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Copyright © 2017 American Scientific PublishersAll rights reservedPrinted in the United States of America

ArticleJournal of

Nanoscience and NanotechnologyVol. 17, 5189–5192, 2017

www.aspbs.com/jnn

Enzyme Logic Gate with Summation FunctionBased on Putidaredoxin Reductase/Cytochrome c

Enzyme Reaction

Si-Youl Yoo1, Junhong Min2, Tomoaki Haga3, Hidehiko Hirakawa3,Teruyuki Nagamune3, and Jeong-WooChoi1�4�∗

1Department of Chemical and Biomolecular Engineering, Sogang University, 35 Baekbeom-ro, Mapo-gu, Seoul 121-742, Republic of Korea2School of Integrative Engineering, Chung-Ang University, Heukseok-dong, Dongjak-gu, Seoul 156-756, Republic of Korea3Department of Chemistry and Biotechnology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku Tokyo 113-8656, Japan

4Department of Biomedical Engineering, Sogang University, 35 Baekbeom-ro, Mapo-gu, Seoul 121-742, Republic of Korea

Various kinds of biologic gates have been developed to realize biocomputing system with enzymereactions. However, logic gate with summation function based on putidaredoxin reductase andcytochrome c has not been reported yet. Here, we developed biologic gate with summation function.To fabricate the biologic gate system, putidaredoxin reductase was immobilized on two gold elec-trodes for input system and cytochrome c was immobilized on one gold electrode for reading outputsignal. Then polydimethylsiloxane based microfluidic chip was fabricated on the gold electrodes andeach electrode was connected with microfluidic channels. To operate the biologic gates, two ANDgates with putidaredoxin reductase and one OR gate with cytochrome c was checked by confirmingenzyme reactions firstly. And then, we checked summation function with fabricated two AND gatesand one OR gate. The benzoquinone injected to each putidaredoxin reductase was acted as inputand it was defined to ‘0’, ‘1’ and ‘2’ by different concentrations. Two input signals were flowed tocytochrome c and summated signal was checked by cyclic voltammetry. The summated output sig-nals were defined from ‘0’ to ‘4’ with various thresholds of current intensity. Proposed enzyme logicgate with summation function can be applied to bioprocessing and biocomputing system.

Keywords: Bioelectronic Device, Enzyme Logic Gate, Putidaredoxin Reductase, Cytochrome c.

1. INTRODUCTIONLogic gates were widely researched in molecular elec-tronics field with electron transfer and redox property ofsingle molecule and Boolean logic system was usuallyrealized. Among the various logic gates, biologic gatesusing protein, DNA, and cell were researched with bio-logical specific functions like enzyme reactions, moleculerecognition, cell metabolism. For example, Wang et al.proposed AND and OR Boolean logic using enzyme inputsand pH for output.1 Zhang et al. developed DNA elec-tronic logic gates and realized AND, NAND, and NORBoolean logic based on metal-ion-dependent induction ofoligonucleotide structural motifs.2 Moon et al. suggestedAND logic gate in single cell with genetic programs.3

∗Author to whom correspondence should be addressed.

Also, biologic gate have various applications.4�5 Stracket al. developed biomolecular keypad lock security systemwith enzyme-based logic gates.6 Tam et al. proposed bio-fuel cell with enzyme logic network.7 However, biologicgate with summation function based on putidaredoxinreductase and cytochrome c enzyme reaction was notreported yet.In this study, enzyme reaction based biologic gate sys-

tem with summation function composed of putidaredoxinreductase and cytochrome c was developed for the firsttime (Fig. 1). To fabricate the biologic gate system, poly-dimethylsiloxane (PDMS) based microfluidic chip wasfabricated with Au patterned chip. To operate the biologicgate system, first, each enzyme reaction was confirmed.And, we confirmed summation function with fabricatedtwo AND gates and one OR gate.

J. Nanosci. Nanotechnol. 2017, Vol. 17, No. 8 1533-4880/2017/17/5189/004 doi:10.1166/jnn.2017.14170 5189

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Enzyme Logic Gate with Summation Function Based on Putidaredoxin Reductase/Cytochrome c Enzyme Reaction Yoo et al.

Figure 1. (a) Schematic diagram of enzyme logic gate with putidare-doxin reductase (Pdr) and cytochrome c (Cyt c). (b) Expected results ofsummation function.

2. EXPERIMENTAL DETAILS2.1. MaterialsCytochrome c, 2-mercaptoacetic acid (2-MAA), nicoti-namide adenine dinucleotide (NADH) and benzoquinonewere purchased from Sigma-Aldrich (Sigma ChemicalCompany, St. Louis, USA). The patterned Au electrodeswere purchased from National Nanofab Center (Korea) forcyclic voltammetry. And Au (43 nm)/Cr (2 nm)/SiO2/Siwafers were purchased from Inostek Inc. (Korea) forscanning tunneling microscopy. A sylgard 184 siliconelastomer kit (Dow Corning, USA) containing a poly-dimethylsiloxane (PDMS) base and a curing agent wasused for device fabrication.

2.2. Fabrication of Biologic Gate DeviceThe Au patterned electrode was produced with film maskand microfluidic device was produced by using PDMS.The microfluidic channel was made with two PDMS lay-ers of valve layer and fluidic layer. After device fabri-cation, 2-MAA was self-assembled on Au electrode forimmobilization. Thiol group (–SH) of 2-MAA connectedto Au substrate and carboxyl group (–COOH) connectedamine group of protein electrostatically. Finally, putidare-doxin reductase and cytorhcome c were immobilized ontwo electrodes and one electrode, respectively.

2.3. Morphology Analysis by AFMThe surface morphology of enzyme immobilized on Auelectrode was checked by atomic force microscopy (AFM)with a Nanoscope (R) III instrument (Digital Instru-ments CA, USA). And the AFM silicon probes were pur-chased from Veeco Instruments (USA). AFM can show

the morphology in nanometer scale. The AFM imageswere obtained by varying the frequency of its cantilever intapping mode. A cantilever (257–319 kHz resonance fre-quency) fabricated using phosphorus (n) doped silicon wasutilized. The tip velocity was 2.0 �m s−1 and the scan sizewas 500 nm×500 nm. The AFM images were used for theevidence of immobilization of enzyme on Au electrodes.

2.4. Confirming Electrochemical Property andBiologic Gate System by CV

The electrochemical property of enzyme was checked bycyclic voltammetry (CV).8–10 The CV experiment wasdone by using an electrochemical analyzer (CH Instru-ments Inc., TX, USA) with general purpose electro-chemical analysis software. Three-electrode system wasused and 4-(2-hydroxyethyl)-1-pierazineethanesulfonicacid (HEPES) buffer of pH 7.0 was used as theelectrolyte.11�12 The enzyme immobilized Au electrode,patterned Au electrode and Ag/AgCl in saturated KCl(Bas, USA) were used as working, reference and counterelectrodes, respectively. The scan range of voltage was400 mV to −300 mV and scan rate was 50 mV/s.

3. RESULTS AND DISCUSSION3.1. Confirmation of Device FabricationThe biologic device was fabricated with microfluidic chan-nel and it was shown in Figure 2(a).The surface morphology of fabricated enzyme logic gate

was checked by AFM images. In Figure 2(b), compar-ing bare gold surface and enzyme immobilized surface,the size of clusters are changed. Bare gold was 50 nmto 100 nm, but in case of putidaredoxin reductase andcytochrome c immobilized surface, the lumps of enzymeare shown. And it indicates well immobilization of puti-daredoxin reductase and cytochrome c on Au surface bychemical linker 2-MAA.

3.2. Confirmation of Biologic Gate SystemThe electrochemical biologic gate system was confirmedwith CV technique. First, we confirmed enzyme reactionas input NADH, benzoquinone, and NADH with benzo-quinone, respectively. As shown in Figure 3, putidaredoxinreductase reacted when both inputs were injected and peakcurrent was decreased because benzoquinone was reducedto semiquinone. And, when only NADH or benzoquinonewas injected, the peak current was not decreased evidently.From the result, putidaredoxin reductase electrode wasacted was AND gate.Next, enzyme chain reaction of putidaredoxin reduc-

tase and cytochrome c was confirmed with CV tech-nique and the results are shown in Figure 4. First, inputmaterial was injected to putidaredoxin reductase immo-bilized electrode and reacted during 20 min. Then, thesolution was transferred to cytochrome c immobilizedelectrode by microfluidic channel and reacted during20 min. The change of peak current of cytochrome c

5190 J. Nanosci. Nanotechnol. 17, 5189–5192, 2017

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Yoo et al. Enzyme Logic Gate with Summation Function Based on Putidaredoxin Reductase/Cytochrome c Enzyme Reaction

Figure 2. (a) Fabrication of biologic gate with microfluidic channel. (b) Surface morphology analysis by AFM (scale bar: 100 nm).

Figure 3. Electrochemically confirmed enzyme reaction of putidare-doxin reductase by measuring CV of putidaredoxin reductase electrode.(a) Change of CV graph when before input (black line), input NADH andbenzoquinone (red line), after 20 min from input (blue line). (b) Com-paring peak current change during reaction (20 min) when input NADHonly, benzoquinone only, NADH and benzoquinone together.

Figure 4. Enzyme chain reaction of putidaredoxin reductase andcytochrome c by measuring CV of cytochrome c electrode. (a) Changeof CV graph when not transferred to cytochrome c (black line), trans-ferred to cytochrome c (red line), after 20 min from transferred (blueline). (b) Comparing peak current change when input NADH only, ben-zoquinone only, NADH and benzoquinone together.

J. Nanosci. Nanotechnol. 17, 5189–5192, 2017 5191

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Enzyme Logic Gate with Summation Function Based on Putidaredoxin Reductase/Cytochrome c Enzyme Reaction Yoo et al.

Figure 5. Confirmation of summation function. Numbers of right sidefrom ‘0’ to ‘4’ are determined thresholds.

immobilized electrode was compared as input combi-nation. When NADH and benzoquinone were injectedtogether, semiquinone was oxidized to benzoquinone againwith enzyme reaction of cytochrome c and peak currentwas increased. From Figures 3 and 4, benzoquinone wasreduced when reacted with putidaredoxin reductase and re-oxidized when reacted with cytochrome c as expected. Theelectrode of cytochrome c was acted as OR gate becausethe reaction was occurred when we input A, B, A and B.

3.3. Processing the Summation FunctionWe designed biologic device with summation function byenzyme chain reactions. We defined the benzoquinone con-centrations from ‘0’ to ‘2’ as inputs and cytochrome cpeak currents from ‘0’ to ‘4’ as outputs. So, the devicecould summate two input values from ‘0’ to ‘2’ and theresults were from ‘0’ to ‘4’ as shown in Figure 5. Finallywe confirmed biologic device which has summationfunction.

4. CONCLUSIONIn this study, biologic gate device was fabricated withmicrofluidic technique. Putidaredoxin reductase acted as

AND gate because it reacted only when two inputs wereinjected together. Differently, cytochrome c acted as ORgate because it could react with only one input channel.And we also proposed biologic gate system with summa-tion function. Proposed biologic gate system with sum-mation function can be applied to realize biocalculator,bioprocessor and biocomputer.

Acknowledgment: This research was supported by theLeading Foreign Research Institute Recruitment Pro-gram, through the National Research Foundation of Korea(NRF), funded by the Ministry of Science, ICT and FuturePlanning (MSIP) (2013K1A4A3055268) and Basic Sci-ence Research Program through the National ResearchFoundation of Korea (NRF) funded by the Ministry ofEducation (2016R1A6A1A03012845). This joint researchproject was also supported by JSPS and NRF under theJapan—Korea Basic Scientific Cooperation Program.

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Received: 3 June 2016. Accepted: 30 August 2016.

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