.5. Electrochemical tests

All electrochemical measurements were performed using a CHI 660d electrochemical

workstation (CH Instruments, Shanghai, China). The electrochemical experiments were carried out

using a conventional three-electrode with the modified electrodes obtained via the above methods

as the working electrode (GCE, 4.0 mm), a platinum wire as the counter electrode, and an Ag/AgCl

(saturated KCl) electrode as the reference electrode, respectively. The cyclic voltammetric

measurements were taken in an unstirred electrochemical cell. A magnetic Teflon stirrer provided

the convective transport during the amperometric measurements. Before electrochemical

measurements, all the electrodes were immersed into acetate buffer for 30 min to remove the

residual components. The 0.1 M acetate buffer solution was used as electrolyte and its volume was

20 mL. All the experiments were performed at room temperature.

2.6. Characterizations

Morphologies of different electrospun nanofibrous mats were observed by SEM (S-4800,

Hitachi, Tokyo, Japan). The elemental compositions of the AgNPs-CMC/cellulose composite

nanofibers were analyzed by EDS (EDAX-TSL, AMETEK, USA). The AgNPs-CMC/cellulose

composite nanofibers were also examined by TEM (JEOL/JEM-2100, Japan). FTIR spectra were

recorded using a Nicolet iS10 FT-IR spectrometer (Thermo Fisher Scientific) at the wave-number

range of 4000-500 cm-1 under ambient conditions.

3. Results and Discussions

3.1. Structural characterization of cellulose and CMC-modified cellulose nanofibers

The electrospun CA nanofibers were regenerated to cellulose nanofibers via deacetylation in

0.05 M NaOH/ethanol solution. Due to the irreversible adhesion between CMC and cellulose [17,

20], the carboxyl groups were then installed on the cellulose nanofibrous mats via CMC adsorption

in the presence of NaCl for subsequent formation of AgNPs. FTIR and SEM were used to

characterize cellulose and CMC-modified cellulose (CMC/cellulose) nanofibrous mats.

FTIR spectra of CA nanofibers, regenerated cellulose and CMC/cellulose nanofibers are

presented in Fig. 1. Shifts in three absorption bands were observed at 1747 cm-1

(vC=O), 1378 cm-1

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(vC-CH3) and 1227 cm-1 (vC-O-C), which were attributed to the vibrations of the acetate group from the

CA samples [14]. The three peaks disappeared and the peak at ca. 3350 cm−1 (vO–H) had an obvious

increase for the regenerated cellulose nanofibers, indicating that the acetate group was eliminated

[16]. Clearly, the spectrum of CMC/cellulose nanofibers appeared similar to that of cellulose

nanofibers. The peaks of C=O bond from CMC, at ca. 1730 cm-1

, were not prominent. This result

indicated that a low amount of CMC adsorption to cellulose nanofibers was obtained in this case.

(Fig. 1)

After electrospinning and deacetylation, smooth and continuous cellulose nanofibers with

random orientation were obtained and the electrospun nanofibers possessed a mean diameter of 370

±174 nm (Fig. 2a and Fig. 2b). The cellulose nanofibrous mats exhibited a three–dimensional

structure with pores in micrometer and sub-micrometer sizes. It was clear that the porous structure

and fiber shape were well maintained when CMC was deposited onto the cellulose nanofibrous

mats. Compared with cellulose nanofibers, CMC/cellulose nanofibers (Fig. 2c and Fig. 2d) had a

larger mean diameter (410 ± 195 nm), which was caused by the adsorption of CMC. The thickness

of CMC layer could be estimated to be about 40 nm.

(Fig. 2)

3.2. Structural characterization of AgNPs-CMC/cellulose nanofiberous mats

The CMC/cellulose nanofibrous mats were then immersed into AgNO3 solution to complex the

Ag ions with carboxyl groups for the formation of AgNPs. AgNPs were formed on the

CMC-modified cellulose nanofibrous mats with the addition of the reducing agent NaBH4.

AgNPs-CMC/cellulose nanofiberous mats were characterized by SEM, TEM, EDS and FTIR.

It is clear that AgNPs were synthesized and uniformly immobilized onto the surface of fibrous

mats, as illustrated in Fig. 3a. AgNPs-CMC/cellulose nanofiberous mats also maintained a porous

nanofibous structure with a smooth surface. The TEM image in Fig. 3b shows that

AgNPs-CMC/cellulose composite nanofibers had an apparent core-shell structure, which was

7

attributed to the adsorption of CMC on the cellulose nanofibers. Meanwhile, AgNPs were dispersed

on the surface of CMC layer and showed a relatively uniform distribution. The elemental silver

signals in the EDS spectrum (inset in Fig. 3b) demonstrated that AgNPs were synthesized and

immobilized onto CMC/cellulose nanofibers. The presence of oxygen in the EDX spectrum was

attributed to cellulose, CMC or dioxygen.

(Fig. 3)

Fig. 4 shows the FTIR spectra of CMC/cellulose and AgNPs-CMC/cellulose composite

nanofibers, respectively. Obviously, both the FTIR spectra displayed the typical absorption bands of

cellulose nanofibers. For the AgNPs-CMC/cellulose composite nanofibers, the band at 3350 cm-1

was due to the stretching vibration of hydroxyl group; the band at 2897 cm-1

was attributed to the

C-H stretching vibration; the bands at 1420 cm-1

and 1320 cm-1

were related to -CH2 scissoring and

-OH bending [29], respectively. The band at 1058 cm-1 was assigned to -CH-O-CH2 stretching

mode from the glucosidic units [30]. Meanwhile, after introduction of AgNPs into the

CMC/cellulose nanofibers, the band at 3350 cm-1

became broader, which was consistent with other

cellulose –base nanocomposites [31, 32].

(Fig. 4)

3.3. Direct electron transfer of Lac/AgNPs-CMC/cellulose/GCE

Fig. 5 shows the cyclic voltammograms (CVs) of different electrodes at a scan rate of 100

mV/s in acetate buffer solution (0.1 M, pH 4.0). No current peak was observed at the bare GCE. A

couple of small redox peaks were observed at Lac/GCE and Lac/CMC/cellulose/GCE. The shape of

the peaks was well-defined, while the currents were weak. The anodic peak potential and cathodic

peak potential were located at 0.403 and 0.293 V, respectively. It can be attributed to the direct

electron transfer between Lac and GCE. The current of Lac/CMC/cellulose/GCE was 2.7 µA，which

was weaker than 3.8 µA of Lac/GCE. It was ascribed to that CMC/cellulose hindered the electron

transfer. Although the redox peaks were similar to those of Lac/GCE and Lac/CMC/cellulose/GCE,

8

Lac/AgNPs-CMC/cellulose/GCE exhibited another pair of redox peaks with the anodic and

cathodic peak potential at 0.294 and 0.151 V, which was due to the presence of AgNPs. Obviously,

the peak current attributed to Lac rose largely to 5.8 µA at the same time. These results indicated

that AgNPs-CMC/cellulose nanofibrous mats can be the effective material for enhancing the

electron transfer. Because of its special structure and properties, CMC was beneficial to the

immobilization of enzyme and the exposure of the electrical activity centre of protein [8]. Besides,

AgNPs played an important role in accelerating the direct electron transfer from Lac to electrode

surface.

(Fig. 5)

Fig. 6 presents the CVs of Lac/AgNPs-CMC/cellulose/GCE at different scan rates from 75 to

225 mV/s in acetate buffer solution (0.1 M, pH 4.0). The formal potential related to the laccase was

independent of the scan rate. The cathodic and anodic peak currents increased linearly with the

increase of the scan rates in that range (Fig. 6 (b)). It indicated that the modified electrode displayed

a typical surface-controlled quasi-reversible electrochemical reaction. It also confirmed that the

immobilized laccase was stable.

(Fig. 6)

3.4. Electrocatalysis of Lac/AgNPs-CMC/cellulose/GCE to catechol

In order to study the electrocatalytic activity of laccase immobilized in cellulose-based

composite nanofibrous mats, the responses of different modified electrodes to catechol were

explored (Fig. 7). In the presence of 1.96 × 10−4

M catechol, a pair of CV peaks was obtained at

Lac/GCE, corresponding to the typical redox reaction of catechol. The redox peak current of

catechol at Lac/GCE was 2.56 µA, with potentials at 0.577 V. When laccase was immobilized in

CMC/cellulose composite nanofibrous mats, an obvious decrease of the response current was

observed, attributing to that the presence of CMC/cellulose nanofibrous mats retarded the

electron-transfer of catechol at the electrode interface. While for Lac/AgNPs-CMC/cellulose/GCE,

9

the redox peak currents caused by catechol increased significantly in comparison with that of

Lac/GCE. Meanwhile, a sharp peak at 0.299 V appeared, which was related to the oxidation of

AgNPs. The CVs indicated that Lac/AgNPs-CMC/cellulose/GCE showed an excellent

electrochemical catalysis for catechol, which may be due to the synergistic effect of biocompatible

CMC/cellulose nanofibers and AgNPs with high conductivity.

(Fig. 7)

3.5. Optimizing the parameters of the biosensing performance

The response of Lac/AgNPs-CMC/cellulose/GCE towards catechol was influenced by various

parameters. These include the pH of the buffer solution, detection potential, the loading amount of

enzyme and the temperature. In this work, the pH of buffer solution and detection potential were

optimized.

The experiments were performed in the pH ranging from 4.0 to 6.5 in 0.1 M acetate buffer

solution containing 1.96 × 10−4 M catechol at the potential of 0.5 V vs. Ag/AgCl (Fig. 8). The

response currents increased with the increase of pH and reached the maximum value at pH 5.0,

which was similar to the optimum pH for laccase in Cu-OMC/CS modified electrode [33], and

smaller than that on cellulose acetate/ionic liquid-modified electrode [34]. Therefore, pH 5.0 was

chosen for the detection of catechol.

As the lowest detection limit can be achieved and the electrochemical interfering species can

be avoided by adjusting the detection potential [35], a further optimized experimental parameter

was the detection potential. The experiments were performed in 0.1 M acetate buffer solution of pH

5.0 containing 1.96 × 10−4

M catechol in the potential ranging from 0.4 V to 0.65 V (Fig. 9). The

response current fluctuated with the change of the detection potential. The maximum response

appeared at the detection potential of 0.55 V, which might be attributed to that the potential of redox

peak of catechol was near 0.55 V. Thus, the detection potential of 0.55 V was selected.

(Fig. 8)

(Fig. 9)

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3.6. Amperometric characteristics of Lac/AgNPs-CMC/cellulose/GCE

The amperometric response of Lac/AgNPs-CMC/cellulose/GCE to the successive addition of

catechol was carried out in solution under the optimal condition. Successive additions of catechol

were performed every 50 s. Immediately after the addition of catechol, the response increased and

reached a steady state very fast. It indicated that the electrode responded rapidly to the change of the

substrate concentration (Fig.10a). The obvious increase of the current could be observed when the

concentration of catechol was as low as 4.98 µM (inset in Fig. 10a). The amperometry result

indicated that the Lac/AgNPs-CMC/cellulose/GCE exhibited high sensitivity. Fig. 10b shows that

the modified electrode linearly responded to the concentration of catechol. The linear response

range of the biosensor to catechol concentration was from 4.98 µM to 3.65 mM, which was

comparable to that obtained at Lac/CNTs–CS/GCE (1.2 µM to 30 µM) [36], Lac-FSM7.0-GCE (2

µM to 100 µM) [37] and Lac/MWCNT/GCE (2 µM to 1 mM) [38], wider than those obtained at

Lac/AP-rGOs/Chit/GCE (15 µM to 0.7 mM ) [39] and MB-MCM-41/PVA/Lac (4 µM to 88 µM)

[40]. The linear regression equation was Iss (µA) = 0.038+0.0115 c (µM), with a correlation

coefficient of 0.999 (n=18). From the slope of 11.5 µA/µM, the detection limit was estimated to be

1.64 µM at a signal/noise (S/N) of 3, which was comparable to that obtained at

Lac/AP-rGOs/Chit/GCE (2 µM) [39], Lac/Cu-OMC/CS/Au electrode (0.67 µM) [33] ,

Lac/MWCNT/GCE (2 µM) [38], better than that obtained at Lac/AP-rGOs/Chit/GCE (7 µM) [39].

The comparisons of linear range and detection limit of different laccase modified electrodes toward

catechol were summarized in Table 1. The results demonstrated that the

Lac/AgNPs-CMC/cellulose/GCE had a wide linear range, low detection limit and reasonable

sensitivity. It is expected to lower the detection limit and improve the sensitivity by increasing the

content of AgNPs attached to the modified cellulose nanofibers.

(Fig. 10)

(Table 1)

3.7. Repeatability, reproducibility and stability of Lac/AgNPs-CMC/cellulose/GCE

When Lac/AgNPs-CMC/cellulose/GCE was scanned repeatedly in 0.1 M acetate buffer

11

solution containing 1.96 × 10−4 M catechol, the relative standard deviation (RSD) of 3.41 % was

obtained for four measurements by the same electrode. The fabrication reproducibility of the

modified electrode was calculated by cyclic voltammetric measurements on the same day. The RSD

for five electrodes which were made independently was 1.57 %, revealing an acceptable

reproducibility in the construction of the electrode. Also, the stability of the electrodes should also

be considered for practical use. When the biosensor was not in use, it was stored in buffer at 4 °C.

The current response still remained 97.6 % of the initial response after three weeks. These results

indicated that Lac could be efficiently immobilized in the network structure of

AgNPs-CMC/cellulose composite nanofibrous mat and retained their bioactivity for almost three

weeks.

To evaluate the selectivity of the proposed biosensor, four possible interfering substances such

as vanillin, guaiacol, phenol and 3, 5-dinitrosalicylic acid were added into the acetate buffer

solution containing 1.96 × 10−4

M catechol. It can be seen from Fig. 11, the current response almost

remained unchanged after adding these interferents. Therefore, the modified electrode showed a

good selectivity for catechol.

(Fig. 11)

4. Conclusions

In summary, a facile method to immobilize AgNPs onto the CMC-modified cellulose

nanofibrous mat were developed and the AgNPs-CMC/cellulose nanofibers showed their

application as an efficient substrate material to fabricate biosensor for catechol detection.

AgNPs-CMC/cellulose composite nanofibrous mat possessed a large specific surface area and

combined the good biocompatibility of cellulose-based materials with the excellent electrical

conductivity of AgNPs, which could keep the immobilized enzyme stable and improve the

sensitivity of biosensors. Therefore, the obtained Lac/AgNPs-CMC/cellulose/GCE exhibited

outstanding electrocatalysis toward catechol with wide linear range and low detection limit.

Meanwhile, the biosensor also showed good repeatability, reproducibility, stability, and selectivity.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China

12

(21201083), National High-tech R&D Program of China (2012AA030313), Changjiang Scholars

and Innovative Research Team in University (IRT1135), the Priority Academic Program

Development of Jiangsu Higher Education Institutions, Industry-Academia-Research Joint

Innovation Fund of Jiangsu Province (BY2012068), Science and Technology Support Program of

Jiangsu Province (SBE201201094), the Innovation Program for Graduate Education in Jiangsu

Province (CXZZ13_07 and KYLX_1133), and Hubei Key Laboratory of Low Dimensional

Optoelectronic Material and Devices (13XKL01002).

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16

Figure Caption

Scheme. 1. Schematic illustration of immobilizing AgNPs onto CMC-modified cellulose nanofibers.

Fig. 1. FTIR spectra of CA (a), regenerated cellulose (b) and CMC/cellulose nanofibers (c).

Fig. 2. SEM (a) images and the diameter distribution (b) for regenerated cellulose nanofibers; SEM (c) images

and the diameter distribution (d) for CMC/cellulose nanofibers.

Fig. 3. SEM (a) and TEM (b) images of Ag NPs-CMC/cellulose composite nanofibers. Inset: EDS spectrum of

AgNPs-CMC/cellulose composite nanofibers.

Fig. 4. FTIR spectra of CMC/cellulose (a) nanofibers and AgNPs-CMC/cellulose (b) composite nanofiber

Fig. 5. CVs of bare GCE (a), Lac/GCE (b), Lac/CMC/cellulose/GCE (c) and Lac/AgNPs-CMC/cellulose/GCE (d)

in 0.1 M acetate buffer solution (pH 4.0) at a scan rate of 100 mV/s.

Fig. 6. CVs (a) of Lac/Ag NPs-CMC/cellulose/GCE in acetate buffer solution (0.1 M , pH 4.0) at scan rates from

the inner to the outer: 75, 100, 125, 150, 175, 200, 225 mV/s and calibration plots (b) of anodic and cathodic peak

current vs. scan rates.

Fig. 7. CVs of Lac/GCE (a), Lac/CMC/cellulose/GCE (b) and Lac/AgNPs-CMC/cellulose/GCE (c) in 0.1 M

acetate buffer solution (pH 4.0) containing 1.96 × 10−4

M at a scan rate of 100 mV/s.

Fig. 8. The dependence of the response of Lac/AgNPs-CMC/cellulose/GCE to an input of 1.96 × 10−4

M catechol

on the pH of the buffer. Conditions: detection potential, 0.55 V versus Ag/AgCl reference. The error bars indicate

the standard error for triplicate measurements with the same electrode.

Fig. 9. The dependence of the response of Lac/AgNPs-CMC/cellulose/GCE to an input of 1.96 × 10−4

M catechol

on detection potential. Conditions: pH 5.0. The error bars indicate the standard error for triplicate measurements

with the same electrode.

17

Fig. 10. Amperometric response curves (a) of the Lac/AgNPs-CMC/cellulose/GCE for successive additions of

different concentration of catechol at 0.55 V, 0.1 M acetate buffer solution (pH 5.0), inset: calibration curve (b) of

the current vs the concentration of catechol.

Fig. 11. Amperometric response of the Lac/AgNPs-CMC/cellulose/GCE upon subsequent additions of (a) catechol,

(b) vanillin, (c) guaiacol, (d) phenol and (e) 3, 5-dinitrosalicylic acid at 0.55 V, 0.1 M acetate buffer solution (pH

5.0).

Table 1. biosensing performances of different laccase modified electrodes toward catechol

18

19

20

21

22

23

24

25

26

27

28

29

30

Table 1. biosensing performances of different laccase modified electrodes toward catechol

Electrode description Linear range (µM) Detection limit (µM) reference

Lac/CNTs–CS/GCE 1.2-30 0.66 [36]

Lac-FSM7.0-GCE 2-100 2 [37]

Lac/MWCNT/GCE 2-1000 2 [38]

Lac/AP-rGOs/Chit/GCE 15-700 7 [39]

MB-MCM-41/PVA/Lac 4-87.98 0.331 [40]

Lac/Cu-OMC/CS/Au 0.67-15.75 0.67 [33]

Lac/AgNPs-CMC/cellulose/GCE 4.98-3650 1.64 This work

31

Graphical abstract

32

Highlights

� A facile approach to produce AgNPs-CMC/cellulose composite nanofibrous mats

has been developed.

� Laccase biosensor based on AgNPs-CMC/cellulose composite nanofibrous mats

for catechol detection was developed in this work.

� The detection limit of the obtained biosensor to catechol is 1.64 µM.

� The composite nanofibers could provide a new platform for other redox proteins

immobilization.