lucstrith

inYue-Zhong Xian , Li-Tong Jin *, Katsunobu Yamamoto

a Department of Chemistry, East China Normal University, Shanghai 200062, PR China

proposed ow-injection analysis with the NRDS-enzyme electrode array system enables simultaneous monitoring of trace levels of

osine production is central to its function in the nervous

system and can therefore be expected to dier consider-

[8]. In addition, the use of microdialysis probes permits

the release of active agents to the monitoring site and the

calculation of local metabolic turnover rates of the tissue

via clearance methods [9]. In many cases, this sampling

method is generally coupled to high-performance liquid

.

* Corresponding author. Fax: +86 21 6223 2627.

E-mail address: fenfenzhang@yahoo.com.cn (L.-T. Jin).

Journal of Electroanalytical Chem

Journal ofElectroanalytical0022-0728/$ - see front matter 2004 Elsevier B.V. All rights reservedglucose, LL-glutamate, lactate and hypoxanthine in rat striatum.

2004 Elsevier B.V. All rights reserved.

Keywords: Neutral red-doped silica nanoparticle; In vivo microdialysis sampling; Rat striatum; Flow-injection analysis; Enzyme electrode array

1. Introduction

Rapid measurement of glucose, LL-glutamate, and lac-

tate is important in understanding the dynamics of the

energy balance in brain tissue [1,2]. LL-glutamate is alsothe main excitatory neurotransmitter [1,3], while hyp-

oxanthine is a major metabolite in the degradation of

adenine nucleotide [4,5], and the accumulation of aden-

ably from those of conventional neurotransmitters [6].

Therefore, the simultaneous monitoring of glucose, lac-

tate, LL-glutamate and hypoxanthine would be of great

benet for studies on the energy metabolites and neu-

rons communicating in the brain. However, their con-centrations in the extracellular brain environment are

still poorly documented [7]. Microdialysis is a powerful

tool for minimally invasive probing of such metabolismsb Department of Electronic Engineering, East China Normal University, Shanghai 200062, PR Chinac BAS Co., Ltd. No. 36-4, 1-Chome, Oshiage, Sumida-Ku, Tokyo 131, Japan

Received 4 June 2004; received in revised form 23 July 2004; accepted 27 July 2004

Available online 18 October 2004

Abstract

A ow-injection enzyme electrode array system with in vivo microdialysis sampling is proposed for the simultaneous measure-

ment of cerebral glucose, lactate, LL-glutamate and hypoxanthine concentrations. The enzyme electrode array system was based on

neutral red-doped silica (NRDS) nanoparticles as the electrocatalyst. These uniform NRDS nanoparticles (about 50 3 nm) were

prepared by a water-in-oil microemulsion method, and characterized by the transmission electron microscopy technique. The inside

neutral red dopant maintained its high electron-activity, while the outside nano silica surface prevented neutral red from leaching

out into the aqueous solutions and showed high biocompatibility. These nanoparticles were then mixed with the glucose oxidase,

lactate oxidase, LL-glutamate oxidase or xanthine oxidase, and immobilized on the four carbon electrode array, respectively. A thin

Naon lm was coated on the enzyme layer to prevent interference such as from ascorbic acid and uric acid in the dialysate. TheSimultaneous monitoring of ghypoxanthine levels in rat

enzyme electrode array system w

Fen-Fen Zhang a, Qiao Wan a, Chen-Xadoi:10.1016/j.jelechem.2004.07.039ose, lactate, LL-glutamate andiatum by a ow-injectionin vivo microdialysis sampling

Li a, Xiao-Li Wang a, Zi-Qiang Zhu b,a, c

www.elsevier.com/locate/jelechem

istry 575 (2005) 17

Chemistry

chromatography (HPLC) or capillary electrophoresis

(CE) [1012]. Nevertheless, microdialysis combined with

an enzymatic electrode possesses simplicity of operation

and substrate selectivity of the enzyme [13].

Neutral red has been reported to act as the electrocat-

alyst for NADt/NADH regeneration [1416]. NR alsoshows catalytic activity toward DNA [17]. Compared

to the traditional methods for dye immobilization onto

the electrode surface (electropolymerization [14], adsorp-

tion [17]), dye doped silica nanoparticles synthesized by

using a water-in-oil (W/O) microemulsion method are

advantageous, providing both prolonged long-term sta-

bility and showing high biocompatibility [18].

In the present work, we have rst developed novelelectro-active neutral red-doped silica (NRDS) nanopar-

ticles [1921] in a four enzyme electrode array with high

sensitivity and long-term stability. The experiments indi-

cated that the inside neutral red dopant maintained its

high electron-activity as an electrocatalyst, while the

outside nano silica surface prevented NR from leaching

2. Experimental

2.1. Reagents

Glucose oxidase (GOD, from Aspergillus niger, EC

1.1.3.4. 150 000 U g1), LL-glutamate oxidase (LL-GLOD,EC 1.4.3.11, from Streptomyces sp., 10.8 U mg1), lac-tate oxidase (LOD, from Pediococcus species, 37

U mg1), xanthine oxidase (XOD, EC 1.1.3.22, GradeI from Buttermilk, 1 U (mg protein)1, 34.7 mg pro-tein ml1), b-DD-glucose, LL-glutamate, lactate, hypoxan-thine, ascorbic acid, uric acid and Naon (1% methyl

alcohol) were purchased from Sigma Chemical Co.

Tetrathyl orthosilicate (TEOS, 98%) was purchasedfrom United Chemical Technologies (Bristol, PA). Bo-

vine serum album (BSA) was obtained from Huamei

Biochemical (Shanghai, China). Glucose stock solution

was allowed to mutarotate for 24 h before use. Other

reagents were of at least analytical-reagent grade. All

solutions were prepared using twice distilled water.

2 F.-F. Zhang et al. / Journal of Electroanalytical Chemistry 575 (2005) 17out into the aqueous solutions and showed high biocom-

patibility. For in vitro monitoring of brain dialysatewith electrochemical biosensors, LL-ascorbic acid (LL-

AA) causes major interference. Naon lm was

dripped on the four sensor array to exclude such electro-

oxidizable interferants [2123]. The ow-injection per-

formance of this newly prepared NRDS-enzyme

electrode array based on these uniform nanoparticles

and the application to simultaneous determination of

glucose, lactate, LL-glutamate and hypoxanthine in in vi-tro rat brain dialysate were investigated. The FIA sys-

tem with the NRDS-enzyme electrode array showed

high sensitivity, wide ranges of response, and was with-

out electroactive interferences.Fig. 1. Schematic illustration of th2.2. Apparatus

The ow-injection system consisted of a LC-10 AS

eluent delivery pump and an SILL-6B injector equipped

with a 20 ll sample loop (Shimadzu, Tokyo, Japan).Flow-injection amperometric data were collected using

a CHI 1030 workstation (CH instruments, Inc.).

The homemade thin-layer cell consisted of an SCE as

the reference electrode, a gold ake as the counter elec-trode and four NRDS-enzyme modied carbon-disk

electrodes, 300 lm in diameter, as the working electrodearray. The structure of the homemade thin-layer cell is

shown in Fig. 1. Parts A, B and C were made of Teon,e homemade thin-layer cell.

taxic frame. A microdialysis probe was implanted into

the left striatum (coordinates with the skull leveled be-

tween bregma and lambda, were x = +3.0, y = +0.6,

z = 7 mm) [26]. Dialysate samples were discarded overthe rst 90 min to allow recovery from the acute eects

of the implantation procedure. Samples were then col-lected continually in 20 ll sample receivers, and injectedinto the sample loop by switching the value. In this man-

ner, four analytes in the dialysates were detected simul-

taneously at a radial stream NRDS-enzyme electrode

array. The standard addition method was also used by

injecting increasing concentrations of the four analyte

mixed solution.

3. Results and discussion

troanalytical Chemistry 575 (2005) 17 3and they were xed with four screws to prevent weeping.

Before use, the carbon disc electrode array was succes-

sively polished with emery paper and 0.5 lm aluminapowder, and sonicated in twice distilled water.

The microdialysis system consisted of a CMA/101

microdialysis pump (Sweden) and a CMA/11 microdialy-sis probe (Sweden) with amembrane diameter of 0.24mm

and a length of 3.0 mm. Ringers solution was used as theperfusion solution at the rate of 1.0 ll min1. The compo-nents were 140 mmol l1 NaCl, 1.0 mmol l1 MgCl2, 1.2mmol l1 CaCl2 and 5.0 mmol l

1 NaHCO3, pH 7.4.Transmission electron microscope (TEM) images

were recorded by a JEOL JEM-100CX-II Electron

Microscope (Japan).

2.3. Synthesis NRDS nanoparticles

Silica nanoparticles were prepared according to the lit-

erature [24]. The W/O microemulsion was prepared rst

by mixing 7.5 ml of cyclohexane, 1.8 ml 1-hexanol and

1.77 ml of Triton X-100 completely. Then 400 ll neutralred solution (1.0 102 mol l1) was added slowly to theabove mixed solution in an ice cooled ultrasonicator

bath. In the presence of 100 ll TEOS, a polymerizationreaction [25] was initiated by adding 60 ll NH4OH.The reaction was allowed to continue for 24 h. After

the reaction was completed, the NRDS nanoparticles

were isolated from the microemulsion with acetone,

and washed thoroughly (56 times) with both ethanol

and water to remove any surfactant molecules or anyphysically adsorbed NR from the particle surfaces.

2.4. Construction of a Naon/NRDS-enzyme electrode

array

A typical NRDS-modied GODLODLL-GLOD

XOD four enzyme sensor array was prepared as follows.

A solution of 50 ll PBS with NRDS nanoparticles in 50ll PBS containing 10 mg BSA and 1 mg GOD for theGOD electrode (0.4 mg LOD for the LOD electrode,

0.2 mg LL-GLOD for the LL-GLOD electrode, or 10 llXOD for the XOD electrode), was mixed with 20 ll5% glutaraldehyde solution. About 1 ll of the resultingenzyme solution with NRDS nanoparticles was coated

onto the CE surface and air-dried at room temperature.

The enzyme and NRDS nanoparticle coated carbonelectrode array was further modied with a thin layer

of Naon by dripping 1 ll of 1% (w/v) Naon/metha-nol solution and allowing the solvent to dry in air. When

not in use, it was stored in PBS in the dark at 4 C.

2.5. FIA with a Naon/NRDS-enzyme electrode array

system for simultaneous detection of biological samples

A male SD rat weighing about 250 g was anesthetized

F.-F. Zhang et al. / Journal of Elecwith urethane (1.5 g kg1, i.p.) and placed in a stereo-3.1. Characterization and electrochemical behavior of

nanoparticles

Neutral red-doped silica nanoparticles prepared by

the microemulsion method were extremely uniform insize, 50 3 nm in diameter, and were characterized by

TEM (Fig. 2). Based on a calculation done on 60 indi-

vidual nanoparticles, the relative standard deviation

(RSD) of their size distribution was less than 2.8%.

Fig. 3 depicts cyclic voltammograms (CV) of the bare

GCE (a) and the Naon/XOD-NRDS sensor (b) in

PBS (pH 6.9). At a scan rate of 100 mV s1, almost sym-metric waves and 70 mV peak-to-peak separations be-tween the potentials of the anodic (Epa) and the

cathodic peaks (Epc) exhibited the features of rapid

charge transfer at surface-bound species [21]. The results

showed that NRDS nanoparticles kept their high elec-

tron-transfer eciency. In addition, no obvious of the

peak currents was observed after 50 cycles, indicating

that NR is not leached out from the SiO2 network under

these conditions. Furthermore, in the 5.0 106 mol l1Fig. 2. TEM image of neutral red-doped silica nanoparticles (NRDS).

tively; despite the use of the same operating potential,

there was no apparent cross reactivity between the four

sensing parts, which gave four reproducible peaks simul-

taneously. In the absence of the NRDS-modied four

enzyme sensor array, the amperometric response for

5.0 103 mol l1 glucose decreased to 23%, 2.0 103

mol l1 lactate decreased to 66%, 5.0 103 mol l1 LL-glutamate lactate decreased to 12% and 1.0 103

mol l1 hypoxanthine lactate decreased to 37%(Fig. 4). This indicated that the NRDS nanoparticles

present electrocatalytic activity toward the four ana-

lytes. The catalytic eect might be attributed to a high

eciency of the electron-transfer mediator NRDS nano-

particles. On the other hand, it might be due to thehydrophobic silica nanoparticles providing a biocom-

patible environment and improving the enzyme activity

[18,28,29].

3.4. Linearity, detection limits of Naon/NRDS-enzyme

electrode array by FIA system

Typical ow-injection responses for the Naon/NRDS-enzyme electrode array with an applied potential

4 F.-F. Zhang et al. / Journal of Electroanalytical Chemistry 575 (2005) 17hypoxanthine solution, a striking change in the vol-tammogram occurs, Fig. 3(c). Both anodic and cathodic

currents were increased. The increases of the peak cur-

rents were dependent on the hypoxanthine concentra-

tion. The other three sensors showed CV responses

similar to that of Naon/XOD-NRDS. This is the

main characteristic of the electrocatalytic reaction by

mediators [27].

3.2. Optimization of ow-injection analysis

In the FIA system, the ow rate used for measure-

ment the four analytes is an important parameter since

the process involves the enzymatic reaction kinetics

and the diusion of the glucose, lactate, LL-glutamate

and hypoxanthine and their products through the

NRDS-modied enzyme electrode array. An optimalow rate of 1.0 ml min1 was obtained by evaluatingthe analytical performance of the sensor array, peak

0.5 0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3

-18

-12

-6

0

6

12

c

b

a

-I /

nA

E / V vs. SCE

Fig. 3. Cyclic voltammograms for: (a) bare GCE in PBS; (b) Naon/

XOD-NRDS/GCE in PBS; (c) Naon/XOD-NRDS/GCE in

5.0 106 mol l1 hypoxanthine solution. Scan rate: 100 mV s1.width and the measurement sensitivity.

The eect of the detection potential was assessed

from the ow injection hydrodynamic voltammogram.

The glucose anodic response started at +0.10 V, rose

sharply to +0.70 V, and leveled o at higher values

(not shown). All subsequent work, thus, employed adetection potential of +0.70 V. A similar potential (on

the current plateau) was used for the detection of lac-

tate, LL-glutamate and hypoxanthine too.

3.3. Amperometric response on Naon/NRDS-enzyme

electrode array and Naon/enzyme electrode array

When a mixed solution (5.0 103 mol l1 glucose,2.0 103 mol l1 lactate, 5.0 103 mol l1 LL-gluta-mate and 1.0 103 mol l1 hypoxanthine) was injectedinto the sample loop, the sensing parts of the Naon/

NRDS-enzyme electrode array responded selectively to

glucose, lactate, LL-glutamate and hypoxanthine, respec-Fig. 4. Amperometric response on the Naon/NRDS-enzyme elec-

trode array (a) and Naon/enzyme electrode array (b) by the FIA

system with 20 ll injections of: (A) 5.0 mM glucose, (B) 2.0 mMlactate, (C) 5.0 mM LL-glutamate and (D) 1.0 mM hypoxanthine.

Applied potentials (for four sensor array), +0.70 V vs SCE. The mobile

phase was 0.1 M phosphate buer pH 6.9. The ow rate was 1ml min1.

of 0.7 V is shown in Fig. 5 The four sensor array re-

sponded rapidly to injections of the corresponding tar-

get analytes, with a nearly instantaneous rise in the

current. The linear calibration curves, correlation coe-

cients and detection limits of the four analytes are sum-

marized in Table 1. The sensitivities for the four analytesare superior to FIA-UV, for which the analytical data

are not shown here.

3.5. Reproducibility and stability of Naon/NRDS-

enzyme electrode array

The reproducibility is ascertained by monitoring the

current response for ten replicate injections of the four

analyte mixture consisting of 1.0 103 mol l1 glucose,2.0 103 mol l1 lactate, 5.0 103 mol l1 LL-gluta-mate and 1.0 103 mol l1 hypoxanthine. The relativestandard deviations (RSD) of the peak currents are 2.5%

for glucose, 3.1% for lactate, 2.4% for LL-glutamate and

4.6% for hypoxanthine, indicating a good reproducibil-

ity and stability of Naon/NRDS-enzyme electrode ar-

ray for FIA.

In addition, the sensitivity of the Naon/NRDS-en-

Fig. 5. Simultaneous ow-injection analysis of glucose + lactate + LL-

glutamate + hypoxanthine mixed solutions. (A) Response to increasing

levels of glucose: 0.5 mM (a), 2.0 mM (b), 4.0 mM (c), 6.0 mM (d), 8.0

mM (e), and 10.0 mM (f). (B) Response to increasing levels of lactate:

0.5 mM (a), 2.0 mM (b), 4.0 mM (c), 6.0 mM (d), 8.0 mM (e), and 10.0

mM (f). (C) Response to increasing levels of LL-glutamate: 1.0 mM (a),

2.0 mM (b), 4.0 mM (c), 6.0 mM (d), 8.0 mM (e), and 10.0 mM (f).

(D) Response to increasing levels of hypoxanthine: 0.1 mM (a), 0.2

mM (b), 0.4 mM (c), 0.6 mM (d), 0.8 mM (e), and 1.2 mM (f). Other

conditions as in Fig. 4.

Table 1

Analytical data of the four analytes in FIA with the Naon/NRDS-enzym

Analytes Regression equation (ax + ba)B Correlation coecient

Glucose y = 6.5536x + 0.1781 0.9988

F.-F. Zhang et al. / Journal of Electroanalytical Chemistry 575 (2005) 17 5y = 1.7299x + 0.4555 0.9967

Lactate y = 1.1502x + 0.2158 0.9949

y = 0.6132x + 0.2906 0.9970

LL-glutamate y = 17.843x + 1.4845 0.9902

y = 1.5557x + 1.8773 0.9981

Hypoxanthine y = 0.0861x + 0.2488 0.9923

y = 0.5314x + 0.2042 0.9974

A FIA conditions as in Fig. 5.

B Where y and x represent the peak current and the concentration of thezyme electrode array shows no observable change after

two weeks of storage in PBS at 4 C or after successivepotential cycles, indicating that this array is very stable

and shows long shelf-life.

3.6. Study on the interference

In the Naon/NRDS-enzyme electrode array, theoutside Naon lm could prevent both anionic electro-

active interferences from reaching the electrode surface

and fouling of the array [2123]. The presence of 0.2

mM ascorbic acid and 0.2 mM uric acid in the buer

containing 1.0 mM glucose, 1.0 mM lactate, 5.0 lM LL-glutamate and 5.0 lM hypoxanthine did not aect theFIA response current, suggesting that, especially at

low glucose, lactate, LL-glutamate and hypoxanthine con-centrations, there was little interference from AA, etc.,

which increased the sensitivity in measuring the concen-

trations of the four analytes in in vitro dialysate

samples.

3.7. Determination of glucose, lactate, LL-glutamate and

hypoxanthine level in living samples

The microdialysis probe was implanted into the brain

of an anesthetized S.D. rat of about 250 g. The dialysate

was collected at a perfusion rate of 2.0 ml min1 after

e electrode array systemA

(r) Linear range (mol l1) 107 Detection limit/mol l1 (r = 3)

1.0 1061.0 104 5.05.0 1041.0 102

1.0 1068.0 105 5.01.0 1041.0 102

5.0 1072.0 105 2.05.0 1051.0 102

5.0 1075.0 105 2.01.0 1041.2 103analytes, respectively.

troanalytical Chemistry 575 (2005) 176 F.-F. Zhang et al. / Journal of Elec1.5 h. Fig. 6. shows the FIA responses to glucose, lac-

tate, LL-glutamate and hypoxanthine of the dialysate

sample. Recovery studies were also carried out by add-

ing known amounts of glucose, lactate, LL-glutamate

and hypoxanthine mixture to the dialysate sample. The

results showed good recoveries, ranging from 97.1% to

103.8% for the four analytes (see Table 2), which corre-

late well with those in the literature [1,3033].

4. Conclusions

It was found from the results that the present FIA

with a Naon/NRDS-enzyme electrode array system

[5] L.Q. Mao, Katsunobu Yamamoto, Anal. Chim. Acta 415 (2000)

143.

10.

[11] J.X. Zhou, D.M. Heckert, H. Zuo, C.E. Lunte, S.M. Lunte,

Fig. 6. Typical FIA response of Naon/NRDS-enzyme electrode

array in the brain dialysate collected from rat striatum (a), and

addition of (A) 2.0 mM glucose, (B) 2.80 mM lactate, (C) 0.70 mM LL-

glutamate and (D) 0.10 mM hypoxanthine mixture standard solutions

(b). Other conditions as in Fig. 4.

Table 2

Content of the four analytes in rat striatum dialysate sample and

recoveriesa

Analytes Detected/

lmol l1Added/

mmol l1Found/

mmol l1Recovery/

%

Glucose 412 2.00 2.48 102.8

Lactate 685 2.80 3.41 97.8

LL-glutamate 1.06 0.70 0.68 97.1

Hypoxanthine 5.91 0.10 0.11 103.8

a The values shown are calculated from the calibration curves and

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toring of glucose, lactate, LL-glutamate and hypoxan-

thine levels in in vitro dialysate samples. Uniform

NR-doped silica nanoparticles retained a high elec-

tron-transfer eciency and showed electrocatalytic

activity toward the four analytes. There was negligibleinterference from oxidizable species (such as ascorbate,

and urate) in the extracellular space of rat brain, and

the system was useful to the study of brain metabolism

and neuron communication. The system has the poten-

tial to be applied to monitor the four analytes in other

parts of living cells, such as hepatic tissue and the blood

stream.

Acknowledgments

Financial support is acknowledged from the National

Natural Science Foundation of China (No. 20175006,

20305007) and the specialized Research Fund for Nano-

technology from Shanghai (No. 0214nm078 and No.

0359nm002).

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F.-F. Zhang et al. / Journal of Electroanalytical Chemistry 575 (2005) 17 7

Simultaneous monitoring of glucose, lactate, l-glutamate and hypoxanthine levels in rat striatum by a flow-injection enzyme electrode array system with in vivo microdialysis samplingIntroductionExperimentalReagentsApparatusSynthesis NRDS nanoparticlesConstruction of a Nafion reg /NRDS-enzyme electrode arrayFIA with a Nafion reg /NRDS-enzyme electrode array system for simultaneous detection of biological samples

Results and discussionCharacterization and electrochemical behavior of nanoparticlesOptimization of flow-injection analysisAmperometric response on Nafion reg /NRDS-enzyme electrode array and Nafion reg /enzyme electrode arrayLinearity, detection limits of Nafion reg /NRDS-enzyme electrode array by FIA systemReproducibility and stability of Nafion reg /NRDS-enzyme electrode arrayStudy on the interferenceDetermination of glucose, lactate, l-glutamate and hypoxanthine level in living samples

ConclusionsAcknowledgmentsReferences