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Analytica Chimica Acta 588 (2007) 140–146

Determination of various alcohols based on a new immobilizedenzyme fluorescence capillary analysis

Yong-Sheng Li a, Xiu-Feng Gao b,∗a School of Chemical Engineering, Sichuan University, Chengdu 610065, China

b West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China

Received 23 August 2006; received in revised form 24 December 2006; accepted 29 January 2007Available online 7 February 2007

bstract

A novel method for the determination of ethanol in tequila based on the immobilized enzyme fluorescence capillary analysis (IE-EFCA) haseen proposed. Alcohol dehydrogenase (ADH) was immobilized in inner surface of a capillary and an immobilized enzyme capillary bioreactorIE-ECBR) was formed. After nicotinamide adenine dinucleotide (NAD+) as an oxidizer is mixed with alcohol sample solution, it was sucked intohe IE-ECBR. The fluorescence intensity of the mixed solution in the IE-ECBR was detected at λex = 350 nm and λem = 459 nm. The experimentalonditions are as follows: The reaction time is 20 min; temperature is 40 ◦C; the concentrations of phosphate buffer solution (pH 7.5) and NAD+ are

.1 mol L−1 and 5 mmol L−1, respectively; immobilization concentration of ADH is 10 U L−1. The determination range of ethanol is 2.0–15.0 g L−1

F = 10.44C + 6.6002, r > 0.9958); its detection limit is 1.11 g L−1; and relative standard deviation is 1.9%. IE-EFCA method is applicable for theetermination of the samples containing alcohol in medicine, industry and environment.

2007 Elsevier B.V. All rights reserved.

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eywords: Immobilized enzyme; Fluorescence capillary analysis; Ethanol dete

. Introduction

Ethanol is a product used in many industrial processes, likeeverage industry, food industry, fermentation and the pulpndustry. It is also an important chemical substance used as aolvent, as a fuel for buses and cars, in antifreeze products ands a disinfectant in a wide variety of industrial processes andlinics [1].

Traditional determination for ethanol is the gravimeterethod. But its accuracy is affected greatly by the distilled

rocess and thermostat condition. The sensitivity and the repro-ucibility of this method are poor and time-consuming. For theravimeter method and the potassium dichromate oxide titrationethod, the samples must be pre-treated; namely, ethanol in the

amples should be pre-separated by distillation process beforehe determination. The procedure is very complex. The potas-ium dichromate oxide titration method uses too much reagents.

∗ Corresponding author. Tel.: +86 28 85501721.E-mail addresses: lysgxf2005@yahoo.com.cn,

iufengg@163.com (X.-F. Gao).

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tion

Besides, there are many other methods to determine themount of ethanol, such as gas chromatography [2], high perfor-ance liquid chromatography [3], capillary electrophoresis [4],odular Raman spectrometry [5], colorimetric methods [6] andany more. These methods require expensive equipment and/or

ood skills.Recently, Svensson et al. [7] have developed an amperomet-

ic biosensor for rapid determination of ethanol, using liquidlcohol dehydrogenase (ADH) as a recognition element. In theresence of ADH, ethanol reacts with nicotinamide adenine din-cleotide (NAD+) and produces reduced nicotinamide adenineinucleotide (NADH) and aldehyde. NADH is oxidized on thelectrodes and produces electrical current signal. Ethanol wasndirectly quantified by the electrical signal. Methanol gave noignal at all, but higher alcohols, such as propanol, pentanolnd hexanol, gave significant signals, decreasing with increasingength of the carbon chain.

Azevedo et al. [8] immobilized alcohol oxidase (AOD) onto

ontrolled-pore glass (CPG) beads [9], and have made up aini packed-bed bioreactor to monitor ethanol concentration

n a flow-injection analysis (FIA) [10,11] system. Its reactionrinciple is that in the catalysis of AOD, ethanol reacts with

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issolved oxygen (O2) and forms hydrogen peroxide (H2O2) andorresponding aldehyde. Further, under catalysis of horseradisheroxidase (HRP), H2O2 is reduced by phenol-4-sulfonic acid inhe presence of 4-aminoantipyrine (4-AAP) and produces a vio-et red quinoneimine dye [12]. The dye was detected at 490 nmavelength.Rangel et al. [13,14] immobilized ADH on alkylaminated

PG and made immobilized enzyme reactor. The enzyme reac-or was connected to a FIA spectrophometric or a SIA [15]ystem, and then absorbance of NADH was detected at 340 nm,nd the aim of determining ethanol in wines was achieved.esides, a lot of immobilized enzyme-FIA methods based on

pectrophotometric [16,17], electrochemical [18–22], chemi-uminometric [23–26] and spectrofluorimetrie [27] detectionsave been reported for the determination of alcohol.

Besides, Cosford and Kuhr [28] had attempted applying aort of fused silica capillaries to fluorescence analysis. Theirethod is that glutamate dehydrogenase (GDH) was immo-

ilized to the inner surface of a capillary (i.d.: 0.075 mm).n the catalysis of GDH, glutamate reacts with NAD+ andonverts to �-ketoglutarate while simultaneously reducingAD+ to NADH. The excitation beam from a laser (325 nm,e/Cd) transmits a filter coated UG-11 and dichroic mirrors

reflects 325 nm, transmits 442 nm) and focuses on a 50 �mpot on the capillary wall. Fluorescence produced from theADH passed through the dichroic mirrors and a 380 nm fil-

er according to former beam road and was collected in anpilluminescence configuration. The concentration of gluta-ate was quantified indirectly with the fluorescence intensity ofADH.

Fluorescence capillary analysis (FCA) [29] is a new methodeveloped by the authors, based on routine medical capillary andommon spectrofluorimetry. The FCA can make fluorescencenalysis to realize the micro-dosage of reagents and populariza-ion and can promote the micromation of traditional fluorescencenalysis instruments.

In the research, based on the combination of the immobilizednzyme technology (IE) and the FCA method [30], an IE-FCAethod was proposed and used for the determination of ethanol.his new method has many merits as follows: it can overcome

he deficiencies as mentioned above, simplify operating proce-ure, save lots of enzyme reagents, decrease the waste, increaseelectivity and sensitivity of the method, realize trace analysiso a micro-volume sample, and it can be applied to rapid deter-

ination of discontinuous batch samples, and the monitoring ofhe samples in the producing fields.

. Experimental

.1. Assay principle

Reaction principle to be used for the determination of ethanols shown in Eq. (1). In the presence of ADH, ethanol reacts

ith NAD+ as an oxidizer. This reaction produces aldehyde

nd NADH. After the NADH is excitated at 350 nm, it willmit 459 nm of fluorescence. Consequently, the content of thethanol can be quantified by detecting the fluorescence intensity

2

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ica Acta 588 (2007) 140–146 141

f NADH.

H3CH2OH + NAD+ADH−→CH3CHO + NADH + H+ (1)

.2. Chemicals and apparatus

All chemicals were of analytical-reagent grade. Reagentssed in this experiment are as follows: ethanol (v/v, 100%),a2HPO4, KH2PO4, 50% glutaraldehyde and Tris were pur-

hased from Baoxin Bioengineering Co., �-NAD+, BSA andDH were purchased from Sigma Chemical Co. The alco-ol samples used for determination are Langjiu (53%, v/v),iannanchun (38%, v/v), Changcheng Ganhong (12%, v/v) andingdao beer (3.8%, v/v) made in China.Instruments used in this experiment are a type of RF-5000

pectrofluorimeter (Shimazu Company); a capillary holder usedor the spectrofluorimeter was homemade. The capillaries usedi.d.: 0.9 mm, o.d.: 1.1 mm) were purchased from Drummondcientific Co.

.3. Preparations of reagent solutions

Ultra purified water (conductivity: 0.065 �S cm−1) was usedor the preparation of the following solutions.

.3.1. 1.0 mol/L phosphate buffer solutions (pH 8.0/7.5/7.0)These solutions were prepared by weighing 358.15 g of

a2HPO4 and 136.10 g of NaH2PO4 in two 1000 mL of cali-rated flasks, respectively, dissolving and diluting to the markith water. Preparations of buffer solutions with pH 8.0, 7.5

nd 7.0 were completed by mixing two phosphate solutionsccording to different proportions, respectively.

.3.2. 10 mmol/L NAD+ solution26.50 mg of NAD+ was dissolved in water and diluted to

.0 mL by using phosphate buffer solution (pH 7.5).

.3.3. Ethanol stock solution (310 g L−1)The solution was prepared by taking 200 mL of pure ethanol

olution (100%) in a 500 mL calibrated flask and completing theolume to the mark with pH 7.5 of PBS.

.3.4. ADH solution (300 U L−1)1.0 mg of ADH (1.5 U mg−1) was weighed exactly, dissolved

n water and diluted to 5.0 mL with pH 7.5 of PBS. 10 U L−1 ofDH solution used in the research has been prepared by diluting

he 300 U L−1 of ADH solution with the 0.1 mol/LPBS (pH 7.5).

.3.5. Gultaraldehyde solution (2.5%, v/v)The solution was prepared by taking 2.5 mL of gultaralde-

yde solution (50%) in a 50 mL calibrated flask and completinghe volume to the mark with water.

.3.6. 2% BSA solution (w/v)The solution was prepared by weighing 0.20 g of dry BSA in

10 mL of calibrated flask, dissolving and diluting to the markith 0.05 mol L−1 of PBS (pH 7.0).

1 Chimica Acta 588 (2007) 140–146

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.4. Pre-treatment of capillary

First the inner and outer surfaces of the capillaries wereashed with an ethanol liquid containing 2 mol L−1 of NaOH,

hen flushed with distilled water and ultrapurified water sequen-ially. At last, these capillaries were put in a thermostat for dryingt 40 ◦C.

.5. Ethanol capillary bioreactor

First, the capillaries pre-treated by the above-mentionedethod were marinated in 2.5% gultaraldehyde for 1 h on the

acuum condition and washed by using 0.1 mol L−1 of phos-hate buffer solution (PBS, pH 7.0). Secondly, ADH solutionere sucked into the capillaries and kept overnight. Thirdly,

he capillaries were marinated in 2% bovine serum albuminor about 1 h, and the immobilized enzyme ethanol capillaryioieactors (IE-ECBRs) were made up. Finally, IE-ECBRs weretored in 0.05 mol L−1 of PBS (pH 7.0) [31] till use.

.6. Determination procedure

The FCA method has a capillary and a holder. Whennalyzing, the holder is inserted into the light path of a fluo-ophotometer. Due to the capillarity, the sample is sucked intohe capillary (reagent or enzyme is immobilized on its innerall). Then, the capillary is vertically inserted into the holder.hen an emitted beam (λex) passes through the capillary, the

uorescent matter formed in the sample is excited, and thus itmits fluorescence (λem). The fluorescence with vertical direc-ion of λex enters into a monochromatic filter and reaches theetector. At the same time, the fluorescence intensity is detected.he fluorescence intensity is proportional to the content of someaterial in the sample to be determined. The capillary is not only

he container of the sample and reagent but also the immobiliza-ion carrier of enzyme and the place of chemical reaction. Theapillary can be made of quartz glass, optical glass, transparentlastic and so on.

In the study, after mixing the ethanol sample and NAD+

olution, it was sucked into the IE-ECBR. The fluorescencentensity (Fs) of the IE-ECBR was detected at the wavelength ofex/λem = 350/459 nm. And then the reagent blank (Fb) (NAD+

olution without the sample) was detected by using the sameE-ECBR, and �F (=Fs − Fb) was obtained. Finally, the con-ent of ethanol was obtained by putting �F value in a regressionquation of ethanol standard solution.

. Results and discussion

.1. Choose of the excitation and emission wavelengths

After the fluorescence intensity of NADH was scannednder different excitation and emission wavelengths, it wasound that its intensity reaches to the maximum whenex/λem = 350/459 nm (slit 10 nm). So λex/λem = 350/459 nmas selected in the experiment.

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Fig. 1. Effect of different reaction time on fluorescence intensity.

.2. Effect of reaction time and liquid ADH concentration

In this experiment, the concentration of NAD+ was fixed at.2 mmol L−1 (pH 8.8) and 78 g L−1 of ethanol standard solutionas used as the test sample. The effect of the reaction time on

he fluorescence intensity was investigated under different ADHoncentrations (1.5, 7.5, 15, 22.5, 30 and 37.5 U L−1) at roomemperature.

When testing, first of all, a mixed solution of ethanol, NAD+

nd ADH was sucked into the ECBR and then the fluorescencentensity of the ECBR was detected under different reactionimes at 5, 10, 15, 20, 25 and 30 min. The results obtained arehown in Fig. 1. It can be seen that the ECBR’s fluorescencentensity reached the maximum when the reaction time was at0 min. Therefore, the reaction time was selected to be 20 min.

Besides, the fluorescence intensity increased sharply withncreasing concentration of liquid ADH between 7.5 and0 U L−1, but decreased when the ADH concentration was over0 U L−1. Consequently, 30 U L−1 of ADH concentration wassed for subsequent contrast with its immobilized concentration.

.3. Effect of temperature

The fluorescence intensities of a series of the same mixedolutions were determined at different temperatures. The exper-mental conditions were as in Section 3.2, except that the reactionime of 20 min was selected and the ADH solution was used0 U L−1 in the mixed solution.

A small vessel containing the mixed solution was put in a ther-ostat when its temperature became to constant at 25 ◦C. And

hen after sucking the mixed solution, the ECBR was insertedn the light path of the fluorophotometer, and its fluorescencentensity was detected at 5, 15 and 30 min, respectively. The

ame procedures were repeatedly done when the temperaturesere 30, 35, 40 and 45 ◦C. The results are shown in Fig. 2. Theuorescence intensity of the mixed solution increases with the

ncreasing temperature between 25 and 40 ◦C, and decreases

Y.-S. Li, X.-F. Gao / Analytica Chimica Acta 588 (2007) 140–146 143

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bove 40 ◦C. The reason that fluorescence intensity decreasesver 40 ◦C may be because of enzyme deactivation.

Besides, under different reaction times, variety of trends ofhe fluorescence intensity with the change of the reaction tem-erature was similar. Consequently, the reaction temperaturesed for the enzyme catalysis was selected to be 40 ◦C in thexperiment.

.4. Effect of pH

The experimental conditions were as in Section 3.3, excepthat the reaction temperature used was 40 ◦C in the mixed solu-ion. Different mixed solutions were prepared by using the PBSuffer solution with different pH (pH 6.0, 6.5, 7.0, 7.5, 8.0 and.0). And then the effect of pH of the mixed solution on its flu-rescence intensity was examined. Obtained result is shown in

ig. 3. It can be seen that the fluorescence intensity reaches theaximum when pH was at 7.5. So this pH value of the mixed

olutions was selected.

Fig. 3. Effect of pH of reaction solution on fluorescence intensity.

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Fig. 4. Effect of NAD+ concentration on fluorescence intensity.

.5. Effect of NAD+ concentration

NAD+ reacts with ethanol under catalysis of ADH and pro-uces NADH with fluorescence. The content of ethanol can beuantified indirectly by detecting its fluorescence intensity. Con-equently, the concentration of NAD+ will affect on the enzymeeaction for determination of ethanol.

In the experiment, the other conditions were fixed; theuorescence intensity was detected under different NAD+ con-entrations (1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 mmol L−1). Thebtained result is shown in Fig. 4. It can be seen that underhe precondition of the excessive of ethanol concentration, theuorescence intensity of the mixed solution was proportional

o the NAD+ concentrations between 1.0 and 5.0 mmol L−1

nd reaches the maximum at 5.0 mmol L−1. But between 5.0nd 7.0 mmol L−1, its fluorescence intensity decreases with thencreasing of the concentration. The reason may be that theigh concentration of NAD+ affects the activation of ADHnzyme. So, 5.0 mmol L−1 of NAD+concentration was selectedor use.

.6. Effect of the kind and concentration of buffer solution

The experimental conditions were as given in Section 3.5,xcept that the 5.0 mmol L−1 of NAD+ solution was usedn the mixed solution. The pH of the mixed solutions weredjusted to 7.5 by using 0.1 mol L−1 of PBS and 0.1 mol L−1

f Tris buffer solution, respectively, and their fluorescencentensities were detected. The obtained results show that flu-rescence intensities of PBS and Tris buffer solutions arelmost the same. Because PBS is cheap, it was used in thexperiment.

Besides, the effect of the buffer concentration on the enzy-atic reaction was examined by using 0.01, 0.05, 0.1, 0.15 and

−1

.2 mol L of PBS, and its result is shown in Fig. 5. Whenhe PBS concentration is 0.1 mol L−1, the fluorescence intensityeaches the maximum. 0.1 mol L−1 of the PBS concentrationas selected for use.

144 Y.-S. Li, X.-F. Gao / Analytica Chimica Acta 588 (2007) 140–146

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Fig. 5. Effect of PBS solution on fluorescence intensity.

.7. Effect of ADH concentration for immobilization

In order to examine effects of the ADH concentration usedor immobilization on the fluorescence intensity, a series of IE-CBR were made by using five sets of capillaries in whichifferent concentrations of ADH (5, 10, 20, 40, 80 U L−1) weremmobilized.

Then three different concentrations of ethanol standard solu-ions (4.0, 9.0 and 15.0 g L−1) were sucked into IE-ECBR,espectively, and their fluorescence intensities were detectedfter the solution reacted for 20 min. The obtained curves arehown in Fig. 6. They show that when the ADH concentrationor immobilization exceeds 10 U L−1, the change trend of theuorescence intensity basically remains constant. To decrease

he test cost and save the enzyme amount, the ADH concentra-ion for immobilization was selected as 10 U L−1 for subsequent

nalyses.

ig. 6. Effect of ADH concentration for immobilization on fluorescence inten-ity.

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Fig. 7. Stability test of immobilization ADH.

.8. Stability of IE-ECBR

To examine the stability of the IE-ECBR, 10 U L−1 of ADHas been immobilized in it, and the fluorescence intensities wereetected repeatedly by using 12 g L−1 of ethanol standard solu-ion as the test sample. The obtained result is shown in Fig. 7.

ith the increase in time, the fluorescence intensity of the IE-CBR tends to decrease and its fluorescence attenuates to 60%fter 31 days.

.9. Comparison between liquid enzyme and immobilizednzyme

To examine the catalysis efficiency of the liquid and immo-ilized ADH enzymes, the following experiment was done. Themmobilized ADH concentration was 10 U L−1 and the liquid

A series of the ethanol standard solutions were used as theest samples. Under the same experimental conditions the fluo-escence intensity of the ECBR containing the ethanol sample

ig. 8. Effect of emzymes under liquid and solid states on fluorescence intensity.

Chimica Acta 588 (2007) 140–146 145

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nly or both ADH and ethanol sample was detected. The resultsbtained are showed in Fig. 8. It can be seen that the linearities ofhe two methods (IE-ECBR: CH3CH2OH + NAD+ and ECBR:DH + CH3CH2OH + NAD+) are all good. But when the ADHas immobilized (solid dots), its sensitivity was better than thatf the liquid ADH (hollow dots).

So after the ADH was immobilized in ECBR (IE-ECBR), itad not only better selectivity, but also better catalysis efficiencyhan the liquid ADH. Besides, the IE-ECBR can be used repeat-dly, possess good economy benefit and its operation procedures simple and convenient. On comparing with the liquid enzyme

ethod, the IE-ECBR can also save a lot of enzyme reagent andurther result in the decrease of the determination cost.

. Determination of the sample

.1. Determination of the sample and recovery by theE-FCA

The optimum conditions used in the method are as follows:he ADH concentrations for immobilized and liquid methodsre 10 U L−1 and 30 U L−1, respectively; the concentration ofAD+ is 5.0 mmol L−1; the buffer solution is 0.1 mol L−1 ofBS (pH 7.5); reaction time is 20 min; reaction temperature is0 ◦C; the excitation and emission wavelength of NADH are50 nm and 459 nm, respectively.

Under the conditions, a series of the standard ethanol solu-ions were determined by using the ECBR. The resulting graph ishown in Fig. 9. The linear range for the determination of ethanols 2.0–15.0 g L−1, and the relative equation of ethanol concen-ration (C) and fluorescence intensity (F) is F = 10.44C + 6.6002r = 0.9950).

Subsequently, four commercial alcohols were chosen in theesearch as test samples. According to the percentage containing

he alcohol (%, v/v), their corresponding fluorescence intensi-ies were detected by using IE-FCA method. Table 1 shows theesults. The data shows that the percentages of the ethanol con-ent in the commercial products were the same as the determined

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able 1etermination of alcohol content in samples by using IE-FCA (n = 3)

amples (%, v/v) Dilution multiple

angjiu (53) 50iannanchun (38) 30hangcheng Ganhou (12) 10ingdao Beer (3.8) 4

able 2ecovery determination of alcohol samples by using IE-FCA

amples Sample conc. (g L−1) Added conc. (g L

angjiu 8.37 10iannanchun 10.0 10hangcheng Ganhou 9.48 10ingdao Beer 7.5 10

Fig. 9. Caliberation curve of determination of ethanol based on IE-FCA.

esults. It proves that the accuracy and credibility of this methods good.

To further examine the credibility of the method, 10 g L−1

f the ethanol standard solution was added to the above alcoholamples, respectively. The recovery of the alcohol samples wasetermined. Table 2 shows the result. The recoveries of the fourlcohol samples are in the range 101–103%, and the results areery satisfactory. Therefore, this method can be used as a newethod to determine ethanol contents in various alcohols.

.2. Detection limit and the reproducibility

In order to examine the accuracy of this method, the repro-ucibility was determined. In the linear range, the lower and theigher concentrations of ethanol were used as detected samples.

able 3 shows the result. Obtained relative standard deviationR.S.D.) was about 2.0%. The accuracy of this method wasontent with the determination demand. The detection limit ofE-ECBR was 1.11 g L−1.

Found cone, (g L−1) Determined result (%, v/v)

8.46 (8.93, 8.16, 8.29) ± 0.336 53.5 ± 0.0429.63 (9.85,9.02, 10.0) ± 0.443 36.6 ± 0.0569.27 (9.43, 10.1, 8.28) ± 0.750 11.7 ± 0.0957.6 (7.52,7.62, 7.94) ± 0.179 3.79 ± 0.023

−1) Found conc. (g L−1) Recovery (%)

9.01 (9.04, 9.10, 8.99) ± 0.06 98.010.26 (10.14, 10.27 10.36) ± 0.09 1039.88 (9.75, 9.82, 10.04) ± 0.12 1019.01 (9.01, 8.96, 9.04) ± 0.03 103

146 Y.-S. Li, X.-F. Gao / Analytica Chim

Table 3Reproducibility test of alcohol samples by using IE-FCA (n = 11)

Sample conc, (g L−1) Mean value (F) S.D. (F) R.S.D. (%)

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. Conclusions

In the research, by combining the immobilized enzymeethod with the FCA, a new method has been proposed for

he determination of ethanol. The determination range of thethanol is 2.0–15 g L−1 (y = 10.44x + 6.6002, r > 0.9958). Theetection limit of ethanol is 1.11 g L−1, with R.S.D. < 1.9%. Thexperimental conditions for ethanol determination are as fol-ows: its reaction time is 20 min, test temperature is 40 ◦C, theoncentrations of PBS (pH 7.5) and NAD+ are 0.1 mol L−1 andmmol L−1, respectively; the ADH concentration for immo-ilization is 10 U L−1. The reproducibility and sensitivity ofE-EBCR is better than that of liquid enzyme method. Becauset can be used repeatedly, the IE-ECBR can save lots of expen-ive enzyme and decrease the waste and realize trace analysis tomicro-volume sample. Its operation procedure and the appa-

atus to be used are simple, so it can be easily popularized.he IE-EFCA method is applicable for the determination of thelcohol samples in medicine, industry and environment. Thisethod is suitable for batch of low-concentration sample deter-ination and field monitoring. The analytical products that areade based on this method are easy to carry and store, and can

e used directly.The content of this paper was a prophase study result. Conse-

uently, we had first focussed on the investigation of conditionsor liquid enzyme, and under its optimized condition the exam-nation and confirmation of the feasibility of the IE-ECBR wasonducted and the purpose of determining ethanol in the samplesf distilled spirit etc. have been achieved. Further, the opti-ization of the conditions of enzyme immobilized capillary

ioreactor is done.

cknowledgement

The authors are grateful to the Promotion Program Founda-ion of the Sichuan University of China for the financial supportf the project (No. 0082204127067).

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ica Acta 588 (2007) 140–146

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