Biosensors & Bioelectronics 16 (2001) 819–825

Rectified photocurrent of molecular photodiode consisting ofcytochrome c/GFP hetero thin films�

Jeong-Woo Choi a,*, Yun-Suk Nam a, Sei-Jeong Park a, Won-Hong Lee a,Dongho Kim b, Masamichi Fujihira c

a Department of Chemical Engineering, Sogang Uni�ersity, CPO Box 1142, Seoul 100-611, South Koreab National Creati�e Research Initiati�es Center for Ultrafast Optical Characteristics Control Spectroscopy Laboratory,

Korea Research Institute of Standards and Science, PO Box 102, Yusong, Taejeon 305-600, South Koreac Department of Biomolecular Engineering, Tokyo Institute of Technology, Yokohama 227, Japan

Received 25 May 2000; received in revised form 10 January 2001; accepted 23 February 2001

Abstract

Photoinduced electron transfer in the molecular electronic device consisting of protein-adsorbed hetero Langmuir–Blodgett(LB) film was investigated. Three kinds of functional molecules, cytochrome c, viologen, and green fluorescent protein (GFP) wereused as an electron acceptor, a mediator, and a sensitizer, respectively. The hetero-LB film was fabricated by subsequentlydepositing cytochrome c and viologen onto the pretreated ITO or quartz glass. GFP adsorbed hetero-LB films were prepared bysoaking the hetero-LB films into the buffer solution containing GFP. The MIM (metal/insulator/metal) structured moleculardevice was constructed by depositing aluminum onto the surface of the GFP-adsorbed hetero LB films. Due to the excitation byirradiation with a 460 nm monochromic light source, the photoinduced unidirectional flow of electrons in the MIM device couldbe achieved and was detected as photocurrents. The photoswitching function was achieved and the rectifying characteristic wasobserved in the molecular device. Based on the measurement of transient photocurrent of molecular device, the unidirectional flowof electrons was verified. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Langmuir–Blodgett film; Cytochrome c ; Green fluorescent protein; Molecular photodiode; Photocurrent

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1. Introduction

Photoinduced electron transport processes in naturesuch as photoelectric conversion and long-range elec-tron transfer in photosynthetic organisms are known tooccur not only very efficiently but also unidirectionallythrough the functional groups of biomolecules (Deisen-hofer et al., 1985). The concept or idea in the develop-ment of new functional bioelectronic devices can beinspired from the biological systems such as electrontransfer chain or photosynthetic reaction center. Bymimicking the organization of the functional moleculesin the biological electron transfer system, the artificialbioelectronic device can be realized.

In the initial process of photosynthesis, a biologicalelectron transfer system, photoelectric conversion oc-curs and then long-range electron transfer takes placevery efficiently in one direction through thebiomolecules (Gust and Moore, 1989; Karvanos, 1993).The specific energy and electron transfer take place ona molecular scale due to the redox potential differenceas well as the electron transfer property of functionalmolecules, especially the electron acceptors, sensitizer,and electron donor (Fujihira, 1995).

Various artificial molecular devices have been fabri-cated by mimicking the electron transport function ofbiological photosynthesis. Fujihira et al. (1985) re-ported the electrochemical photodiode using Lang-muir–Blodgett (LB) films of three functional moleculesor an aligned triad on the electrode, which worked inelectrolyte solution (Sakomura and Fujihira, 1994; Fu-jihira, 1995). The optical and electrical characteristics of

� Editors Selection* Corresponding author. Tel.: +82-2-705-8480; fax: +82-2-711-

0439.E-mail address: jwchoi@ccs.sogang.ac.kr (J.-W. Choi).

0956-5663/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S 0 9 5 6 -5663 (01 )00225 -1

J.-W. Choi et al. / Biosensors & Bioelectronics 16 (2001) 819–825820

the molecular device consisting of flavin/porphyrin- orflavin/cytochrome c- hetero–LB films have been inves-tigated (Isoda et al., 1992; Ueyama, 1997). The molecu-lar photodiode consisting of hetero-organic LB film offour functional organic molecules, ferrocene, flavin,viologen, and TCNQ to be used as the electron donor,sensitizer, electron relay and electron acceptor units,respectively, were fabricated and photoinduced electrontransfer was investigated (Choi et al., 1998a,b). Theauthors reported that the fabrication of molecularphotodiode consisting of GFP and viologen was doneand the photoinduced electron transfer of the devicewas observed (Choi et al., 2000). Though the combina-tion of organic molecules and biological molecules hasbeen used to construct the molecular device, thebiomolecular device composed of biological moleculesto be used as a sensitizer and an electron acceptor hasnot been reported yet.

Cytochrome c is one of the most widely studiedproteins due to its stability and solubility in water aswell as availability from many sources. The structure,physiological and physicochemical properties, and ap-plications of cytochrome c, have been extensively stud-ied through the various research fields (Lippard andBerg, 1994). The key feature of cytochrome c, capabil-ity of electron transfer, is driven from the redox statechange and conformational change of heme groupscovalently bound via two thioether linkages formed bytwo cysteine side chains and two axial ligands, histidineand methionine. Since cytochrome c is a constituentacting as an electron transfer protein in the bacterialphotosynthetic reaction center, cytochrome c could beused as an electron acceptor in the development ofmolecular electronic device by mimicking biologicalphotosynthesis mechanism (Ueyama, 1997).

The GFP from the jellyfish Aequorea �ictoria hasbeen widely used as a marker in the determination ofgene expression and protein localization (Li et al.,1997). The purified GFP, a protein of 238 amino acids,absorbs blue light and emits green light (peak emissionat 510 nm). This fluorescence is very stable, and virtu-ally no photobleaching is observed (Chalfie et al.,1994). Since GFP shows very highly fluorescence quan-tum yield, approx. 80%, it is very reasonable approachto use GFP as a sensitizer in the development ofmolecular electronic device by mimicking biologicalphotosynthesis mechanism (Choi et al., 2000).

In this study, the metal/insulator/metal (MIM) struc-tured device was fabricated with GFP/viologen/cy-tochrome c hetero-films to be used as an electronsensitizer, a mediator and an electron acceptor, respec-tively. To investigate the photoindued electron transferof the proposed device, the photo-switching functionand rectifying characteristic of the device were mea-sured. The analysis of film surface by atomic forcemicroscopy (AFM) was carried out to verify the film

construction. To investigate the dynamic process ofcharge transfer and to observe the performance ofhetero-type molecular electronic device as a switchingdevice, the transient photocurrents of the GFP homo-,GFP/viologen hetero- and GFP/viologen/cytochrome chetero-structured MIM devices were measured.

2. Materials and methods

2.1. Materials

Green fluorescent protein (GFP), N-allyl-N �-[3-propylamido-N �,N �-di(n-octadecyl)]-4, 4�-bipyridium di-bromide (viologen), and cytochrome c (extracted fromhorse heart, type VI) were used as an electron sensitizer(S), a mediator (M), and an electron acceptor (A),respectively. Organic mediator, viologen, was synthe-sized according to the method by Tundo et al. (1982),and GFP (rEGFP), cytochrome c was purchased fromClontech Co. (Palo Alto, CA) and Sigma Chemical Co.(St. Louis, MO), respectively. Other reagents were pur-chased from Sigma Chemical Co. and used withoutfurther purification.

2.2. Fabrication of molecular hetero-film

Considering the redox potential of each moleculeobtained by cyclic voltammetry measurement (Choi etal., 1998a, 2000), the A and M molecules were de-posited onto ITO coated glass by LB technique andsubsequently adsorbing S molecules onto the M/A LBfilm surface. Fig. 1 shows the energy diagram of themolecular array consisting of S/M/A hetero film. In thismolecular photodiode, the photoinduced electric signalwas generated by light irradiation, so that the lighttransparency and electrical resistance of the device wereconsidered. The ITO coated glass substrate is adequatefor the bottom electrode of the molecular photodiode,because of mid transparency and low electrical resis-tance less than ca 20.

Fig. 1. Schematic illustration of energy diagram with redox potentialdifference.

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The deposition of LB film was carried out using acircular type Langmuir trough (Type 2022, Nima Tech.,UK). Cytochrome c was mixed with phosphate buffersolution with pH 8. Its concentration is 4.0 �M. Forspreading cytochrome c solution on the subphase, cy-tochrome c was mixed with ethanol diluted with deion-ized distilled water (DDW). The volume ratio ofethanol, cytochrome c and DDW is 2:2:1. Deionizedwater (�18 MW) was used as a subphase. Afterspreading of cytochrome c molecules, monolayers ofcytochrome c on the subphase was compressed to thesurface pressure of 20 mN/m, which was previouslydetermined as the target pressure for the optimal depo-sition condition of its material. Three layers of cy-tochrome c was deposited on ITO-coated glass for thedevice. If the cytochrome c LB layers are thicker (abovethree layers), the surface of cytochrome c LB layersbecomes more irregular. Also, it is too difficult to detectthe photoelectric signal of below three layers of cy-tochrome c. Thus, three layer of cytochrome c wereadequate for the proposed molecular photodiode. Chlo-roform was used as the spreading solvent for the violo-gen molecules. The 10 layers of viologen was depositedon the cytochrome c LB film. In the case of viologen,the target pressure was 37 mN/m. The GFP and cy-tochrome c are water-soluble. So, the GFP monolayercannot be directly deposited onto the cytochrome c LBlayer by LB or self-assembly (SA) method. However,the viologen LB layer is easily formed onto the cy-tochrome c layer by LB method. In pH 8.0, GFP hasthe negative net charge because the isoelectric point (pI)of the GFP is 5.0. In addition, the mediator film surfacehas been positive net charge in pH 8.0. Due to the netcharge difference, two functional molecules were easilyinteracted and the GFP was adsorbed onto the violo-gen. However, the electrostatic force is weak interac-tion. So, the parts of GFP molecules were adsorbedonto the viologen surface, and formed monolayer.

Finally, the SA monolayer of GFP was constructedby dipping the hetero-LB film into the GFP solution.Thus the GFP/viologen/cytochrome c structured molec-ular hetero-film was constructed with the electrostaticforce attraction. After adsorbing of GFP monolayersonto the viologen/cytochrome c LB layer, the GFP/vio-logen/cytochrome c structured hetero-film was driedunder the nitrogen atmosphere at 50 °C because thepresence of adsorbed water can take detrimental effectto the deposition of Al top electrode.

The topographies of hetero-films were obtained byAFM (Auto Probe CP, Park Scientific Instruments,USA). UV-visible absorption and fluorescence emissionspectrum of hetero-film were measured by UV/VIS/NIR Spectrophotometer (V-550 Jasco, Japan) andFluorescence Spectrometer (Photon Tech. Inc., USA).

Fig. 2. Experimental system for the photoelectric response measure-ment.1, 500 W xenon lamp; 2, 460 nm filter; 3, shield box; 4, sampledevice; 5, signal measurement unit (Keithley, SMU Model 236); 6,PC.

2.3. Photocurrent measurement

To measure the photoelectric response of the molecu-lar device, MIM structured device was constructed. Tomake a top electrode, the Al deposition onto the GFP/viologen/cytochrome c (S/M/A) hetero-films was per-formed by vacuum coater (VPC-260F, Sinku Kiko Co.,Japan) with 500 A� thickness. The molecular photodiodehas 2.5×1.5 cm size. The input light of 460 nm wave-length was generated by the 500 W xenon lamp (OrielCo., USA) system, and then the photocurrent producedfrom the molecular electronic device was detect througha current-voltage (I–V) measuring unit (SMU Model236, Keithley, USA) and personal computer. Fig. 2shows the experimental system for the current–voltage(I–V) measurement.

2.4. Transient photocurrent measurement

Fig. 3 shows the experimental system for the tran-sient photocurrent measurement. A light pulse fromlaser system consisting of a self-mode-locked femtosec-ond Ti:sapphire laser (Clark MXR, NJA-5), aTi:sapphire regenerative amplifier (Clark MXR, CPA-

Fig. 3. Experimental system for the transient photocurrent measure-ment. 1, Ti:sapphire laser (400 nm, 400 fs FWHM); 2, boxcar; 3,shield box; 4, Cu; 5, glass; 6, ITO; 7, film; 8, Al; 9, amplifier; 10,signal; 11, trigger; 12, oscilloscope; 13, data acquisition.

J.-W. Choi et al. / Biosensors & Bioelectronics 16 (2001) 819–825822

Fig. 4. Characteristics of UV-visible absorption and fluorescenceemission spectrum of the S/M/A hetero-film; solid line; absorptionspectrum; dotted line; emission spectrum.

of solution state of S, 510 nm. Thus it could beconcluded that the S molecules was successfully ad-sorbed onto the M/A hetero-LB film surface.

3.2. Surface analysis by AFM

To construct the molecular electronic device withfunctional protein films, the formation of aggregatedmolecular films of S and A have been considered as oneof the most important factors dominantly affecting thedevice performance. The film formation of each proteinis done by LB and self-assembly (SA) technique. SAtechnique using electrostatic force difference ofmolecules provided a high degree of density withoutloss of activity. The surface morphology of A homo-film and S/M/A hetero-film were shown in Fig. 5.

Fig. 5. AFM topology of film surface (a) cytochrome c ; (b) GFP/vio-logen /cytochrome c.

1000) pumped by a Q-switched Nd:YAG laser (ORC-1000), a pulse stretcher/compressor OPG-OPA system,and an optical detection system (FWHM 400 fs; wave-length 400 nm; repetition rate 20 Hz) was introduced toexcite the S molecules. With fast electronics using a V/Vamplifier of 300 MHz frequency (Model SR445, Stan-ford Research) and a storage oscilloscope of 500 psresolution (2Gs/s, model 54616B, Hewlett–Packard),the interlayer photocarrier movement was detectedform a 50� strip line geometry in order to acquiresignals with high time resolution.

3. Results and discussion

3.1. Characteristics of UV-�isible absorption andfluorescence spectrum

To verify the adsorption of S molecules onto theM/A hetero-LB film and to find the maximum emissionwavelength, the UV-visible absorption and fluorescenceemission spectrum of S/M/A hetero-film deposited ontothe quartz were investigated. As shown in Fig. 4, noabsorption peaks except the peaks of acceptor andmediator were observed. Peak at 280 nm and 410 nmwere the absorption peak of M and A, respectively.Absorption wavelength of S molecule was 480 nm butwas not shown in Fig. 4. Since the monolayer of Smolecule was formed onto the M/A hetero-film surface,the absorption peak was too weak to be detected.Therefore, the S film formation was verified with thefluorescence emission of S/M/A hetero- film. At 510nm, the peak related with the fluorescence emissionwavelength of S was observed in Fig. 4. This resultcorresponds with the fluorescence emission wavelength

J.-W. Choi et al. / Biosensors & Bioelectronics 16 (2001) 819–825 823

Fig. 6. (a) Photocurrent– time curves of the MIM device: a, 2 V; b, 0V. (b) I-V characteristics of the MIM device: (�) photocurrent; (�)darkcurrent.

When a forward bias was applied in accordance withthe energy level profile in the MIM device, the pho-tocurrent was generated. With repeated step illumina-tion, the reproducible photocurrent was generatedaccordingly. The results indicate that the photoswitch-ing function of the MIM device was achieved. In theproposed molecular device, the photoinduced unidirec-tional flow of electrons could be achieved due to theredox potential difference as well as electronic couplingbetween the functional molecules as shown in Fig. 1. Itwas also observed that the intensity of the photocurrentwas dependent on the external bias voltage. As theexternal bias voltage increased, higher photocurrentswere generated.

As shown in Fig. 6(b), the rectifying characteristicwas also observed from the measurement of current–voltage characteristics. When a 460 nm monochromaticlight was irradiated, the photocurrent was generatedwith the appropriate bias voltage. On the other hand,the current was less generated in the dark environment(without light irradiation) even though the bias voltagewas increased. In photo state, when a +LV(forward)bias was applied to the molecular photodiode, thephotocurrent was generated with 5mA. Otherwise a−LV(reverse) bias was applied to the molecular photo-diode, the photocurrent was 0 mA. This property wascalled rectification of photodiode. From these results, itcan be concluded that the photodiode characteristics ofthe proposed device was verified and the proposedmolecular array mimicking photosynthetic reaction cen-ter can be usefully applied as a model system for thedevelopment of the biomolecular photodiode.

3.4. Transient photocurrent measurement of de�ice

In order to investigate the charge transfer in themolecular device, the transient photocurrent profile wasanalyzed as shown in Fig. 7. By light illumination, theelectrons of S molecules were excited from their groundstate to excite state (S*). The photo-excited electronsreturned to their ground state and then green fluores-cent emitted at 510 nm. However the electron acceptorwas exposed to excited sensitizer, some of photoexcitedelectrons of S* were separated (S+/M−/A) and trans-ferred (S+/M/A−) to A molecule via their redox poten-tial difference. Thus the photoinduced one-way electrontransfer could be generated. By measuring the transientphotocurrent, the charge transfer rate of S molecule toA molecule could be calculated and the photoinducedone-way electron transfer could be verified.

Fig. 7 shows the transient photocurrent of S homo-junction, S/M heterojunction, and S/M/A heterojunc-tion. Although the initial rise of the transientphotocurrent has concerned with the charge separationrate, the charge separation rate cannot be calculateddue to the sub-nanosecond order of time constant. The

As shown in Fig. 5(a), the height of A film was about40 A� and the size of protein aggregation was about 0.3�m. As shown in Fig. 5(b), the spherical type of aggre-gations of S molecules was observed, which indicatedthat the S molecules were partially adsorbed onto theM film surface. It is intimated that the GFP moleculeswere aggregated on the viologen surface. In Fig. 5(b),the aggregation size of S molecules on M film surfacewas about 0.1–0.2 �m.

3.3. Photocurrent response of MIM de�ice

The photocurrent– time response of the proposeddevice with the irradiation of a 460 nm monochromiclight by a xenon lamp was shown in Fig. 6(a). Thephotocurrent of the device consisted of S/M/A hetero-film was generated by light excitation in Fig. 6(a).

J.-W. Choi et al. / Biosensors & Bioelectronics 16 (2001) 819–825824

charge transfer rate could be calculated with the decayprofile of transient photocurrent due to the nanosecondorder of time constant. The decay of transient pho-tocurrents of S homojunction, S/M heterojunctions,and S/M/A heterojunctions were fitted with single ex-ponential function, in which the exponent was thedecay time constant. The decay time constant of pho-tocurrent was about 4273 ns for S homo-film device,

1134 ns for S/M hetero-film device and 487 ns forS/M/A hetero-film device, respectively. From these timeconstants the charge transfer rate in the molecularhetero-film could be calculated since the inversion oftime constant was the charge transfer rate.

In Fig. 8, the faster electron transfer with S/M/Aheterojunction was observed compared with that inS/M heterojunction based on the charge separationrate. The photo-excited electrons of S* are transportedto A and M, but some of electrons in the A and M canreturn to S0, which is called the charge recombination.The charge recombination caused the increment of thedecay time. In the S/M/A structured heterojunction, theefficient charge transport from M to A reduced thecharge recombination from M to ground state S0, andcharge recombination from A to S0 could be reducedalso due to the longer distance between S and A by theexistence of the mediator molecules. Thus in S/M/A theunidirectional charge transfer was improved and chargetransfer rate became higher than S homojunction andS/M heterojunction. By addition of A molecules, thedirectional flow of photocurrent through their redoxpotential difference occurred efficiently.

4. Conclusions

The biomolecular device composed of functionalproteins based on the electron transport in the biologi-cal photosynthesis process was investigated. The MIMstructured device was fabricated with the hetero-typefilms consisting of GFP, viologen and cytochrome c,which were used as a sensitizer, an electron mediatorand an electron acceptor, respectively. Based on thetopologies by AFM measurement, the deposition ofcytochrome c LB film and the adsorption of GFP filmonto viologen/cytochrome c hetero-films were verified.The photodiode characteristic and the photoswitchingfunction of the proposed device were verified by cur-rent–voltage characteristic measurement and photocur-rent response with the repeated step illumination. Inorder to investigate the charge transfer in the moleculardevice, the transient photocurrent profile was measuredand analyzed. The directional flow of photocurrentthrough their redox potential difference occurred effi-ciently in the proposed molecular device. The diodecharacteristic of the proposed device was verified andthus the proposed molecular device composed ofbiomolecules could be applied as biomolecularphotodiode.

Acknowledgements

This work was supported by grants from KoreaScience and Engineering Foundation (KOSEF: 98-

Fig. 7. Transient photocurrent profile of the MIM device: (a) Shomojunction; (b) S/M heterojunction; (c) S/M/A heterojunction.

J.-W. Choi et al. / Biosensors & Bioelectronics 16 (2001) 819–825 825

Fig. 8. Comparison of the decay component of transient photocurrent of the MIM device: a, S homojunction; b, S/M heterojunction; c, S/M/Aheterojunction.

0502-08-01-3) and supported by the national programfor Super Intelligence Chip of the Ministry of Com-merce, Industry and Energy as one of the new technol-ogy of the next generation.

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