Biosensors and Bioelectronics 19 (2003) 277�/282

www.elsevier.com/locate/bios

Preparation of poly(thionine) modified screen-printed carbonelectrode and its application to determine NADH in flow injection

analysis system

Qiang Gao, Xiaoqiang Cui, Fan Yang, Ying Ma, Xiurong Yang *

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Renmin Street 159,

Changchun, Jilin 130022, China

Received 16 November 2002; received in revised form 2 April 2003; accepted 15 May 2003

Abstract

A poly(thionine) modified screen-printed carbon electrode has been prepared by an electrooxidative polymerization of thionine in

neutral phosphate buffer. The modified electrodes are found to give stable and reproducible electrocatlytic responses to NADH and

exhibit good stability. Several techniques, including cyclic voltammetry, X-ray photoelectron spectroscopy (XPS) and scanning

electron microscopy (SEM), have been employed to characterize the poly(thionine) film. Further, the modified screen-printed

carbon electrode was found to be promising as an amperometric detector for the flow injection analysis (FIA) of NADH, typically

with a dynamic range of 5�/100 mM.

# 2003 Elsevier B.V. All rights reserved.

Keywords: Electropolymerization; Thionine; NADH; Screen-printed carbon electrode; FIA

1. Introduction

The determination of reduced b-dihydronicotinamide

adenine dinucleotide (NADH) is very important in

enzyme assays, due to its participation in the enzymatic

catalysis of more than 250 dehydrogenases useful both

in bioprocesses and analytical applications (Katakis and

Dominguez, 1997; Lobo et al., 1997). The electrochemi-

cal oxidation of NADH has been studied (Gorton, 1986)

and attempts have been made to use surface-modified

electrodes in order to speed up the rate of heterogeneous

electron transfer between an electrode and NADH in

solution (Gorton and Dominguez, 2002).The use of electropolymerization to prepare modified

electrodes is a practical approach to the direct formation

of films on small or irregularly shaped electrodes,

especially microelectrodes or microarray electrodes

(Emr and Yacynych, 1995). The method has also been

used to prepare modified electrodes with electrocatalytic

* Corresponding author. Tel.: �/86-431-568-9278; fax: �/86-431-

568-9711.

E-mail address: xryang@ns.ciac.jl.cn (X. Yang).

0956-5663/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0956-5663(03)00212-4

activity toward NADH oxidation (Pariente et al., 1995;

Lobo et al., 1996; Bartlett et al., 1997; Zhou et al., 1998;

Cai and Xue, 1997; Silber et al., 1996; Chen and Lin,

2001). Thionine, as a redox dye, has been studied

extensively due to its potential utility in photogalvanic

cell (Albery et al., 1979) and sensor (Ruan et al., 1998;

Xiao et al., 1999) applications. Albery et al. (1979)

reported on the modification of platinum and tin

dioxide electrodes with thionine. They applied a positive

voltage for 15 min (1�/1.5 V vs. SCE) on electrodes

immersed in a thionine solution containing 0.05 M

H2SO4, and estimated the organic coverage to be of the

order of 20 monolayers. The thionine film was, however,

immediately disordered when the pH value of the

solution was increased to higher than pH 4. Another

way for the preparation of poly(thionine) modified

electrodes is potential-sweep electrolysis in the potential

range of �/0.2 to �/1.4 (vs. Ag/AgCl) in an acetonitrile

solution containing NaClO4 and thionine. Using this

method, modified electrodes with catalytic activity for

oxidation of NADH have been prepared on the basal-

plane pyrolytic graphite electrode (Ohsaka et al., 1993)

and microband gold electrode (Cai et al., 1995). In

Q. Gao et al. / Biosensors and Bioelectronics 19 (2003) 277�/282278

addition, Yang et al. (1999) have presented a two-step

method for the electropolymerization of thionine in

neutral phosphate buffer on glassy carbon electrodes

and constructed a H2O2 biosensor.Screen-printed electrode has combined advantages of

being simple, inexpensive and versatile (Alvarez-Icaza

and Bilitewski, 1993). Reports about the use of electro-

polymerization of redox dyes on screen-printed carbon

electrodes are very rare. Much effort has been directed

towards the goal of preparing new electrode materials

that can exhibit a facile electrochemical oxidation of

NADH (Wang et al., 1998; Sprules et al., 1995;Aviamescu et al., 2002). We found that the poly(thio-

nine) modified screen-printed carbon electrodes, which

can be prepared by the electropolymerization of thio-

nine in neutral phosphate buffer, considerably electro-

catalyzes the oxidation of NADH and have a good

stability.

In the present paper, we reported on the preparation

of the poly(thionine) modified screen-printed carbonelectrodes and its electrocatalysis for NADH oxidation.

X-ray photoelectron spectroscopy (XPS) and scanning

electron microscopy (SEM) were employed to charac-

terize the modified electrodes. The determination of

NADH using the modified electrodes was also per-

formed in a flow injection analysis (FIA) system.

2. Experimental

2.1. Chemicals and reagents

Reduced b-dihydronicotinamide adenine dinucleotide

(NADH, AMRESCO) was used as received. Thionine

(dye content 85%) was obtained from Aldrich. Ascorbic

acid, uric acid and dopamine were purchased from

Sigma. Carbon ink (Electrodag 423SS) was obtainedfrom Acheson Colloids (Japan).

Unless otherwise stated all chemicals and reagents

used are of analytical grade. Buffers were prepared using

water from a Milli-Q ultra-pure water system. 0.2 M

phosphate buffer (pH 6.9) was prepared by mixing the

stock solution of potassium dihydrophosphate and

sodium hydroxide.

2.2. Apparatus

Amperometric and cyclic voltammetric experiments

were performed with a CHI 832 (Shanghai, China). All

experiments were carried out with a conventional three-

electrode system with the screen-printed carbon elec-

trode as working electrode, a platinum foil as counter

electrode, and an Ag/AgCl (saturated potassium chlo-ride) as reference electrode. All solutions tested were

thoroughly deoxygenated by bubbling pure nitrogen,

and a continuous flow of nitrogen was maintained over

the solution during experiments at room temperature

(239/2 8C).

SEM was performed on a JXA-840 (JEOX, Japan)

apparatus. The acceleration voltage was 20 kV. Sampleswere gold sputtered prior to SEM measurements.

XPS measurements were conducted with an ESCA-

LAB MK II spectrometer (VG Co., UK) with a Mg Karadiation (hg�/1253.6 eV) as the X-ray source and a

pass energy of 20 eV. The pressure inside the analyzer

was maintained at 6.5�/10�7 Pa. Data analysis was

based on deconvolution of a high-resolution composite

XPS peak into peaks of the individual species indifferent states. The deconvolution was carried out by

a nonlinear regression analysis provided by the instru-

ment, with position, height, and width of each individual

Gaussian peak as variable parameters. The criterion for

the best fit was the good agreement between the

experimental points and fitted curves.

The flow injection system comprised of an ISIF-C

brainpower flow injection analyzer (Ruimai Company,Xi’an, China) equipped with two three-channel peristal-

tic pumps, a rotary injection valve with a 50 ml of sample

loop and a home-made flow cell.

2.3. Preparation of base electrodes and poly(thionine)-

modified screen-printed carbon electrodes

The screen-printed carbon electrodes used as a base

electrodes were prepared by the method described in thepaper (Zhang et al., 2001). The poly(thionine) modified

electrodes were prepared by a potential-sweep electro-

lysis at 50 mV s�1 in the potential range of �/0.5 to 1.1

V (vs. Ag/AgCl) in phosphate buffer containing 0.1 mM

thionine. The electrodes thus prepared were treated in

an ultrasonic bath for a few minutes to dissolve thionine

monomer adsorbed on the electrode surface or trapped

in the polymer matrix. The surface concentration of theelectroactive moieties within the poly(thionine) films

could be controlled by appropriately choosing the

number of the cyclic scans. Unless otherwise stated,

the poly(thionine) modified electrodes with cyclic scans

of 25 times were used.

2.4. Flow injection analysis

A home-made three-electrode flow cell (20 ml volume),

equipped with a poly(thionine) modified screen-printed

carbon electrode, a stainless steel counter electrode and

an Ag/AgCl reference electrode, was used. The

poly(thionine) modified screen-printed carbon electrode

was inserted into the flow-through cell and connected

finely with a CHI 832 Potentiostat. Then, a carrier

electrolyte (0.2 M phosphate buffer pH 6.9) wasdelivered and a potential (0.1 V vs. Ag/AgCl) was

applied to obtain a stable baseline. Amperometric

signals were recorded using a PC 586 when NADH

Q. Gao et al. / Biosensors and Bioelectronics 19 (2003) 277�/282 279

solution was injected with a flow rate of 0.5 ml min�1.

Peak height of the oxidation current was used as a signal

and three injections were made for each concentration of

NADH standard.

3. Result and discussion

3.1. Properties of the poly(thionine) modified screen-

printed carbon electrode

Cyclic voltammograms (Fig. 1) showed the growth

process of the poly(thionine) film with consecutivecyclic. The first cyclic voltammogram (initial potential

of 0.4 V) showed a pair of sharp reversible peaks at the

region of the monomer redox peak, which could be due

to thionine monomer redox on the screen-printed

carbon electrode. On further potential scanning, a

well-defined shoulder peak was observed at a peak

potential of 0.9 V (vs. Ag/AgCl). The irreversible anodic

reaction corresponded to the oxidation of the �/NH2

groups of the thionine molecule. During the thionine

electropolymerization, a pair of new reversible peaks

with a peak potential of 0.1 V grew up, which increased

gradually and shifted to a cathodic peak potential (Epc)

of �/0.1 V and an anodic peak potential (Epa) of 0.27 V

with increasing scan number. This demonstrated that

the polymer film was formed on the screen-printed

carbon electrode.The potential sweep range, especially the upper

potential limit, was most important factor for preparing

poly(thionine) modified electrode. If the potential sweep

was confined to the range that upper limit was below 0.8

V, then a simple CV corresponding to the two-electron

reduction of thionine to leucothionine was observed

while polymerized thionine was not obtained. The

Fig. 1. Cyclic voltammograms recorded during the electropolymeriza-

tion of thionine in phosphate buffer (pH 6.9) containing 0.1 mM

thionine. Scan rate, 50 mV s�1. Initial potential, 0.4 V (vs. Ag/AgCl).

mechanism of thionine electropolymerization has been

proposed and confirmed (Bauldreay and Archer, 1983;

Hutchinson and Hester, 1984). Schlereth and Karyakin

(1995) have reported that the cation-radical species were

formed at about 0.8 V for the electropolymerization of

phenoxazines or phenothiazines if the parent monomer

has primary (or secondary) amino groups as ring

substituents. On the bases of these discussions, it was

suggested that the oxidation potential not less than 0.9 V

was mainly based on the same reason. In order to

achieve the formation of the poly(thionine) film, the

electrode potential must be larger than the potential

where the oxidation of NH2 groups of the thionine

molecule (probably to the cation radicals, Bauldreay

and Archer, 1983) occur, as the usual case for most

NH2-group containing aromatic compounds (Ohsaka et

al., 1984, 1991; Kunimura et al., 1988).

Fig. 2 showed the cyclic voltammograms of the

poly(thionine) modified screen-printed carbon electrode

at various scan rates. The relationship between peak

current and scan rate was found to be linearly with v1/2,

which indicates that the charge-transport process within

the film was diffusion-controlled. Further successive

potential scanning at 100 mV s�1 for 1 h caused a

decrease in current response less than 5%.

The electrochemical behavior of poly(thionine) as

other polymer dyes was affected by pH (Torstensson

and Gorton, 1981). The anodic and cathodic peak

potentials shifted negatively with increasing pH in

phosphate buffer. The E8 value decreased by 55 mV/

pH between pH 6 and 8 for the immobilized poly(thio-

nine), which was close to the expected Nernstian value

of 59 mV for a two-electron, two-proton process.

It was reported that the screen-printed electrode and

conventional electrode differed in their electrochemical

behavior (Yoon and Kim, 1996). They were suggested

Fig. 2. Cycle voltammograms of the poly(thionine) modified screen-

printed carbon electrode at various potential scan rates. Potential scan

rates; 10, 20, 40, 60, 80, 100 mV s�1.

Fig. 3. Scanning electron micrograph of poly(thionine) modified

screen-printed carbon electrode. The modified electrode was prepared

by repeating 100 times of potential sweep in phosphate buffer (pH 6.9)

containing 0.1 mM thionine. Magnification was 1000.

Q. Gao et al. / Biosensors and Bioelectronics 19 (2003) 277�/282280

that the difference might be attributed to the increased

surface area of the screen-printed electrode due to rough

and porous structure. Fig. 3 showed the typical electronmicrograph of the screen-printed electrode at low

magnification (1000-fold), a rough and jagged structure

with randomly distributed carbon particles was ob-

served, which supports the presumption that the thick

film electrode acted like a huge array of microelectrodes.

Fig. 4. (A) XPS spectra of N1s for electropolyzerated thionine (a) and

adsorbed thionine (b). (B) Resolved XPS peaks of electropolymer-

izated thionine. The poly(thionine) modified screen-printed electrode

was prepared by repeating 100 times of potential sweep in phosphate

buffer (pH 6.9) containing 0.1 mM thionine.

3.2. X-ray photoelectron spectroscopy (XPS) analysis

High-resolution XPS spectra of N1s were recorded

and the binding energy of the nitrogen of poly(thionine)

has been investigated. Fig. 4 showed the XPS peaks of

N1s for thionine molecules in the adsorbed film (Fig.

4A.b) and the polymer film (Fig. 4A.a). For the

adsorbed thionine molecules on the electrodes, a

screen-printed carbon electrode was immersed in thethionine-containing phosphate buffer for an hour.

Adsorption process could be followed by cyclic voltam-

metry with potential range from �/0.5 to 0.6 V (vs. Ag/

AgCl), which was characterized by the fact that the

cyclic voltammograms reached a steady-state. The

detailed analysis of the N1s peak of poly(thionine)

showed three different peaks, as shown in Fig. 4B,

indicating that three chemical states of nitrogen atomsexisted in the polymer film. The one at 400.1 eV (42.9%)

was corresponding to the amino nitrogen atoms, while

another at 398.1 eV (29.5%) was ascribed to the

heterocyclic nitrogen atoms. The third one at 399.3 eV

(27.6%) came from a new state of nitrogen atoms.

According to Kessel and Schultze (1990) and Camalli et

al. (1990), this kind of nitrogen atoms might be

originated from the bonding via an amino nitrogenatom in one thionine molecule to a carbon atom in

another one. Thus, the electropolymerization of thio-

nine took place under our experimental conditions.

3.3. Electrocatalysis of poly(thionine)-modified

electrodes for oxidation of NADH

For further electrochemical measurements, the elec-

trode was then transferred into the phosphate buffer

with or without NADH. Fig. 5 showed typical voltam-

mograms demonstrating the electrocatalytic activity of

the poly(thionine) modified screen-printed carbon elec-

trodes for the oxidation of NADH. The reversible redox

response of the poly(thionine) film can be observed over

a wide range of potentials ca. �/0.2 to 0.2 V (vs. Ag/

AgCl) and its center was located ca. 0.05 V (vs. Ag/

AgCl) as shown in voltammogram a. Voltammogram b

observed in the presence of NADH showed an enhanced

oxidation current and a large negative shift in the anodic

peak potential of about 300 mV, compared with that

obtained at the bare screen-printed carbon electrode

(voltammogram c). These results demonstrated the

Fig. 5. Cyclic voltammograms of (a, b) poly(thionine) modified screen-

printed carbon electrode in the absence (a) and presence (b, c) of 1 mM

NADH. Voltammogram (c) was obtained at a bare screen-printed

carbon electrode. Scan rate, 10 mV s�1.

Q. Gao et al. / Biosensors and Bioelectronics 19 (2003) 277�/282 281

electrocatalytic oxidation of NADH by the poly(thio-

nine) film.

3.4. Detection of NADH in flow injection system

The flow-rate dependence of the current response at aconstant concentration of NADH was examined by

recording the peak currents at different flow rates. The

current response was found to decrease with increasing

flow rate. At a flow rate of 0.5 ml min�1, the

determination of NADH can be performed in 3 min

including the sampling and washing, giving a through-

out of about 20 samples per hour with a relative

standard deviation (R.S.D.) of 4.5%.

Fig. 6. Flow-injection responses to NADH injection at poly(thionine)

modified screen-printed carbon electrode. The concentration of

NADH solution was increased from 5�/10�6 to 1.5�/10�3 M.

Applied potential, 0.1 V (vs. Ag/AgCl). Flow rate, 0.5 ml min�1.

Electrolyte and carrier, phosphate buffer (pH 6.9).

Fig. 6 displayed the flow-injection response of the

poly(thionine) modified screen-printed carbon electro-

des for NADH solution of increasing concentration

from 5 mM to 1.5 mM. Well-defined and sharp peakswere observed using a detection potential of 0.1 V (vs.

Ag/AgCl). The flow injection peak currents were

proportional to the NADH concentration. At higher

concentrations, there appeared to be a leveling off of the

response, likely due to kinetic limitation. At the lower

concentrations, a linear correlation was observed. The

present modified electrodes possessed a dynamic range

of 5�/100 mM. The resulting calibration plot had a slopeof 1.14 mA mM�1 and correlation coefficient, 0.999. A

detection limit of 3�/10�6 M can be estimated on the

basis of the signal-to-noise (S/N�/3). In all cases the

response was rapid and reproducible. After an initial

loss activity, the electrodes exhibited a very stable

response during hours of continuous flow injection.

The current response of the modified electrodes in the

presence of some interferent compounds was alsoexamined. The concentration of ascorbic acid, uric

acid and dopamine were 0.05, 0.32 and 0.02 mM,

respectively, which are their individual values in human

serum samples. Dopamine and uric acid exhibited no

response. On the contrary, a large current response was

observed for ascorbic acid. Thus, a preseparation or

preoxidation of ascorbic acid (Shin et al., 2001) was

required for use of poly(thionine) modified electrodes asan electrochemical detector for NADH.

The sensing elements were found to be stable for at

least 1 week and maintained 90% of their activity during

that period while being stored at 4 8C between measure-

ments.

4. Conclusion

In this paper, a poly(thionine) film on the screen-

printed carbon electrode was prepared by the oxidative

electropolymerization of thionine in neutral phosphate

buffer, and the resulting poly(thionine) modified elec-

trodes exhibited a good electrocatalytic activity for

NADH oxidation and good stability. In a FIA, the

limit of detection of NADH was estimated to be of theorder of 3�/10�6 M. The modified screen-printed

carbon electrodes can be base electrodes for dehydro-

genase enzyme biosensor.

5. Supporting information available

XPS characterization of N1s. This material is avail-

able free of charge via the Internet at http://pubs.ac-

s.org.

Q. Gao et al. / Biosensors and Bioelectronics 19 (2003) 277�/282282

Acknowledgements

Support of this study by the National Natural Science

Foundation of China (No. 20299030) is gratefullyacknowledged.

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