Fiber-optic biosensor for the detection of organophosphorus compoundsusing AChE-immobilized viologen LB films

Jeong-Woo Choi*, Junhong Min, Jung-Won Jung, Hee-Woo Rhee, Won Hong Lee

Department of Chemical Engineering, Sogang University, C.P.O. Box 1142, Seoul 100-611, South Korea

Abstract

Acetylcholinesterase (AChE)-immobilized viologen Langmuir–Blodgett (LB) film was fabricated for the development of a fiber-opticbiosensor to detect organophosphorus compounds in contaminated water. AChE was adsorbed onto viologen monolayer by electrostaticforce. The optimal amount of the dissolved enzyme for the LB film formation was determined based on the enzyme adsorption isotherm andrelative enzyme amount adsorbed. The surface characteristics of AChE-immobilized viologen LB film were analyzed at various conditionsof surface pressure with atomic force microscopy. The optimal number of AChE-immobilized viologen LB film was determined based onthe enzyme activity. The signal output of the sensor composed of AChE–viologen hetero LB film was obtained for the various concentra-tions of organophosphorus compounds. The response time to the steady signal and the detection range of the sensor were 5 min and 0–2.0ppm, respectively. 1998 Elsevier Science S.A. All rights reserved

Keywords:Fiber-optic biosensor; Acetylcholinesterase; Organophosphorus compound; Langmuir–Blodgett film; Atomic force microscopy

1. Introduction

The detection of the ground-water contamination due tothe wide use of pesticides has been needed for the qualitycontrol of water supplied. Paraoxon, an organophosphorusplant protective, is a widely used commercial insecticidewith a very wide range of activity. It is known that acetyl-cholinesterase (AChE), the essential enzyme in the nervetissue, is inhibited by organophosphorus compounds widelyused as pesticides and nerve toxin for military purposes [1].The biosensors using the enzyme reaction have been devel-oped due to reasons such as easy handling, no need forexpensive equipment, and capability of the simultaneousmeasurement of a great number of samples. Several workshave been published about the development of biosensor forthe analysis of organophosphorus compounds using immo-bilized enzymes [2,3]. The authors reported that the fiber-optic biosensor consisting of a tubular enzyme reactor andtwo sensing parts was developed for the detection of orga-nophosphorus compounds [4]. The reaction part of the bio-

sensor consists of a silicon tube containing a coordinated setof enzymes entrapped by Ca-alginate gel on the inner sur-face [4].

The Langmuir–Blodgett (LB) film technique has beenused for the formation of organized molecular film for thesensor device [5,7]. The advantages of this technique are theultrathin film deposition, highly ordered molecular array,easiness of packing and stacking molecules, low tempera-ture and biomimetic membrane fabrication [8,9]. It is neces-sary to investigate the microscopic morphology of theenzyme-immobilized LB films using atomic force micro-scopy (AFM) to optimize the LB film formation conditionfor the enhancement of the sensor signal.

The objective of this study is to develop a fiber-opticbiosensor using enzyme–viologen hetero LB films for thedetection of organophosphorus compounds. To optimize theformation condition of the AChE-immobilized LB film forthe improvement of the sensor signal output, the optimalspreading amount of enzyme, the effect of dipping pressure,the topology observation of the LB films by AFM, and theeffect of the number of LB layers on enzyme activity wereinvestigated. The signal output of the sensor composed ofAChE–viologen hetero LB films was measured at variousconcentrations of organophosphorus compounds.

Thin Solid Films 327–329 (1998) 676–680

0040-6090/98/$ - see front matter 1998 Elsevier Science S.A. All rights reservedPII S0040-6090(98)00739-1

* Corresponding author. Tel.: +82 2 7058480; fax: +82 2 7110439;e-mail: jwchoi@ccs.sogang.ac.kr

2. Experimental

2.1. Materials

Acetylcholinesterase (AChE, EC 3.1.1.7: V-s, from elec-tric eel) with a specific activity of 1000 u/mg and Paraoxonwere obtained from Sigma (St. Louis, MO). Viologen with along chain for LB films was synthesized according to theRef. [10]. C18N was mixed with C20Me (molar ratio of 1:4)to form a monolayer of C18N0.2 that has a positive charge ofhead group [5,6]. Chloroform was used as the spreadingsolvent for the lipid and viologen. The topography ofAChE-containing LB film was obtained by AFM (Autop-robe CP, Park Scientific Instruments, USA).

2.2. Sensor configuration

The configuration of sensor system is schematicallyshown in Fig. 1. The three solutions (distilled water, litmusand acetylthiocholine iodide solutions, and sample solution)were prepared. Substrate and dye solution with potassiumphosphate buffer (pH 7.2) and distilled water were mixedusing a peristaltic pump (Masterflex, Cole-Parmer Inst.) forinvariant concentration of substrate when the sample wasintroduced and flowed to the enzyme reactor at first. Afterthe reaction period, the distilled water was exchanged withsample solution, which contained various concentrations ofParaoxon. The mixing ratio of the sample to mixed solutionof dye and substrate was 1:128 (v/v). The AChE convertedthe acetylthiocholine iodide into acetic acid and thiocholine.The selection of dye was done based on the absorptionspectra to the 633 nm line of He-Ne laser and the transmis-sibility of fiber. The blue color of litmus dye was changed tored by the pH decrease due to the formation of acetic acid.The difference of absorption at 633 nm, i.e. the difference ofa red color formation of litmus, by enzyme reaction repre-sented the product formation. As the inhibitor was intro-duced, decrease of the absorption difference occurred due

to the inhibition of inhibitor on the AChE, which was pro-portional to inhibitor concentration. By monitoring thetransmittance at 633 nm, concentration of the organopho-sphorus compounds could be determined.

2.3. The preparation of AChE-immobilized LB films

For enzyme immobilization, three compartment LBtroughs of Fromherz’s type (Nima Tech, Coventry, UK)was used. 1 mM HEPES buffer was used as the subphaseat pH 7.0, which was adjusted with 1 M NaOH solution.Lipid and viologen were dissolved in chloroform to 1 mM,respectively, which were then used to form the monolayer.The solid substrate, quartz and mica, were prepared to makea hydrophobic surface by dipping in 0.2%n-octadecyltri-cholorosilane toluene solution for 30 min and then rinsingwith fresh toluene.

The AChE-immobilized lipid monolayer was prepared byFromherz’s method using a multicompartment trough. Theprepared lipid or viologen solution was spread and left for20 min in one compartment. After evaporation of chloro-form, the lipid or viologen monolayer was formed and thenwas compressed to a surface pressure of 20–40 mN/m. Theenzyme solution prepared by dissolving the enzyme in thesame buffer was injected to another compartment. The bar-riers were moved to the enzyme compartment keeping thelipid or viologen monolayer and left for 60 min for theadsorption of enzyme molecules onto the layer. After form-ing the AChE-immobilized lipid or viologen monolayer, itwas moved from the enzyme compartment to the dippingcompartment and dipping was then performed at a speed of5 mm/min.

2.4. Relative amount of AChE adsorbed

For measurement of adsorbed enzyme amount in the LBfilms, o-phthaldialdehyde and 2-mercaptoethanol wereused. The measurement of fluorescence intensity for thecomplex of o-phthaldialdehyde and amino group ofAChE, the fluorescence material, at 455 nm was done toestimate the adsorbed AChE amount [5].

3. Results and discussion

3.1. Surface–pressure isotherm

p–A isotherms of the lipid monolayer, viologen mono-layer, AChE-immobilized lipid monolayer and AChE-immobilized viologen monolayer are shown in Fig. 2.Based on the electrostatic force C18N0.2 was chosen as thelipid to be used for the adsorption of AChE. It was reportedthat the electrostatic force between enzyme and lipid layerdominates the adsorption process [5,7]. Since AChE has thenet negative charge at pH 7 due to the isoelectric point ofAChE, 5.5 [11], it can be considered that C18N0.2 with posi-

Fig. 1. The experimenal setup for the biosensor: 1, power supply; 2, He-Nelaser; 3, sample; 4, distilled water; 5, dye and substrate; 6, pump; 7,reference probe; 8, optical fiber; 9, indicator probe; 10, phototransistor;11, amplifier/converter; 12, reactor containing AChE LB film; 13, compu-ter; 14, matrix molecules; 15, bacteriorhodopsin molecules.

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tive surface charge was the suitable lipid for the AChEadsorption. In the AChE-immobilized lipid monolayer, thephase regions were not clearly distinguished. The area permolecule of the lipid was 20 A˚ 2 smaller than that of theAChE-immobilized lipid, 24 A2, as shown in Fig. 2. It canbe considered that the increase of area per molecule was dueto the adsorption and permeation of enzyme molecules intothe pure lipid monolayer [5]. Therefore, the result indicatesthat the AChE was adsorbed to the lipid molecules. But theincrease of area per molecule was very small, which meansthat the adsorbed amount of enzyme was very small.

In the AChE immobilized viologen monolayer, the phaseregions were not clearly distinguished, as shown in Fig. 2.The area per molecule of the viologen monolayer was about4 A2 and that of AChE-immobilized viologen monolayerwas about 100 A˚ 2. The increase of surface area of viologenis larger than that of lipid, which means more adsorption ofenzyme. It is considered that the head group of viologencontains the positive charge derived from two nitrogenatoms, which enhances the adsorption amount of enzyme.Therefore, the viologen was chosen as the more suitablematerial for adsorption of AChE than the lipid from theresults of the large increment in area per molecule.

3.2. Optimal condition of enzyme adsorption

The coverage of AChE adsorbed on the viologen mono-layer could be considered as a function of the amount ofdissolved enzyme in subphase. The surface pressureincrease by permeation and adsorption of enzyme indicatesthe adsorbed amount of enzyme to viologen monolayer atthe air–water interface. The surface pressure of viologenmonolayer at the air–water interface with constant areawas increased in proportion to the amount of dissolvedenzyme up to 300mg, as shown in Fig. 3. The measurementof fluorescence of a complex ofo-phthaldialdehyde and

amino group of AChE at 455 nm could estimate theadsorbed AChE amount [5]. Based on the surface pressureincrease by AChE adsorption and the relative amount ofamino groups of the adsorbed enzyme in LB films, the opti-mal amount of dissolved AChE on subphase was deter-mined as 300mg. The determined optimal amount wasverified by observing the topography of enzyme–viologenmonolayer using AFM. As shown in Fig. 4, AChE was notuniformly adsorbed on the monolayer of viologen. Theaggregates of AChE and viologen were enlarged as theamount of dissolved AChE in subphase was increased upto 300mg. The image of hetero LB film for 500mg of AChEdissolved (Fig. 4d) was very similar to that for 300mg ofAChE dissolved (Fig. 4c).

The dipping surface pressure of AChE adsorbed in theviologen monolayer is one of the key operating variables inthe deposition process by the LB technique. The dipping

Fig. 2. p–A isotherm: (a) pure lipid monolayer; (b) AChE-absorbed lipidmonolayer; (c) pure viologen monolayer; (d) AChE-absorbed viologenmonolayer.

Fig. 3. The absorption isotherm of AChE on the viologen monolayer: (W)pressure increment; (X) relative protein amount.

Fig. 4. The AFM images of AChE adsorbed on the viologen monolayer tothe various AChE amounts in subphase: (a) 100mg, (b) 200mg, (c) 300mg,(d) 500mg.

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surface pressure representing a molecular density of mono-layer is proportional to the surface charge density capable ofadsorbing AChE. Fig. 5 shows that the activity of AChEimmobilized in LB films was increased by up to 30 mN/m inthe dipping surface pressure. But the relative amount ofamino groups of the enzyme adsorbed in LB films waslinearly increased as the surface pressure was increased toover 30 mN/m. This result might be due to the aggregationof AChE adsorbed at the high surface pressure. Fig. 6 showsthe surface topographies of AChE-immobilized LB filmsrepresenting the AChE adsorbed in the viologen monolayer.In the topographies, the higher part corresponds to theenzyme of AChE-immobilized LB films. The aggregatesof AChE adsorbed onto the substrate were increased asthe surface pressure of viologen monolayer was increased.The coverage of AChE on the viologen monolayer at 40mN/m in surface pressure (Fig. 6d) was more enlarged

than that at 35 mN/m (Fig. 6c). It might be consideredthat the larger aggregate of AChE adsorbed prevents thereaction of AChE in the films over 35 mN/m of surfacepressure. Based on the enzyme activity, the optimum dip-ping surface pressure to enhance the sensor outputs wasdetermined as 35 mN/m.

3.3. Sensor signal

To enhance the sensor signal, the extent of reaction ofAChE immobilized in LB film was investigated by consid-ering the relationship between the number of enzyme–LBlayers and enzyme activity. The effect of number of AChE-adsorbed LB layers is shown in Fig. 7. In Fig. 7a, the filmswere deposited asZ-type with weak tail–tail interaction ofviologen due to the effect of AChE adsorbed on the headgroup. The activity of AChE adsorbed in the LB films wasmeasured as a function of stroke number, as shown in Fig.7b. The activity was linearly increased up to 30 strokes andthen the extent of activity increment was decreased as thestroke number was increased further. This result might bedue to the adsorption of AChE on the viologen with type of

Fig. 5. The effect of dipping surface pressure on the AChE-viologen films:(X) activity; (W) relative protein amount.

Fig. 6. The AFM image of AChE adsorbed on the viologen monolayer tothe various dipping surface pressures: (a) 25 mN/m, (b) 30 mN/m, (c) 35mN/m, (d) 40 mN/m.

Fig. 7. (a) The deposition type of AChE–viologen monolayer: (X) to +,downstroke;+ to (X), upstroke. (b) The effect of the stroke number on theactivity of AChE.

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cluster. Based on the above result, the optimal stroke num-ber for the deposition of AChE-adsorbed viologen LB filmswas determined as 60.

The signal output of the sensor composed of AChE–vio-logen hetero LB films deposited at the optimal operatingconditions was obtained with the various concentrationsof organophosphorus compound, as shown in Fig. 8. Theresults showed that signal was proportional to the concen-tration of organophosphorus compound, and the detectiontime was 5 min. The detection range of the sensor represent-

ing the linear relationship between the signal output andanalyte concentration was about 0–2.0 ppm.

Acknowledgements

This work was supported by the Biochemical Engineer-ing Fund of Korea Ministry of Education (1996).

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Fig. 8. The signal of the fiber-optic biosensor.

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