Optical biosensor consisting of glutathione-S-transferase fordetection of captan

Jeong-Woo Choi a,*, Young-Kee Kim b, Sun-Young Song a, In-ho Lee a,Won Hong Lee a

a Department of Chemical Engineering, Sogang University, C.P.O. Box 1142, Seoul 100-611, South Koreab Department of Chemical Engineering, Hankyong National University, Sukjung-dong 67, Ansung, Kyonggi-do 456-749, South Korea

Received 17 May 2001; received in revised form 3 September 2002; accepted 8 March 2003

Abstract

The optical biosensor consisting of a glutathione-S-transferase (GST)-immobilized gel film was developed to detect captan in

contaminated water. The sensing scheme was based on the decrease of yellow product, s-(2,4-dinitrobenzene) glutathione, produced

from substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), due to the inhibition of GST reaction by captan.

Absorbance of the product as the output of enzyme reaction was detected and the light was guided through the optical fibers. The

enzyme reactor of the sensor system was fabricated by the gel entrapment technique for the immobilized GST film. The immobilized

GST had the maximum activity at pH 6.5. The optimal concentrations of substrates were determined with 1 mM for both of CDNB

and GSH. The optimum concentration of enzyme was also determined with 100 mg/ml. The activity of immobilized enzyme was

fairly sustained during 30 days. The proposed biosensor could successfully detect the captan up to 2 ppm and the response time to

steady signal was about 15 min.

# 2003 Elsevier Science B.V. All rights reserved.

Keywords: Glutathione-S-transferase; Captan; Gel entrapment; Optical biosensor; 1-Chloro-2,4-dinitrobenzene; Glutathione

1. Introduction

Captans are non-systemic fungicides used to control

diseases of many fruit, ornamental, and vegetable crops

like as pathogenic mold and bacteria. And the captans

are widely used in farming for the plant protection

purposes. However, captan is known as a potential

carcinogen and a harmful chemical to a water ecosystem

(Mao et al., 1998; Martens and Bremner, 1997; Mueller

et al., 1999). Therefore, the rapid and simple detection

of captan in the contaminated water is required in water

supplies (Wittmann and Schmid, 1993; Wolfbeis and

Koller, 1989; Wolfgang et al., 1993). The sustaining

application of captans and the growing concern with the

potential contamination of water requires the develop-

ment of fast and sensitive detection method. The

conventional measuring methods, such as gas chroma-

tography (GC) and high performance liquid chromato-

graphy (HPLC), are very expensive and time consuming,

because they require sophisticate laboratory equipment

(Ingram et al., 1997). By the way, biosensors using

enzyme reaction are easy to handle, cheap equipment

and allow the simultaneous measurement for a great

number of samples, so many researchers developed

enzyme biosensor with various transducer (Choi et al.,

2001; Wolfbeis and Koller, 1989; Wolfgang et al., 1993).

However, the optical enzyme biosensor to detect captan

has not been studied. Optical sensor systems have many

advantages compared with the other sensor systems due

to their capability of remote and multiple sensing

(Klaimer et al., 1993; Trettnak et al., 1991). Optical

sensors are not interfered with an electric field and are

easy to miniaturize, which can lead the development of

very small, light and flexible sensors (Carome et al.,

1993; Trettnak et al., 1993).

In this study, optical biosensors to detect the captan

have been developed based on the inhibition of glu-

* Corresponding author. Tel.: �/82-2-705-8480; fax: �/82-2-711-

0439.

E-mail address: jwchoi@ccs.sogang.ac.kr (J.-W. Choi).

Biosensors and Bioelectronics 18 (2003) 1461�/1466

www.elsevier.com/locate/bios

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

doi:10.1016/S0956-5663(03)00124-6

tathione-S-transferase (GST) by captan. The amounts of

captan were determined by measuring the absorbance

change of products amounts, which caused by the

inhibition of immobilized enzyme by inhibitors (captan).The sensing scheme is based on enzyme reaction that

GST converts the substrates, 1-chloro-2,4-dinitroben-

zene (CDNB) and glutathione (GSH), into yellow

products, s-(2,4-dinitrophenyl) glutathione (Antolini et

al., 1995; Dillio et al., 1996; Hansson et al., 1999). In the

absence of inhibitors, the substrates are completely

converted into yellow products, while in the presence

of inhibitors, the amounts of yellow product arereduced. This absorbance change, due to the different

amounts of yellow products, can be related to the

amounts of captan in a proposed sensor system. The

reaction characteristics of GST-immobilized gel film

were analyzed. The detection range and response time to

the steady signal was also investigated.

2. Materials and methods

2.1. Materials

GST (EC 2.5.1.18, from human placenta) with a

specific activity of 125 U/mg, the substrates (CDNB and

GSH), and the inhibitor (captan) were purchased from

Sigma chemical company (St. Louis, MO, USA). To

immobilize GST, enzyme was entrapped in the gel ofsodium alginate formed by 1.5% w/w of sodium alginate

solution and 1.5% w/w of CaCl2 solution.

2.2. Sensor system configuration

The configuration of sensor system is schematically

shown in Fig. 1. The three kinds of solutions (distilled

water, substrates (CDNB and GSH) and sample solu-tions) were prepared. The CDNB and GSH were

prepared in a potassium phosphate buffer (pH 6.5, 4

mM) and were mixed with distilled water using a

peristaltic pump (Model 7017, Marubishi, Japan) for

invariant concentration of substrate when the sample

was introduced to the enzyme reactor. After a steady

enzyme reaction was achieved, the distilled water was

exchanged with sample solution containing variousconcentrations of captan. The mixing ratio was 1:1 (v/

v) for the substrates solution and distilled water or

sample solution. The mixing was achieved by in-line and

peristaltic pump, and the flow rates of mixtures were in

a range of 1.5�/3.3 ml/min. The substrates were con-

verted into yellow products, s-(2,4-dinitrophenyl) glu-

tathione, by the GST reaction. Light emitting from a Xe

arc lamp (300 W, Model 6258, Oriel Instruments, CT,USA) was filtered by optical band-pass filter (400 nm)

and was guided through optical fibers to the two parts

of an enzyme reactor (light path 20 mm, volume 30 ml).

Transmitted lights were guided through optical fibers to

photodiodes. In the absence of inhibitors, the substrates

were completely converted while the amounts of yellow

product were reduced in the presence of inhibitors. Thisabsorbance change at 400 nm could be detected with a

proposed detection system. The absorbance was mea-

sured at two points. The first (reference) signal was

measured at an inlet of reactor before the enzyme

reaction. And the second signal was measured after

enzyme reaction at an outlet of reactor. The sensor

signal was represented as a difference between two

values. By monitoring the transmittance at 400 nm,the concentration of captan could be determined.

3. Results and discussion

3.1. Detection principle

The GST converted the CDNB and GSH into yellow

products, s-(2,4-dinitrophenyl) glutathione. The absorp-tion spectra of substrates, product, and captan solution

were shown in Fig. 2. It was shown that the largest

difference of absorbance between the substrates and

products was detected at 335 nm. However, the sig-

nificant absorbance of captan solution was detected in

this wavelength. Also, in order to fabricate fiber-optic

sensor system to detect the absorbance at a wavelength

of 335 nm, a fused silica fiber should be used as the lightguide but this material was a very expensive. Therefore,

an inexpensive plastic fiber (diameter 3 mm) with the

application range from 400 to 700 nm was used to

fabricate the economical biosensor system, and all

experiments were carried out at the wavelength of 400

nm.

The difference of absorbance at 400 nm represented

the product formation due to enzyme reaction. In theabsence of inhibitors, the substrates were completely

converted. As the inhibitors were introduced, the

absorbance decreased due to the inhibition of GST

reaction and this absorbance change was proportional

to the inhibitor concentration.

3.2. Effect of pH on immobilized enzyme reaction

The pH of the substrates solutions can affect overall

enzymatic activity because enzymes have a native

tertiary structure like all natural proteins that are

sensitive to pH and the denaturation of enzymes can

occur at extreme pH levels. Thus, the appropriate pH

range for a specific enzyme should be determined

empirically. The effect of pH on the reaction of the

immobilized enzyme was examined with the use ofpotassium phosphate buffer over the pH range of 5.0�/

7.0 by measuring the absorbance of products using an

UV�/vis spectrophotometer (Fig. 3). The potassium

J.-W. Choi et al. / Biosensors and Bioelectronics 18 (2003) 1461�/14661462

phosphate buffers in the range of pH 5.0�/7.0 were

prepared with respective mixing ratio of NaH2PO4 and

Na2HPO4 �/7H2O and these buffers had a sufficient

buffering capacity. It was observed that immobilized

GST had the maximum activity at pH 6.5.

3.3. Effect of substrate concentration on enzyme reaction

The substrates, CDNB and GSH, are converted into

yellow products, S-(2,4-dinitrophenyl) glutathione byGST reaction. Fig. 4 shows the magnitude of an

absorbance, which was measured using an UV�/vis

spectrophotometer. Fig. 4(a) shows the absorbance

change when the concentrations of GSH were varied

from 0 to 2 mM in 1 mM CDNB concentration. Fig.

4(b) shows the absorbance change when the concentra-

tions of CDNB were varied from 0 to 2 mM in 1 mM

GSH concentration. In Fig. 4(a) and (b), the magnitude

of absorbance, which represents the product amounts of

enzyme reaction, increased as the substrate concentra-

tions increased until the reaction rate was saturated. The

saturated reaction rate was essentially defined by the

used enzyme amounts and also could be affected by

diffusion limitation in gel membrane. From these

results, it was observed that both of the CDNB and

GSH concentrations in 1 mM showed the maximum

enzyme reaction rate.

Fig. 1. The experimental set-up of biosensor system; (1) sample; (2) distilled water; (3) substrate; (4) peristaltic pump; (5) reference; (6) optical fiber;

(7) detector; (8) power supply; (9) Xe-lamp; (10) optical band pass filter (400 nm); (11) photodiode; (12) multimeter; (13) computer; (14) GST

immobilized gel film.

Fig. 2. The absorption spectra of substrates mixture, product, and

captan solution (solid line: product, dashed line: substrates mixture,

and dash�/dot line: captan solution).

Fig. 3. The effect of pH on the activity of immobilized GST reaction.

J.-W. Choi et al. / Biosensors and Bioelectronics 18 (2003) 1461�/1466 1463

3.4. Effect of immobilized enzyme amounts on sensor

signal

The effects of amounts of immobilized enzymes on

sensor signal are shown in Fig. 5. It was observed that

the increment of the enzyme amounts resulted in the

increment of sensor signal. However, enzymes are the

most expensive material in sensor system, so the

optimization of enzyme amounts should be conducted.From 10 to 100 mg/ml of the enzymes amounts, the

sensor signal increased rapidly and the signal increased

slightly over 100 mg/ml. From these results, optimum

enzyme concentration was determined to 100 mg/ml with

respect to economic aspect.

3.5. Effect of retention time on sensor signal

The magnitude of sensor signal was investigated with

the sample retention time of 9�/20 min in flow cell. Fig. 6

shows that the magnitude of sensor signal increased

slowly with the increment of retention time up to 13.5

min, and increased rapidly from 13.5 to 17 min. These

results showed that the fast flow rates resulted in smaller

signal because the contacting time between substratesand enzymes was not sufficient to enzyme/substrate

reaction. Therefore, the retention time of 15 min was

determined as an optimum retention time, from the

viewpoint of sufficient measurable signal and fast

detection time.

3.6. Sensor signal analysis

When the substrates solution entered into a flow cell

with GST-immobilized gel film, the substrates was

converted into yellow products by GST. The transmit-

tances were measured at two parts of flow cell.

Reference signal was maintained throughout the experi-

ment, but reaction signal varied. After reaction signalreached steady value, inhibitor (captan) was injected at

600 s. After the inhibitor injection, reaction signal

increased from 1100 to 1200 s, and then the significant

change of reaction signal was not observed (Fig. 7).

From these results, it was observed that inhibition

reaction required 600 s in a proposed sensor system.

Fig. 4. The effect of substrates concentration on GST activity. (a)

CDNB concentration: 1 mM; (b) GSH concentration: 1 mM.

Fig. 5. The effect of immobilized GST amounts on sensor signal

(CDNB concentration: 1 mM and GSH concentration: 1 mM). Fig. 6. The effect of retention time on sensor signal.

J.-W. Choi et al. / Biosensors and Bioelectronics 18 (2003) 1461�/14661464

The sensor signal was calculated as a difference between

reference signal and reaction signal. The sensor signals

were obtained with the various concentrations of captan

as shown in Fig. 8 (data obtained from the three

replicated experiments). These results showed that

sensor signal was proportional to the concentration of

captan. The detection range of a proposed biosensor

was determined in a range of 0�/2.0 ppm captan because

the linear relationship between the sensor signal and

captan concentration was obtained in this range.

3.7. Stability of immobilized enzyme

Because the enzymes are biological materials, enzyme

activity decreases with time. Therefore, the sustainment

of enzyme activity is important for enzyme biosensors.

Fig. 9 shows the stability of enzymes immobilized by gel

entrapment technique. The GST-immobilized film

stored in a freezer of �/20 8C during the experiment.

In Fig. 9, absorbance measured by an UV�/vis spectro-

photometer was sustained up to 30 days and rapidly

decreased after that.

4. Conclusions

The optical biosensor consisting of GST-immobilized

gel film was constructed for the simple and direct

detection of captan in contaminated water. The absor-

bance change of product to be induced directly by the

inhibition of captan on immobilized GST was success-fully detected by a proposed sensor system. The

immobilized GST had the maximum activity at pH

6.5. The optimum concentrations of substrates were

determined with 1 mM in both of CDNB and GSH. The

optimum amounts of enzyme were determined as 100

mg/ml. And the stability of immobilized enzyme was

sustained up to 30 days. The proposed biosensor could

successfully detect the captan up to 2 ppm and theresponse time to steady sensor signal was about 15 min.

It was known that GST used in this study has resistance

to most of other toxic substances, especially insecticides

(Perry et al., 1998). Therefore, a proposed GST im-

mobilized biosensor system can be used for analysis of

captan in real contaminated samples. In conclusion,

captan in contaminated water could be detected easily

and cheap by a proposed sensor system compared withconventional analysis method (Ingram et al., 1997;

Wittmann and Schmid, 1993).

Acknowledgements

This work was supported in part by the Korea

Ministry of Agriculture and Forestry through theAgricultural R&D Promotion Center (2991)16-3) and

by the Korea Science and Engineering Foundation

(KOSEF) through the Advanced Environmental Mon-

Fig. 7. Typical sensor signal analysis for a proposed sensor system.

Fig. 8. Calibration curve of captan concentration vs. sensor signal (bar

represents the standard error of three experiments).

Fig. 9. The stability of immobilized GST activity.

J.-W. Choi et al. / Biosensors and Bioelectronics 18 (2003) 1461�/1466 1465

itoring Research Center at Kwangju Institute of Science

and Technology.

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