molecular pattern formation using chemically modified cytochrome c

Colloids and Surfaces B: Biointerfaces 23 (2002) 295–303

Molecular pattern formation using chemically modifiedcytochrome c

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

a Department of Chemical Engineering, Sogang Uni�ersity, C.P.O. Box 1142, Seoul 100-611, South Koreab Department of Biomolecular Engineering, Tokyo Institute of Technology, Nagatsuta, Yokohama, Japan

Received 25 August 2000; accepted 23 January 2001

Abstract

Molecular pattern formation using chemically modified cytochrome c and green fluorescent protein (GFP) waspresented for the application as a bioelectronic device. A protein conjugate was synthesized by the formation ofdisulfide bridges. In order to make molecular assembly onto the gold-coated substrate, cytochrome c was cross-linkedwith N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). After the modification of cytochrome c, it was sponta-neously deposited, so that it could be adsorbed onto the gold-coated substrate by self-assembly (SA) technique. Usingthe cellulose membrane, cytochrome c molecules were deposited onto the gold-coated substrate with the spatialresolution of ca. 0.2 �m. In order to verify the modified cytochrome c, UV absorption spectrum was measured. GFPwas adsorbed onto the cytochrome c monolayer by electrostatic force. Fluorescence emission spectrum wasinvestigated to verify the existence of the GFP molecule onto the cytochrome c monolayer. To verify the adsorptionof cytochrome c molecules onto the gold-coated substrate and GFP molecules onto the cytochrome c monolayer, theatomic force microscopy and lateral force microscopy investigations were performed. Molecular pattern formation ofcytochrome c and GFP molecules were successfully performed by chemical means and electrostatic force. © 2002Elsevier Science B.V. All rights reserved.

Keywords: Cytochrome c ; Green fluorescent protein; N-succinimidyl-3-(2-pyridyldithio)propionate; Dithiothreitol

www.elsevier.com/locate/colsurfb

1. Introduction

Various artificial molecular devices have beenfabricated by mimicking the electron transportfunction of biological photosynthesis [1–4]. Isoda

et al. investigated the optical and electrical char-acteristics of the molecular photodiode consistingof flavin–porphyrin hetero-LB films [5]. Fujihiraet al. also reported the electrochemical photodi-ode using LB films of three functional moleculesor an aligned triad on the electrode, whichworked in an electrolyte solution [6–8]. We havebeen investigating the molecular photodiode con-sisting of hetero-organic LB film of four func-tional organic molecules, ferrocene, flavin,

* Corresponding author. Tel.: +82-2-705-8480; fax: +82-2-711-0430.

E-mail address: [email protected] (J.-W. Choi).

0927-7765/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.

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J.-W. Choi et al. / Colloids and Surfaces B: Biointerfaces 23 (2002) 295–303296

viologen and TCNQ used as an electron donor,sensitizer, relay and acceptor units, respectively[9–11].

Though molecular pattern formation or fabri-cation is an important factor for molecular elec-tronic device composed of biological molecules, ithas been rarely investigated. Piner et al. developedthe dip-pen nanolithography using atomic forcemicroscopy (AFM) tip for molecular patterning[12]. Also, Sarikaya et al. reported the proteinpatterns for an electronic device using geneticallyengineered bacterial protein that could bind totiny semiconductor and metal particles [13]. How-ever, molecular pattern formation with cy-tochrome c and GFP using cellulose membranehas not been investigated yet.

Horse heart cytochrome c is a small hemeprotein with 104 amino acid residues and is animportant electron transfer protein in the respira-tory chain [14]. The key feature of cytochrome c,capability of electron transfer, is driven from theredox state change and conformational change ofheme groups covalently bound via two thioetherlinkages formed by two cysteine side chains andtwo axial ligands, histidine and methionine. Sincecytochrome c is a constituent acting as an electrontransfer component in the bacterial photosyn-thetic reaction center, cytochrome c could be usedas an electron acceptor in the development of amolecular electronic device [15].

The green fluorescent protein (GFP) from thejellyfish Aequorea �ictoria has been widely used asa fluorescent marker in the determination of geneexpression and protein localization [16]. Thepurified GFP, a protein of 238 amino acids, ab-sorbs blue light and emits green light (peak emis-sion at 510 nm). This fluorescence is very stable,and virtually no photobleaching is observed [17].Since GFP shows very high quantum yield, ca.80%, it is a very reasonable approach to use GFPas an electron sensitizer in the development of themolecular electronic device by mimicking the bio-logical photosynthesis mechanism.

In this study, a functional group on the cy-tochrome c surface was modified with N-succin-imidyl-3-(2-pyridyldithio)propionate (SPDP).SPDP, which contains N-hydroxysuccinimide andpyridyl disulfide residues, is the most commonly

used cross-linking reagent and frequently appliedfor the synthesis of protein–protein conjugates[18].

To prove the existence of a modified cy-tochrome c, UV absorption spectrum was mea-sured. Using AFM and lateral force microscopy(LFM), surface analysis was carried out to verifythe deposition of cytochrome c onto the gold-coated substrate. Also, GFP molecules were ad-sorbed on the cytochrome c monolayer byelectrostatic force. To investigate the GFP ad-sorption onto the cytochrome c monolayer,fluorescence emission spectrum was measured andanalyzed. The morphology of the GFP monolayeradsorbed onto the cytochrome c monolayer wasalso observed by AFM.

2. Materials and methods

2.1. Materials

Cytochrome c (extracted from horse heart, typeVI) and GFP (recombinant red shifted GFP,rEGFP) were purchased from Sigma ChemicalCo. (St. Louis, USA) and CLONTECH Co. (PaloAlto, CA, USA), respectively. SPDP and dithio-threitol (DTT), also purchased from Sigma Chem-ical Co., were used as a cross-linking and acleavage reagent, respectively. The gold-coatedsubstrate was made by thermal evaporation on acleaned glass substrate.

2.2. Modification of functional group oncytochrome c surface

For modification of the functional group on thecytochrome c surface, cytochrome c was dissolvedinto a phosphate buffer solution (pH 8, 10 ml).SPDP (3.2 mM, MW=312.4) and DTT (25 mM)were mixed with deionized distilled water (DDW;�18 M�, 10 ml). The cytochrome c solution andSPDP solution were mixed for 30 min at roomtemperature. When the SPDP with disulfidebonds is exposed to the outer surface of cy-tochrome c, the succinimide group of SPDP isremoved, and the hydrogen ion of NH2 on thecytochrome c surface can also be released. Thus,

J.-W. Choi et al. / Colloids and Surfaces B: Biointerfaces 23 (2002) 295–303 297

the NH group on the cytochrome c surface iscross-linked with the sulfur of 2-pyridyldithio-propionamide, and then cytochrome c and SPDPwere cross-linked by a disulfide bond. Fig. 1(a)shows the reaction scheme of cytochrome c andSPDP. Excess reagent and N-hydroxysuccinimidewere removed by gel filtration on a columnpacked with SephaloseCL-6B gel. The cross-linked cytochrome c with SPDP was eluted withphosphate buffer solution (pH 8).

2.3. Clea�age of disulfide bond by DTT

For cleavage of the disulfide bond in 2-pyridyldithio-propionamide conjugated with cy-tochrome c, DTT was used. DTT is also a famousreagent for cleavage of the disulfide bond. Afterthe disulfide bond was cleaved, the functionalgroup on cytochrome c was transformed to thiol

group and then cytochrome c can be adsorbedonto the gold-coated substrate selectively. Fig.1(b) shows the reaction scheme for cleavage of thedisulfide bond by DTT.

2.4. Pattern formation of cytochrome c and GFPonto the gold-coated substrate

The thiol group on the cytochrome c is ad-sorbed onto the gold-coated substrate by chemicalmeans. For molecular pattern formation of thecytochrome c molecule onto the gold-coated sub-strate, a cellulose membrane was used as a patternmask. Since a cellulous membrane has a lot ofpores, of size ca. 0.2 �m, the cytochrome cmolecule can be adsorbed onto the gold-coatedsubstrate by chemical means through the mem-brane and deposited with a special resolution ofca. 0.2 �m. The gold-coated substrate coveredwith a membrane was soaked into the modifiedcytochrome c solution for 24 h at roomtemperature.

The modified cytochrome c-adsorbed gold sub-strate was put into the GFP solution. The GFPmolecules were adsorbed onto the cytochrome cmonolayer by electrostatic attractive force. Fig. 2shows the scheme for the pattern formation of themodified cytochrome c molecule onto the gold-coated substrate (a) and GFP onto the modifiedcytochrome c monolayer (b).

3. Results and discussion

3.1. In�estigation of modified cytochrome c byUV–�isible spectroscopy

To verify the modified cytochrome c, it wascompared with the UV absorption spectrum ofcytochrome c, SPDP, and modified cytochrome cfilm deposited onto the quartz. As shown in Fig.3, the maximum absorbance peak of SPDP was280 nm (a) and that of cytochrome c was 410 nm(b). Fig. 4 shows the comparison of absorbanceintensity between SPDP (a), cytochrome c (c), andthe cytochrome c conjugated with 2-pyridyldithio-propionamide (b), according to the elution by gel

Fig. 1. Total scheme for the modification of the functionalgroup on the cytochrome c surface: (a) the reaction scheme ofsynthesis of cytochrome c and SPDP; and (b) the reactionscheme for cleavage of the disulfide bond by DTT.


Fig. 2. Scheme for the molecular pattern formation of cytochrome c and GFP: (a) modified cytochrome c was deposited onto thegold-coated substrate by self-assembly technique; and (b) GFP molecule was adsorbed onto the cytochrome c monolayer by theelectrostatic force.

filtration. The maximum absorbance peak of cy-tochrome c conjugated with 2-pyridyldithio-propi-onamide was 340 nm. Also, Fig. 4 shows that theabsorbance intensity of cytochrome c and SPDPis higher than the cytochrome c conjugated with2-pyridyldithio-propionamide, but the absorbanceintensity of the cytochrome c conjugated with2-pyridyldithio-propionamide is higher than cy-tochrome c and SPDP as time goes on. Therefore,it is considered that cytochrome c was conjugatedwith SPDP successfully.

3.2. Characteristics of fluorescence of GFPadsorbed onto cytochrome c

To verify the maximum emission wavelength ofGFP onto the cytochrome c monolayer, fluores-cence emission spectrum was investigated. Ab-sorbance of the GFP molecule is 480 nm, but theabsorption wavelength of GFP is not shown. Be-cause the GFP was formed onto the cytochromec monolayer, the absorption peak is too weakto detect. Therefore, the adsorption of GFP


molecules onto the cytochrome c monolayer wasverified with the fluorescence emission. As shownin Fig. 5, the emission peak of GFP is at 510 nm.This indicates that the GFP molecules were suc-cessfully adsorbed onto the cytochrome cmonolayer.

3.3. Analysis of morphology using AFM andLFM

To construct the molecular electronic deviceswith functional protein, the molecular pattern for-

mation has been considered as one of the mostimportant factor dominantly affecting thedevice performance. Self-assembly (SA) techniqueusing electrostatic attractive force or chemicalbond between the molecule and the substrateprovides a high degree of density without loss ofactivity.

Since the modified cytochrome c has a thiolgroup, cytochrome c molecules can be easily ad-sorbed onto the gold-coated substrate by SA tech-nique. The morphology of cytochrome c adsorbedonto the gold-coated substrate was observed usingAFM. Fig. 6 shows the surface morphology ofmodified cytochrome c molecules deposited ontothe gold-coated substrate. As shown in Fig. 6, theheight of the modified cytochrome c aggregates isabout 170 A� and the size of the modified cy-tochrome c aggregates is about 0.3 �m. For thepattern formation of the modified cytochrome c,porous cellulose membrane was used as a mask.Because the pore size of the membrane is about0.2 �m in size, the modified cytochrome cmolecule adsorbed onto the substrate is 0.2 �m orbelow. Fig. 7 shows the pattern formation of themodified cytochrome c molecules onto the gold-coated substrate using cellulose membrane as apattern mask. As shown in Fig. 7, spherical typeof aggregates of the modified cytochrome c wasobserved. This result indicates that the modifiedcytochrome c molecules are partially depositedonto the gold-coated surface.

Fig. 8 shows the AFM image (a) and LFMimage (b) of the pattern formation of the cy-tochrome c adsorbed onto the gold-coated sub-strate. The bright regions in the LFM imageindicate the modified cytochrome c depositedonto the gold substrate. Difference in brightnessarose from the differences in interaction in eachregion. Changes in friction between the tip andthe surface result in changes in the torsion of thecantilever in the LFM image. Thus, the tip experi-enced higher friction in the regions terminated bycytochrome c particles than in the regions of baregold-coated substrate.

As shown in Fig. 9, aggregates of GFPmolecules were observed. This result indicates

Fig. 3. UV–visible spectra: (a) UV absorbance of SPDP at 280nm; and (b) UV absorbance of SPDP at 410 nm.

Fig. 4. Variation of UV absorbance vs. elution time: (a) SPDP;(b) cytochrome c conjugated with a 2-pyridyldithio-propi-onamide; and (c) cytochrome c.


Fig. 5. Fluorescence emission spectrum of GFP adsorbed onto the cytochrome c monolayer by the electrostatic force at 510 nm.

Fig. 6. Morphology of cytochrome c molecules deposited onto the gold-coated substrate by AFM: (a) 3×3 �m; and (b) 1×1 �m.

that the GFP molecules are partially adsorbedonto the cytochrome c monolayer. This resultmight be due to the fact that the small aggregates

of GFP molecules were constructed by molecu-lar–molecular interaction in solution by electro-static force.


Fig. 7. Morphology of the patterned cytochrome c molecule adsorbed onto the gold-coated substrate using cellulose membrane asa pattern mask by AFM: (a) 5×5 �m; and (b) 1×1 �m.

Fig. 8. Morphology of cytochrome c molecules deposited onto the gold-coated substrate by AFM and LFM: (a) AFM image with5 �m; and (b) LFM image with 5 �m.


Fig. 9. Morphology of GFP particles adsorbed onto the cytochrome c monolayer by AFM: (a) 5×5 �m; and (b) 1×1 �m.

4. Conclusions

In this study, the functional groups of cy-tochrome c were transformed to a thiol groupusing the SPDP. The morphology of modifiedcytochrome c monolayer deposited onto the gold-coated substrate was investigated using AFM andLFM. Also, GFP molecules were adsorbed ontothe cytochrome c monolayer by the electrostaticforce difference and investigated. The surfaceanalysis of the GFP monolayer was also per-formed using AFM. It is suggested that the pat-tern formation or fabrication of the functionalproteins is an important factor in the fabricationof a molecular electronic device. From this view-point, it is one of the possible candidates to forma molecular pattern of the protein using SDPD asa mediator, for the fabrication of the molecularelectronic device.

Acknowledgements

This work was supported by grants from theKorea Science and Engineering Foundation(KOSEF: 98-0502-08-01-3).

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molecular pattern formation using chemically modified cytochrome c

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