A Biomemory Device Based on Electrically Controlled Hemin ...nbel.sogang.ac.kr/nbel/file/363.pdf · A Biomemory Device Based on Electrically Controlled Hemin/G-Quadruplex Complex

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Copyright American Scientific Publishers

ARTICLE

Copyright copy 2016 by American Scientific Publishers

All rights reserved

Printed in the United States of AmericaScience of

Advanced MaterialsVol 8 pp 767ndash774 2016

wwwaspbscomsam

A Biomemory Device Based on ElectricallyControlled HeminG-Quadruplex ComplexQi Chen1 Yong-Ho Chung3 Si-Youl Yoo1 Sang-Uk Kim2 Junhong Min4lowast and Jeong-Woo Choi1 2lowast

1Department of Chemical and Biomolecular Engineering Sogang University Mapo-guSeoul 121-742 Republic of Korea2Interdisciplinary Program of Integrated Biotechnology Sogang University Mapo-guSeoul 121-742 Republic of Korea3Department of Chemical Engineering Hoseo University Hoseoro79bungil20 Baebang AsanChungnam 336-795 South Korea4School of Integrative Engineering Chung-Ang University Heukseok-Dong Dongjak-guSeoul 156-756 Republic of Korea

ABSTRACT

Various DNA-based circuits that use material inputs and outputs do not properly connect nor co-operate withother electrically controlled systems on a chip Thus the development of a closed system operated by electricalsignals for DNA-based biodevices is needed Here a novel label-free biomemory device was proposed toimplement memory functions for ldquowriterdquo ldquoeraserdquo and ldquoreadrdquo The device was based on structural transformationof the heminG-quadruplex complex via electrical control without using materials Two electrochemical systemsin a single chamber termed ldquocontrollerrdquo and ldquooperatorrdquo were constructed to achieve memory functions Appliedpotentials of minus0054 and minus0339 V in the controller were used to operate the operator with ldquowriterdquo and ldquoeraserdquofunctions respectively These potentials led to H+ and OHminus generation in the controller which resulted inthe formation and deformation of the heminG-quadruplex complex in the operator In a cyclic voltammogramof the operator two different current levels of reduction peaks for the ldquoreadrdquo function were read as ldquo1rdquo andldquo0rdquo with respect to the structural formation and deformation The constructed device was a stable durableand reliable write-once-read-many-times (WORM) memory device These features of the proposed biomemorydevice provide a feasible and promising method for applications in DNA-based biocomputing devices

KEYWORDS Biomemory Device Structural Transformation Electrical Control HeminG-Quadruplex ComplexElectrochemical Systems Write-Once-Read-Many-Times

1 INTRODUCTIONDNA has been employed as a highly versatile andcontrollable material in various molecular computingfields such as logic gates logic circuits calculators andautomatons1ndash3 Considerable efforts have been dedicatedto developing DNA-based computing techniques to fulfillmany types of Boolean logic functions and to constructchemical reaction networks4ndash6 These types of biocomput-ing are governed by various unique properties of DNAsuch as specific recognition toehold-exchange hybridiza-tion enzymatic reaction and structural transformation7ndash10

The G-quadruplex one of the most well-known DNAstructures is highly cation-dependent and can exhibithorseradish peroxidase (HRP)-mimicking DNAzyme

lowastAuthors to whom correspondence should be addressedEmails junmincauackr jwchoisogangackrReceived 27 March 2015Accepted 24 June 2015

activity when complexed with hemin This cation-drivenallosteric regulation of DNA nanomaterials has also beenapplied to the development of various biosensors nano-structures and logic gates In the logic gates that havebeen reported the G-quadruplex structure of G-rich DNAsequences is transformed by the input of additional sub-stances such as oligonucleotides metal ions and hydrogenions11ndash13

Input of materials such as oligonucleotides or chemi-cals into dynamic DNA devices is laborious and requireskinetic control14 Nevertheless control of hydrogen ionconcentration that is pH regulation has been reported asa representative method for switching the G-quadruplexand I-motif structures1516 Moreover an electrical sys-tem was reported to be a rational design that is capa-ble of switching pH accurately and reversibly by usinga feedback circuit this electrical pH-modulating systemwas termed the micro-pH-stat system17 This system wasfurther used in a DNA-based switchable device based on

Sci Adv Mater 2016 Vol 8 No 4 1947-293520168767008 doi101166sam20162674 767



A Biomemory Device Based on Electrically Controlled HeminG-Quadruplex Complex Chen et alARTICLE

the pH-sensitive I motif which requires fluorescent tagsfor detection18 Detection using optical measurements inDNA-based biocomputing studies is unsuitable for connec-tion and co-operation with other electric devices becauseof its requirement of a complex setup Electrochemi-cal methods have incorporated DNA biosensors in lab-on-a-chip devices19 However an electrically controlledDNA-based computing system that properly connects andco-operates with other electrically controlled systems ona chip has yet to be reported Hence in this report themicro-pH-stat system was integrated with an electrochem-ical detecting method to utilize a biomemory device thatuses only electrical signalsWe have developed various electronic biomemory

devices and bioprocessing devices using metallopro-teins such as recombinant azurin and cytochrome c20ndash22

We have also used ssDNA-Cu heterolayers to develop aDNA-based biomemory based on electrostatic adsorptionwithout an electrical control system23 G-rich DNA wasused as a platform to bind iron-containing porphyrin heminby covalent bonds to develop a DNA-based biomemorywith an electrical control systemIn this study a biomemory device was developed based

on the electrically controlled formation and deformation ofthe heminG-quadruplex complex for the first time Mul-tifunction information processing (write-read-erase) wasproposed and a write-once-read-many-times (WORM)biomemory device was constructed The WORM biomem-ory device is a closed system with high stability dura-bility and reliability and uses only electrical signals Thechip platform of the constructed biomemory device canconnect and co-operate with other electrically controlledsystems on a chip A four-bit biomemory system waseasily constructed by cascading the constructed biomem-ory devices Hence our proposed concept provides anovel method for developing DNA-based biocomputingdevices

2 MATERIALS AND METHODS21 MaterialsG-rich DNA with the sequence 5prime-HS-(CH26-TTT TTTTTT TGG GTT AGG GTT AGG GTT AGG G-3prime wassynthesized by Bioneer Co (Korea) Phosphate-bufferedsaline (PBS) solutions at pH 50 and 90 were pre-pared 22-azino-bis(3-ethylbenzthiazoline-6-sulfonic acidABTStrade chromophore) were purchased from EMD Mil-lipore (USA) and used directly Silicon wafers withelectrode deposits were designed and purchased fromLG Siltron Inc (Korea) A Sylgard 184 elastomer kit(Dow Corning USA) composed of polydimethylsiloxane(PDMS) base and curing agent was used Piranha solu-tion composed of 30 vol H2O2 (Duksan Pure Chem-ical Co Ltd Korea) and 70 vol H2SO4 (DaejungChemical Co Ltd Korea) was freshly prepared andused with caution Hemin dithiothreitol (DTT) and other

reagents were purchased (Sigma-Aldrich USA) and useddirectly A working solution of hemin (5 mM) in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)solution (25 mM HEPES pH 74 20 mM KCl 200 mMNaCl 005 (wv) Triton X-100 1 (vv) DMSO) wasprepared

22 Installation of the Biomemory PlatformThe silicon wafer deposited with six patterned elec-trodes was cleaned with piranha solution and then alignedwith the predesigned PDMS compartment as shown inFigure 1(a) Two chambers with AgAgCl electrodes wereseparated from a reaction chamber by using salt bridgescomposed of 1 agarose gel The reaction chamber wascomposed of four electrodes namely iridium (Ir) iridiumoxide (IrOx) Au surface and Pt The deposited AgAgClelectrodes were immersed in 80 mL NaCl (1 M) solutionswhile 1 mL Na2SO4 (10 mM hemin 25 mM) was usedas the electrolyte in the reaction chamber

23 Immobilization of Memory-Active Cores(HeminG-Quadruplex Complex) on Au

The properties of memory-active cores on Au surfaceswere characterized by using bare Au electrodes with anactive area of 025 cm2 The Au surface in the installedbiomemory platform was subsequently modified withmemory-active cores for biomemory functionalizationBare gold electrodes were cleaned with piranha solu-

tion and then incubated with DTT-reduced G-rich DNAsequence (1 M) in PBS (pH 50) at RT for 4 hrssubsequently washed with DI water and dried withN2 gas The G-rich DNA modified Au substrates werethen immersed in the working solution of hemin at RTfor 4 hrs to capture hemin Self-assembled monolayers(SAMs) of the memory-active cores on Au substrates wereprepared

24 Confirmation of Memory-Active Cores onAu by Raman Spectroscopy

Immobilization of the memory-active cores on the Au sur-faces was determined by Raman spectroscopy The instru-ment used for Raman spectroscopy was an NTEGRASpectra (NT-MDT Russia) equipped with a CCD detec-tor cooled with liquid nitrogen and an inverted opticalmicroscope (Olympus IX71) The XYZ three-dimensionalscanning range was 100 mtimes 100 mtimes 6 m The res-olution of the spectrometer was 200 nm in the xy planeand 500 nm along the z axis The incident laser used forobtaining Raman spectra was an infrared laser that emitslight at a wavelength of 785 nm Five 30 sec scans from1000ndash2000 cmminus1 were performed and the mean intensitywas taken as the Raman signal The background was gen-erated subtracted from the signal by using OriginPro 80program

768 Sci Adv Mater 8 767ndash774 2016



Chen et al A Biomemory Device Based on Electrically Controlled HeminG-Quadruplex Complex

ARTICLE

Fig 1 Schematic description of an electrically controlled DNA-based biomemory device (a) The depicted biomemory platform consists of patternedelectrodes and a PDMS compartment (b) Schematic illustration of the electrochemical interactions between two three-electrode electrochemical systems(ldquocontrollerrdquo and ldquooperatorrdquo) in a single chamber (c) Information processing of ldquowriterdquo ldquoeraserdquo and ldquoreadrdquo functions of the constructed biomemorydevice

25 Electrochemical Analysis ofMemory-Active Cores

An electrochemical analyzer (CHI 660 USA) whichwas operated by general-purpose electrochemical analysissoftware was used for the analysis of electrochemicalproperties The SAMs of the memory-active cores onbare Au electrodes were analyzed in an electrochemi-cal cell composed of a 5 mL quartz cuvette The three-electrode system consisted of a platinum counter electrodea AgAgCl double junction reference electrode and amemory-active core modified gold substrate with an areaof 025 cm2 as the working electrode The electrolyte wasPBS (pH 50 or pH 90) The electrochemical characteriza-tion of memory-active cores on Au were carried out at RTat a scan rate of 50 mV secminus1 For the constructed biomem-ory platform in the operator the scan rate was 30 mV secminus1

in coincidence with the decrease volume of electrolytebuffer

26 UV-Vis Spectroscopy for Verification of theFormation of the HeminG-Quadruplex Complex

The Au substrate modified with memory-active core wasimmersed in a 1 mL reaction system (PBS pH 50 in UVcuvette) ABTStrade chromophore (50 L) was added to thereaction system and UV-Vis absorption spectroscopy wasperformed at a wavelength of 405 nm through 6000 secat RT on a Jasco model V-530 spectrophotometer (JascoInternational Co Ltd Tokyo) Full-wavelength scanningof the final product at 200ndash800 nm was also performed

3 RESULTS AND DISCUSSION31 Mechanism of the Biomemory DeviceThe electrically controlled heminG-quadruplex complexthat was used as a biomemory device is schematicallyillustrated in Figure 1The platform was composed of a silicon wafer and a

PDMS compartment (Fig 1(a)) The silicon wafer was

Sci Adv Mater 8 767ndash774 2016 769




patterned with six designed electrodes and three chamberswere constructed by using 1 agarose gel as salt bridges toseparate them Two electrochemical systems namely thecontroller and operator were combined to work togetherby sharing a single chamberThe roles of the electrodes in both electrochemical sys-

tems and the relevant redox reactions are illustrated inFigure 1(b) The controller and operator functioned as asignal generator and as a signal reader respectively andthe heminG-quadruplex complex on the working electrodein the operator functioned as a signal transducer The sig-nal transduction was similar to informational processing inmemoryThe implementation of memory functions ldquowriterdquo

ldquoeraserdquo and ldquoreadrdquo in the proposed biomemory device ispresented in Figure 1(c) The controller which was con-structed from specially designed electrodes was devisedto electrically modulate the ionic conditions in the cham-ber The applied potentials of minus0054 and minus0339 V inthe controller that represent the generation of H+ and OHminus

are defined as ldquowriterdquo and ldquoeraserdquo operations respectivelyG-rich DNA sequences on Au are originally unfoldedin basic condition and ldquoreadrdquo as a cyclic voltammogramin the operator After ldquowriterdquo generation of H+ in thecontroller induces the folding of G-rich DNA sequencesinto the G-quadruplex structure The G-quadruplex subse-quently captures hemin forming the heminG-quadruplexcomplex by stacking and intercalation The ferric ioncenter of hemin underwent reduction or oxidation (Fe3+

Fe2+) in accordance with the potential sweep directionresulting in effective ET The peak cathodic current (ipc)that is detected increases in the cyclic voltammogramusing the operator Alternatively the heminG-quadruplexcomplex after the ldquoeraserdquo function deforms when hemindisassociates from it because of OHminus generation in thechamber The enhanced ipc in the cyclic voltammogramis thus erased when the operator performs ldquoreadrdquo againThe structural transformation of the memory-active coresis electrically controlled and is transduced into two currentlevels of the ipc Upon defining the two current levels of ipcas memory states (ldquo1rdquo and ldquo0rdquo) we realized a biomemorydevice that was electrically controllable and that did notrequire material input or output

32 Setting of the Controller to ModulateIonic Conditions

The controller was designed to modulate H+OHminus concen-trations in the chamber on the basis of results from theaforementioned micro-pH-stat research An unusual three-electrode system that deviates from the rule typical of elec-trochemical measurements was applied as the operatingprinciple of the controller in this research1718

A typical three-electrode cell for electroanalyticalchemistry consists of a reference electrode an auxil-iary electrode and a working electrode in electrolyte

The reference electrode commonly has a stable and well-known electrode potential The potential that is appliedto or measured from the working electrode is defined asa function of the reference The current that originatesfrom the cell reaction flows between the auxiliary elec-trode and the working electrode where half redox reac-tions take place Here the typical potential-stable electrodeAgAgCl (1 M NaCl) was used as the working electrodeA pH-sensitive IrOx electrode was used as the referenceelectrode An auxiliary electrode was constructed from IrAn automatic negative feedback system (the controller)was installed to modulate the H+ and OHminus concentrationsin the chamber Reliable control of the ion concentrationsby the controller was realized because of electrophoresison the Ir auxiliary electrode and pH sensitivity of the IrOxreference electrodeThe high stability of the electrode potential of AgAgCl

saturated in NaCl solution is due to the constant (bufferedor saturated) concentration of each participant in the redoxreaction Characteristic of electrodes with stable poten-tial a small change in overpotential across the electrodecan induce a very large current A AgAgCl electrodecontaining sufficient chloride ions was used as the work-ing electrode in our system The potential of the refer-ence IrOx electrode depended on the environmental pHBecause of a predetermined voltage applied to AgAgClwith respect to the IrOx electrode which was the appliedpotential in the system the potential stability of AgAgClinduced a large current flow between the AgAgCl work-ing electrode and the Ir auxiliary electrode Depending onthe current direction either of the following half-reactionscould occur when the system was functioning Ag+ClminusrarrAgCl+ eminus (oxidation half-reaction) or AgCl+ eminus rarrAg+Clminus (reduction half-reaction) Electrophoresis of water onthe auxiliary electrode occurred in response to the redoxof AgAgCl Hence the corresponding half-reaction thattook place on Ir was 2H2O+ 2eminus rarr 2OHminus +H2 (reduc-tion half-reaction) or 2H2O rarr O2 + 4H+ + 2eminus (oxida-tion half-reaction) The responsive electrophoresis of waterresulted in the accumulation of H+ or OHminus leading tolocal pH variations The amount of H+ or OHminus generatedwas related to the charge passing through the electrodesThe electrode potential of IrOx changed in terms of therelationship between the potential of the pH-sensitive IrOxelectrode and the pH Thus the generation of H+ or OHminus

allowed the recovery of the initial exerted potential and astable state was achieved when the applied potential wasfully recovered Once the pH-stable state was achieved theresulting pH did not change until the applied potential inthe controller was switched to regulate the pH to anothervalue Shutting off the power supply did not change theionic condition in the chamber This negative-feedback cir-cuit provided the ability to accurately obtain the expectedionic conditions in chamber and there was no concernabout the stability of the state





ARTICLE

The performance of the deposited IrOx electrode inthe controller was repeatedly characterized for 3 timesby its pH sensitivity As Figure 2 illustrates the poten-tial response of the deposited IrOx electrodes to pH val-ues was linear with a slope of minus7115plusmn 145 mVpHwhich agrees with the reported pH sensitivity of depositedIrOx electrodes1824 The fitted open circuit potentials thatresponded to pH values of 50 and 90 were minus0054 andminus0033 V respectively

The ability to switch the pH value in the chamber usingthe controller was further confirmed According to thefeatures presented in Figure 2 the potentials of minus0054and minus0339 V were applied for 100 sec each to regu-late the pH to the intended values of 50 and 90 respec-tively We determined the corresponding acidic and basicvalues by using pH indicator strips (not shown) whichdemonstrated the realization of switching pH values in oursystem

33 Self-Assembly of Memory-Active Coreson the Au Surface

The SAMs of the memory-active cores on Au were furtherconfirmed by Raman spectroscopy and a cyclic voltammo-gram (Figs 3(a b))

Assignment of main characteristic peaks was accom-plished as described in the literature2526 Characteristicbands for hemin appeared at 1015 (shoulder) 1033 1056(shoulder) 1115 and 1209 cmminus1 The peak at 1209 cmminus1

was derived from the 1195 and 1222 cmminus1 regions at theshoulder of both sides which were assigned to (pyr) Theband at 1088 cmminus1 in this region was assigned to symmet-ric stretching vibration of the phosphor dioxy group PO2minus

As the 1150ndash1600 cmminus1 region originated from thein-plane vibrations of base residues in Raman spec-troscopy we expected band variation in this region toresult from base-stacking interactions Effective stackingbetween external hemin and guanine bases induced weakbands at 1490 and 1594 cmminus1 while stacking of heminwith the quadruplex structure formed peaks at 1332 13751481 1611 and 1640 cmminus1 The slight shift of peaks is

Fig 2 pH sensitivity of the electrochemically deposited IrOx electrodethat is characteristic of the controller

Fig 3 Fabrication of the constructed biomemory device on Au(a) Raman spectroscopy investigation of the SAMs of the memory-activecores on the Au substrate (b) Electrochemical characterization of theconstruction processes (c) Enzyme kinetics investigation using time-course UV-Vis spectroscopy at a maximum absorbance wavelength of405 nm Inset full-wavelength characterization of the final product

due to the solid state of our samples and the ferric centerof heminThe modification of G-rich DNA sequences on bare

Au was also demonstrated by the existing redox peaksA remarkable increase and shift of the redox peaks mani-fested as faradic currents in the presence of hemin whichindicated an enhanced redox electron transfer (ET)The capture of hemin by the G-rich DNA on the Au

surface can be further confirmed by the DNAzyme activ-ity of the heminG-quadruplex complex Here catalysis ofthe H2O2-mediated oxidation of colorless ABTS2minus into theblue product ABTSbullminus was studied by using the maximumabsorption at 405 nm (Fig 3(c)) Time-resolved UV-Visabsorption spectroscopy revealed the enzyme kinetics Thebiocatalyzed product was further confirmed by a single anddistinguishable absorption peak at 405 nm as shown in





the inset of Figure 3(c) The formation of memory-activecores by binding of hemin to G-quadruplex structures wasreconfirmed

34 Verifying the Functions of the Biomemory DeviceThe states of the memory-active cores on the Au surfacewere controlled by using an electrical method in the pro-posed biomemory deviceAs illustrated in Figure 4(a) the ipc in the operator

decreased and shifted to negative values when the functionof the controller was switched from ldquowriterdquo to ldquoeraserdquoThe generation of OHminus resulted in a basic buffer con-dition in the chamber and led to the deformation of theheminG-quaduplex complex Subsequently the ET thatwas influenced by the distance between the ferric centerof hemin and the Au surface diminished drastically Thememory state of ldquo1rdquo was replaced by ldquo0rdquo When ldquowriterdquowas selected again the effective ET at close proximitywas retrieved because of the generation of H+ and the for-mation of the heminG-quadruplex complex Subsequentlythe memory state ldquo1rdquo was written againThe decreased current of the memory-active cores in the

electrically controlled biomemory device compared withthat in the bulk electrochemical cell resulted from differentelectrochemical cells and different scan ratesFigure 4(b) demonstrates that the memory state in the

operator switched between ldquo1rdquo and ldquo0rdquo representing two

Fig 4 Performance of the constructed biomemory device (a) Cyclicvoltammogram showing the switch between memory states ldquo1rdquo and ldquo0rdquo inthe operator based on the electrical operations in the controller (b) Char-acterization of the reproducibility upon alternative ldquowriterdquo and ldquoeraserdquooperations

ipc levels which is in accordance with the alternativeoperations of ldquowriterdquo and ldquoeraserdquo respectively in thecontroller Thus a resettable two-state system that waselectrically controlled was detected this system includedonly electrical sources Neither material transference norfluorescence labeling was needed The resettable two-state system is in agreement with the theory that theheminG-quadruplex complex is formed and deformedaccording to H+OHminus generation and that this conforma-tional transformation is reversible

35 A WORM Biomemory DeviceThe time-course operations in the controller and the cor-responding ipc detected by the operator are illustrated inFigure 5(a) Upon reversible switching of the input in thecontroller with potentials of minus0054 and minus0339 V toachieve pH 50 and 90 respectively over lt200 sec thedetected ipc in the operator was found to switch betweentwo stable and different levels with hysteresis Similarlymemory states of ldquo1rdquo and ldquo0rdquo were switched at intervalsaccording to the operations in the controller The delayin the current change relative to the application of poten-tials in the controller for the ldquowriterdquo or ldquoeraserdquo operationsresulted from the time required for water electrophoresisin the chamber to generate H+ or OHminus and the transfor-mation of the G-quadruplex with respect to the ionic con-ditions The structural transformation was then convertedinto electrical data (cyclic voltammogram) by the opera-tor the working electrode of which was modified with theSAMs of the memory-active coresThe biomemory device were verified non-volatile by

repeatedly reading the memory state which maintainedthe ldquowriterdquo or ldquoeraserdquo operation in the controller The datashown in Figure 5(b) indicate that the memory states wereretained for at least 500 cycles The biomemory state didnot change until the input in the controller was switchedto reach another pH-stable state Thus the memory stateldquo1rdquo which represents the formation of the memory-activecores can be stably read at any time for multiple repeti-tions until ldquoeraserdquo is operated in the controller The mem-ory state ldquo0rdquo upon operation of ldquoeraserdquo was retained forat least 500 cycles As a result a novel WORM biomem-ory device was establishedThe reproducibility of the constructed WORM biomem-

ory device that consisted of two electrochemical systemswas authenticated by testing 10 constructed devices Theipc data for the devices in the operator over alternatingldquowriterdquo and ldquoeraserdquo operations were averaged and aredepicted in Figure 5(c) By using the least-squares methodthe current after the ldquowriterdquo operations in the biomem-ory devices was found to be 06184plusmn 00039 A (logicstate ldquo1rdquo) whereas the current after the ldquoeraserdquo operationswas verified to be 04202plusmn 00024 A (logic state ldquo0rdquo)The threshold current range for the two-state biomemorysystem at 0519plusmn0019 A was set for recognition





ARTICLE

Fig 5 Electrically controlled biomemory performance of the complete device (a) The operator detected distinct cathodic currents according to thepotential applied in the controller (b) Maintenance of the memory state in the operator after ldquowriterdquo or ldquoeraserdquo operations in the controller (c) Reliabilityof electrically controlled biomemory devices confirmed by 10 devices with alternative ldquowriterdquo and ldquoeraserdquo operations (d) Comparative measurementof the redox peaks of our memory active cores over many cycles and that of previous biomemory research using Cu2+G-rich ssDNA

The effective ET was also confirmed by comparisonwith our previous work23 Compared with the flexiblessDNA assay which had greater potential for electro-static adsorption of copper ions (Cu2+)2728 our devicewith the heminG-quadruplex complex produced highercurrent levels with stability (Fig 5(d)) The signal with

Fig 6 A four-bit biomemory system using cascaded devices (a) A number converted from a binary to decimal value by using four cascaded deviceswith defined operations (ldquowriterdquo or ldquoeraserdquo) (b) The computational ability of four cascaded biomemory devices was verified to code decimal valuesfrom zero to 15 (16 numbers)

higher sensitivity originated from the folded structures ofG-quadruplex DNA which provided the effective ET atclose proximity Peak currents over various cycles revealthat the stability of the proposed covalent bond that formedthe heminG-quadruplex complex was enhanced comparedwith that of the Cu2+G-rich ssDNA biomemory formed





through electrostatic adsorption These results illustratethat the heminG-quadruplex complex has potential use inrigid memory-active coresThus a reliable durable and resettable biomemory sys-

tem involving only electrical sources was constructedIn addition this WORM biomemory could be furtherimproved by designing the patterned electrodes moresophisticatedly The sizes and components of electrodescould be investigated in detail to decrease the operationtime between memory states and enhance the stability ofelectrodes For example the pH sensitivity and stability ofIrOx depends on its components and fabrication methodThe distance between Ir and Au influenced the ion diffu-sion coefficient while their shapes also should be takeninto account The whole setup could also be miniaturizedfor practical usage

36 Multibit SystemFurther application of our WORM biomemory system wasverified for use as a multibit system by cascading A four-bit biomemory system was illustrated by using four cas-caded biomemory devices As shown in Figure 6(a) fourbiomemory devices coded as ldquoArdquo ldquoBrdquo ldquoCrdquo and ldquoDrdquo wereindependently operated by using ldquowriterdquo or ldquoeraserdquo Theldquooperatorsrdquo in these devices exhibited binary data 0110which can be converted into the decimal number 6 Dataaccess with a coding ability of up to 16 was achievedwith this four-bit system as demonstrated in Figure 6(b)This integrated platform which contains a set of our con-structed biomemory chips a more delicate design anda scaled-down version of the biomemory platform ispromising

4 CONCLUSIONSIn this study a reliable DNA-based WORM biomemorydevice with only electrical signals was newly developedbased on the transformation of the heminG-quadruplexcomplex We demonstrated that we transduced the elec-trical input in the controller into electrical output in theoperator by switching between the memory states ldquo1rdquo andldquo0rdquo In addition we found the constructed biomemory chipto be non-volatile over numerous switches The memorystates were reliable in many chips and could be main-tained for a long period until the input signal was changedThis is the first report of a DNA-based biomemory devicethat is controlled with only electrical signals It does notrely on tagging or external chemical inputs and insteaduses only electrical controls This is a closed system withconsiderable stability durability and reliability In addi-tion this device has advantages for further application inconnection and co-operation with other electric systemsA four-bit biomemory system was constructed by usingthis proposed electrically operated device Therefore ourconcept can provide a promising method to construct bio-computing devices in a practical feasible and stable way

Acknowledgments This research was supported bythe Leading Foreign Research Institute Recruitment Pro-gram through the National Research Foundation ofKorea (NRF) funded by the Ministry of Science ICTand Future Planning (MSIP) (2013K1A4A3055268) andby the National Research Foundation of Korea (NRF)grant funded by the Korean government (MSIP) (No2014R1A2A1A10051725) The first author gratefullyacknowledges support from the China Scholarship Council(CSC) for the fellowship provided

References and Notes1 T Li F Lohmann and M Famulok Nat Commun 5 4940

(2014)2 R Pei E Matamoros M Liu D Stefanovic and M N Stojanovic

Nat Nanotechnol 5 773 (2010)3 L Qian and E Winfree Science 332 1196 (2011)4 Y J Chen N Dalchau N Srinivas A Phillips L Cardelli

D Soloveichik and G Seelig Nat Nanotechnol 8 755 (2013)5 D Y Zhang and G Seeglig Nat Chem 3 103 (2011)6 D Y Zhang R F Hariadi H M T Choi and E Winfree Nat

Commu 8 755 (2013)7 J Elbaz O Lioubashevski F Wang F Remacle R D Levine and

I Willner Nat Nanotechnol 5 417 (2010)8 A Kuzuya and Y Ohya Acc Chem Res 47 1742 (2014)9 G Seelig D Soloveichik D Y Zhang and E Winfree Science

314 1585 (2006)10 M N Stojanovic and D Stefanovic J Am Chem Soc 125 6673

(2003)11 Y Guo L Zhou L Xu X Zhou J Hu and R Pei Sci Rep

4 7315 (2014)12 T Li E Wang and S Dong J Am Chem Soc 131 15082 (2009)13 L Olejko P J Cywinski and I Bald Angew Chem Int Ed Engl

53 1 (2014)14 D Monchaud P Yang L Lacroix M P Teulade-Fichou and J L

Mergny Angew Chem Int Ed Engl 47 4858 (2008)15 Y Dong Z Yang and D Liu Acc Chem Res 47 1853 (2014)16 Y Y Yan J H Tan Y J Lu S C Yan K Wong D Li L Q Gu

and Z S Huang Biochim Biophys Acta 1830 4935 (2013)17 K Morimoto M Toya J Fukuda and H Suzuki Anal Chem

80 905 (2008)18 Y Yang G Liu H J Liu D Li C H Fan and D S Liu Nano

Lett 10 1393 (2010)19 M Mir A Homs and J Samitier Electrophoresis 30 3386

(2009)20 T Lee S-U Kim J Min and J-W Choi Adv Mater 22 510

(2010)21 T Lee A K Yagati J Min and J-W Choi Adv Funct Mater

24 1781 (2014)22 A K Yagati T Lee J Min and J-W Choi Biosens Bioelectron

40 283 (2013)23 T Lee W A El-Said J Min and J-W Choi Biosens Bioelectron

26 2304 (2011)24 D O Wipf F Ge T W Spaine and J E Baur Anal Chem

72 4921 (2000)25 G Rusciano A C D Luca G Pesce A Sasso G Oliviero

J Amato N Borbone S DrsquoErrico V Piccialli G Piccialli andL Mayol Anal Chem 83 6849 (2011)

26 C Wei G Jia J Yuan Z Feng and C Li Biochemistry 45 6681(2006)

27 S W Liu J F Chu C T Tsai H C Fang T C Chang and H WLi Anal Biochem 436 101 (2013)

28 R R Machinek T E Ouldridge N E Haley J Bath and A JTurberfield Nat Commun 5 5324 (2014)





the pH-sensitive I motif which requires fluorescent tagsfor detection18 Detection using optical measurements inDNA-based biocomputing studies is unsuitable for connec-tion and co-operation with other electric devices becauseof its requirement of a complex setup Electrochemi-cal methods have incorporated DNA biosensors in lab-on-a-chip devices19 However an electrically controlledDNA-based computing system that properly connects andco-operates with other electrically controlled systems ona chip has yet to be reported Hence in this report themicro-pH-stat system was integrated with an electrochem-ical detecting method to utilize a biomemory device thatuses only electrical signalsWe have developed various electronic biomemory

devices and bioprocessing devices using metallopro-teins such as recombinant azurin and cytochrome c20ndash22

We have also used ssDNA-Cu heterolayers to develop aDNA-based biomemory based on electrostatic adsorptionwithout an electrical control system23 G-rich DNA wasused as a platform to bind iron-containing porphyrin heminby covalent bonds to develop a DNA-based biomemorywith an electrical control systemIn this study a biomemory device was developed based

on the electrically controlled formation and deformation ofthe heminG-quadruplex complex for the first time Mul-tifunction information processing (write-read-erase) wasproposed and a write-once-read-many-times (WORM)biomemory device was constructed The WORM biomem-ory device is a closed system with high stability dura-bility and reliability and uses only electrical signals Thechip platform of the constructed biomemory device canconnect and co-operate with other electrically controlledsystems on a chip A four-bit biomemory system waseasily constructed by cascading the constructed biomem-ory devices Hence our proposed concept provides anovel method for developing DNA-based biocomputingdevices

2 MATERIALS AND METHODS21 MaterialsG-rich DNA with the sequence 5prime-HS-(CH26-TTT TTTTTT TGG GTT AGG GTT AGG GTT AGG G-3prime wassynthesized by Bioneer Co (Korea) Phosphate-bufferedsaline (PBS) solutions at pH 50 and 90 were pre-pared 22-azino-bis(3-ethylbenzthiazoline-6-sulfonic acidABTStrade chromophore) were purchased from EMD Mil-lipore (USA) and used directly Silicon wafers withelectrode deposits were designed and purchased fromLG Siltron Inc (Korea) A Sylgard 184 elastomer kit(Dow Corning USA) composed of polydimethylsiloxane(PDMS) base and curing agent was used Piranha solu-tion composed of 30 vol H2O2 (Duksan Pure Chem-ical Co Ltd Korea) and 70 vol H2SO4 (DaejungChemical Co Ltd Korea) was freshly prepared andused with caution Hemin dithiothreitol (DTT) and other

reagents were purchased (Sigma-Aldrich USA) and useddirectly A working solution of hemin (5 mM) in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)solution (25 mM HEPES pH 74 20 mM KCl 200 mMNaCl 005 (wv) Triton X-100 1 (vv) DMSO) wasprepared

22 Installation of the Biomemory PlatformThe silicon wafer deposited with six patterned elec-trodes was cleaned with piranha solution and then alignedwith the predesigned PDMS compartment as shown inFigure 1(a) Two chambers with AgAgCl electrodes wereseparated from a reaction chamber by using salt bridgescomposed of 1 agarose gel The reaction chamber wascomposed of four electrodes namely iridium (Ir) iridiumoxide (IrOx) Au surface and Pt The deposited AgAgClelectrodes were immersed in 80 mL NaCl (1 M) solutionswhile 1 mL Na2SO4 (10 mM hemin 25 mM) was usedas the electrolyte in the reaction chamber

23 Immobilization of Memory-Active Cores(HeminG-Quadruplex Complex) on Au

The properties of memory-active cores on Au surfaceswere characterized by using bare Au electrodes with anactive area of 025 cm2 The Au surface in the installedbiomemory platform was subsequently modified withmemory-active cores for biomemory functionalizationBare gold electrodes were cleaned with piranha solu-

tion and then incubated with DTT-reduced G-rich DNAsequence (1 M) in PBS (pH 50) at RT for 4 hrssubsequently washed with DI water and dried withN2 gas The G-rich DNA modified Au substrates werethen immersed in the working solution of hemin at RTfor 4 hrs to capture hemin Self-assembled monolayers(SAMs) of the memory-active cores on Au substrates wereprepared

24 Confirmation of Memory-Active Cores onAu by Raman Spectroscopy

Immobilization of the memory-active cores on the Au sur-faces was determined by Raman spectroscopy The instru-ment used for Raman spectroscopy was an NTEGRASpectra (NT-MDT Russia) equipped with a CCD detec-tor cooled with liquid nitrogen and an inverted opticalmicroscope (Olympus IX71) The XYZ three-dimensionalscanning range was 100 mtimes 100 mtimes 6 m The res-olution of the spectrometer was 200 nm in the xy planeand 500 nm along the z axis The incident laser used forobtaining Raman spectra was an infrared laser that emitslight at a wavelength of 785 nm Five 30 sec scans from1000ndash2000 cmminus1 were performed and the mean intensitywas taken as the Raman signal The background was gen-erated subtracted from the signal by using OriginPro 80program





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A Biomemory Device Based on Electrically Controlled Hemin ...nbel.sogang.ac.kr/nbel/file/363.pdf · A Biomemory Device Based on Electrically Controlled Hemin/G-Quadruplex Complex