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A Multifunctional Graphene/DNA-based Platform for the Construction of Enzyme-free Ternary Logic Gates Chunyang Zhou, Dali Liu, Changtong Wu, Shaojun Dong, and Erkang Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09021 • Publication Date (Web): 18 Oct 2016 Downloaded from http://pubs.acs.org on October 23, 2016

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ACS Applied Materials & Interfaces

A Multifunctional Graphene/DNA-based Platform for the Construction of Enzyme-free Ternary Logic Gates Chunyang Zhou,†, ‡ Dali Liu,‡ Changtong Wu,†, # Shaojun Dong†* and Erkang Wang†*

†

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of

Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, P. R. China. E-mail: [email protected], [email protected]. ‡

State Key Laboratory on Integrated Optoelectronics, College of Electronic Science

and Engineering, Jilin University, Changchun, China. #

Department of Chemistry and Environmental Engineering, Changchun University of

Science and Technology, Changchun, China.

ACS Paragon Plus Environment


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ABSTRACT: In this work, we have successfully realized the multi-valued logic circuits including ternary INHIBIT and ternary OR logic gates in an enzyme-free condition by integration of graphene oxide and DNA for the first time. Compared to the binary logic gate with two states of “0” and “1”, the multi-valued logic gate contains three different states of “0” and “1” and “2”, which can increase the information content in a system and further improve the ability of information processing. Such type of multi-valued logic operations provides a wilder field of vision towards DNA-based algebra logical operations to make the applications more accurate with complexity reduction and accelerate the development of advanced logic gates.

KEYWORDS: multi-valued logic, ternary INHIBIT logic, ternary OR logic, graphene oxide, DNA-based


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1. INTRODUCTION Molecules or biomolecules that were used in computing to substitute traditional silicon-based electronic integrated circuits have become an aspiring research field in the past fifteen years and can act as the foundation of bio-computer development.1 Biomolecular logic gates, which can generate the corresponding outputs from its inputs according to the Boolean calculation at molecular level, have gain significant attention in the development of bio-computer.2-7 Up to now, various basic molecular logic gates ,such as AND, OR, XOR, NOR, NAND or INHIBIT, and advanced molecular logic circuits such as half adder, full adder, half subtractor, full subtractor, encoder or decoder and so on all binary logic computing.8-15 However, in some cases these logic gates may encounter some uncertainty and imprecision conditions when processing complex information because they have only two states of on and off, which can often struggle to process information.16 Multi-valued logic gates, in which the digital signals in the information content of a system has been increased to a higher value than binary operation, have been the subject of much research over many years.17-18 However, its application is rarely reported based on DNA used in molecule or biomolecule computing. The multi-valued logic gates involve the switches by using more than two states of false (0, low voltage) or, true (1, high voltage) which make it grateful to bring about more powerful information processing capability.16 However, the implementation of multi-valued logic operations based on the molecular ternary computing is rare, especially the use of nucleic acids. Nie and coworkers have developed a multi-valued logic gates by using a 3D DNA triangular prism to receive



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several kinds of distinguishable outputs produced by different inputs.19 Qu et al. designed two multi-valued logic gates by conjugating upconverting nanoparticles with DNA sequences and exhibited the fluorescent resonant energy transfer with graphene.16 Due to its low-cost, hybridization character, feasible synthesis and special identification of target molecules, nucleic acids, have been confirmed as the most attractive building block for the study and operating of biochemical circuits.20-25 Graphene oxide (GO), a kind of hydrophilic material due to it contains epoxy, hydroxyl, carbonyl and ether groups, and showing great catalytic, optical and electronic properties, has gained wide attention and found versatile bio-applications including cellular growth, differentiation gene and photothermal therapy.26-28 What is more interesting is that GO can adsorb single-stranded DNA (ssDNA) with high affinity through pi-stacking interactions and hydrogen bonds while shows weak affinity toward duplex DNA, which makes it flexible for modulating output signal in bimolecular logic gate.26,

27, 29

Most especially, GO can be regarded as a kind of

nanoquencher for it can quench various fluorescence dyes through fluorescent resonant energy transfer (FRET).29 Based on these properties, GO-based nanotemplate has been widely used for the fluorescent detection of DNA, metal ions, proteins and small molecules.30-31 By the integration of GO and single-stranded DNA, we have successfully realized some advanced logic gates including full adder, full subtractor, voter and reversible logic gates,32-33 however, they are binary logic gates with two states of “0” and “1”. To improve the ability of information processing and speed up


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the development of molecular logic computing, we first realized some multi-valued logic gates including a ternary INHIBIT logic gate and a ternary OR logic gate, which were realized by three different states of “0”, “1” and “2”. The ternary INHIBIT and ternary OR logic gates were realized in an enzyme-free condition, which can reduce the cost, simplify the experimental conditions and get rid of the dependence on experimental environment for enzyme was easy to lose its activity, performing a promising future for the development of molecular logic gates into practical application.34 Such type of multi-valued logic operations can further increase the information content of a system and may have great potential applications in biological computing science and biosensing fields to make the applications much more accurate with complexity reduction.

2. EXPERIMENTAL SECTION All DNA sequences were listed in Table S1. The DNA sequences were dissolved in water and diluted with Tris-HCl buffer (200 mM KCl, 20 mM Tris-HCl, 10 mM MgCl2) for reaction in the logic circuits. Before use, the DNA solutions diluted with Tris-HCl buffer were first heated at 90℃ for about 10 min. After the DNA solutions gradually cooled down to room temperature, the platform were prepared in our logic gate by mixing graphene oxide (GO) (8 µg/mL), 6-carboxyfluorescein (FAM) modified DNA sequence (P-DNA) (100 nM) and N-methylmesoporphyrin IX (NMM) (1 µM) for 15 min. Fluorescence spectra were recorded after added their respective inputs and incubated under room temperature for 30 min to implement the ternary INHIBIT and OR logic operations. The materials and instruments involved in this



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work refer to the previous work. 32

3. RESULTS AND DISCUSSION In this work, we have implemented a GO/P-DNA-based multivalued logic computing system including a ternary INHIBIT and a ternary OR logic gates as shown in Scheme 1 and Scheme 2, which were produced for the first time and demonstrated to be much lower cost and more easily to implement compared with previous works.16,19 The 6-carboxyfluorescein (FAM, emission max at 521 nm) modified single-stranded P-DNA were anchored on the GO due to the π–π stacking effect, acting as the original reacting platform. The fluorescent response of N-methylmesoporphyrin IX (NMM, emission max at 607 nm) acted as the output signal. First of all, the function of GO used in these systems was to quench the fluorescence of FAM that was modified on P-DNA to demonstrate the connection between GO and P-DNA. Secondly, the function of GO can further optimize the concentration of P-DNA (Figure S1 in Supporting Information (SI)). Finally, it also aim to reduce the background signals that induced by NMM for the P-DNA contained the sequence of GGGTGGGTGGGT, which can enhance the fluorescence of NMM to some extent and increase the jamming signals without the existence of GO. As shown in Figure 1A, P-DNA strands were immobilized on the surface of GO due to the π-π stacking effect and the fluorescence of P-DNA was quenched by GO via FRET. Before operating the ternary logic gate, three states were defined as low, medium and high for describing the fluorescence intensity of the output signals, which corresponded to logic values of 0, 1 and 2 shown in the truth table of the


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Figure 1. (A) The working scheme of the GO/P-DNA-based system for the reaction mechanism. (B) The NMM fluorescence response of the ternary INHIBIT logic systems at 607 nm. (C) The truth table of the ternary INHIBIT logic gate. ternary INHIBIT logic gate in Figure 1A. The four designed single-stranded DNAs used in ternary INHIBIT logic gate were acted as the two inputs, input A and input B (INA and INB), respectively. For INA, the logic value [1] represents the introduction of single stranded A1 sequence and value [2] represents the introduction of the mixture of single stranded A1 and A2 sequences to the system. For INB, the value [1] represents the introduction of B2 and value [2] represents the mixture of B1 and B2, respectively. The input value [0] corresponded to none of strands were added. The ssDNA strand of A1 is complementary to B1, and A2 is complementary to B2. The A1 and A2 can hybridize with P-DNA to form the G-quadruplex structure, which can significantly enhance the fluorescence of NMM once binding on G-quadruplex.35 The



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B1 and B2 have no right to hybridize with P-DNA and produce a relative low NMM fluorescence. Based on the above principle, when the addition of A1 and no addition of B1 to the system, A1 can hybridize with P-DNA and then enhance the fluorescence of NMM. However, when the addition of the mixture of A1 and A2 and no addition of the mixture of B1 and B2 to the system, the fluorescence of NMM can also be enhanced based on the G-quadruplex structure. The required ternary INHIBIT logic gate was realized according to the fluorescence of NMM (Figure 2). To establish optimal conditions, the concentrations of GO, FAM modified P-DNA and other DNAs were explored (see Figure S1-S2). At the initial state of GO/P-DNA that represented the inputs of “0/0” in logic gate, the single-stranded P-DNA anchored on the GO, producing a low NMM fluorescence signal with the output of “0” in logic gate, (Figure 2A curve a). The addition of B2 or the mixture of B1 and B2 cannot hybridize with P-DNA and prevent the formation of G-quadruplex, producing a low NMM fluorescence signal of “0” with the input of “0/1” or “0/2” in logic gate (Figure 2A, curve b and c). When the strand A1 (input of “1/0” in logic gate) or the mixture of A1 and A2 (input of “2/0” in logic gate) were introduced, they can hybridize with P-DNA and form the duplex of A1/P-DNA or A1- and A2/P-DNA with G-quadruplex structure to enhance the fluorescence of NMM. The resulted duplex of A1/P-DNA with G-quadruplex structure induced the NMM fluorescence marked as “1” and the resulted mixture of duplex A1/P-DNA and A2/P-DNA can reach the strongest NMM fluorescence marked as “2” in the truth table, the corresponding expressions of NMM fluorescence were shown in Figure 2A (curve d and g). Upon adding A1 and B2 to the


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Figure 2. (A) The ternary INHIBIT logic gate fluorescence emission response profiles of the curve a: no inputs, curve b: B2, curve c: B1+B2, curve d: A1, curve e: A1+B2, curve f: A1+(B1/B2), curve g: A1+A2, curve h: B2+(A1/A2), curve i: (A1/A2)+(B1/B2). (B) The electronic equivalent circuitry of the ternary INHIBIT logic gate. system (input of “1/1” in logic gate), A1 can hybridize with P-DNA, producing the output value of “1”, (curve e). After adding A1 and the mixture of B1 and B2 to the system (input of “1/2” in logic gate), A1 preferred to hybridize with B1, leaving the quenched P-DNA and B2 on the surface of GO, producing the output value of “0”, (curve f). So when the addition of B2 and the mixture of A1 and A2 to the system (input of “2/1” in logic gate), A2 preferred to hybridize with B2 and A1 hybridized with P-DNA, producing the output value of “1”, (curve h). When the addition of the mixture of A1 and A2 and the mixture of B1 and B2 (input of “2/2” in logic gate) to the system, A1 hybridized with B1 and A2 hybridized with B2, producing a low NMM fluorescence signal of “0”, (curve i). The NMM fluorescence at 607 nm was plotted as column bar in Figure 1B to define the output threshold values with an



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undefined range. The output threshold value was defined as “0” when the normalized fluorescence intensity was lower than 0.25 and the “1” was defined when the normalized fluorescence intensity was higher than 0.3 and lower than 0.7. So the output threshold value can be defined as “2” when the normalized fluorescence intensity is higher than 0.75.

Figure 3. (A) Polyacrylamide gel analysis of the ternary INHIBIT logic gate. Different DNA samples were added into lanes 1-13. Lane 1: P-DNA, (P) Lane 2: A1, Lane 3: A1+A2, Lane 4: B2, Lane 5: B1+B2, Lane 6: P+B2, Lane 7: P+B1+B2, Lane 8: P+A1, Lane 9: P+A1+B2, Lane 10: P+A1+B1+B2, Lane 11: P+A1+A2, Lane 12: P+A1+A2+B2, Lane 13: P+A1+A2+B1+B2. (B) The circular dichroism of different DNA inputs. To identify the hybridizations among the designed input DNAs, the native polyacrylamide gel electrophoresis experiments (PAGE) were performed (Figure 3A). From Lane 1 to Lane 5, the belts showed the single DNA strands of P-DNA, A1, the mixture of A1 and A2, B2, the mixture of B1 and B2 in sequence as control. Because of the similarity in DNA sequences of A1 and A2, the belts locations were similar in Lane 3, and so did B1 and B2 in Lane 5. The addition of P-DNA and B2 did not


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generate a new belt because of the two sequence cannot hybridization in Lane 6. And so did Lane 7, the addition of P-DNA and the mixture of B1 and B2 cannot generate a new belt. The hybridization of P-DNA and A1 could create a new belt and form the duplex of P-DNA/A1 in Lane 8. There were two belts in Lane 9, one was the original belt of B2, and another one was a new belt created by the hybridization of P-DNA and A1. The existence of P-DNA, A1 and mixture of B1 and B2 produced a new belt for the preferred hybridization of A1 and B1 in Lane 10. A new belt was also produced because of the hybridization of P-DNA and the mixture of A1 and A2 in Lane 11. The existence of P-DNA, B2 and mixture of A1 and A2 could produce two new belts, one was the preferred hybridization of A2 and B2, and another one was the hybridization of P-DNA and A1 in Lane 12. The coexistence of P-DNA, the mixture of A1 and A2 and the mixture of B1 and B2 generated two new overlapping belts for the hybridization of A1 and B1 and A2 and B2 in Lane 13. All the lanes in PAGE experiments were well demonstrated the reactions among the inputs in the ternary INHIBIT logic gate. The circular dichroism (CD) experiments were operated to identify the formation of G-quadruplex structures in the reacting solution. As shown in Figure 3B, the CD spectrum of P-DNA, A1, the mixture of A1 and A2, B2, the mixture of B1 and B2 were of relatively low amplitude (curve 1-5) in Tris-HCl buffer with GO, indicating random DNA structures. As a result of GO, the formation of G-quadruplex structure was inhibited by the P-DNA with TGGG TGGG TGGG sequence, so the CD spectrum of P-DNA showed relatively low amplitude in curve 1. After mixing the P-DNA and B2, no obvious change was found (curve 6), indicating



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that the mixture does not form G-quadruplex structure. When adding B1 and B2 into P-DNA, still no obvious change was found (curve 7). When adding A1 or the mixture of A1 and B1 to P-DNA, two obvious peak, a positive peak at 263 nm and a negative peak at 243 nm, were found (curve 8 and curve 9), indicating the formation of G-quadruplex structure.36-38 When adding A1 and the mixture of B1 and B2 to P-DNA, A1 and B1 had the priority to hybridize and form the duplex of A1/B1 without the G-quadruplex structure (curve 10). When the mixture of A1 and A2 or the mixture of A1 and A2 and B1 was added to P-DNA, two obvious peaks were all found in each curve (curve 11 and curve 12) respectively, indicating the formation of G-quadruplex structure. However, in the coexistence of P-DNA, the mixture of A1 and A2 and the mixture of B1 and B2 in the system, no obvious peak was found due to their hybridization mentioned above (curve 13), indicating that the mixture does not form G-quadruplex structure. All the results that we obtained in the CD spectrum were corresponding to the PAGE experiment and further confirmed the implementation of INHIBIT logic gate. By changing the original DNA sequences of various inputs, the ternary OR logic gate was operated by utilizing the same platform of GO/P-DNA as the front work’s basis. Before operating the ternary OR logic gate, we defined three different states of the output as low, medium and high, which corresponded to logic values of 0, 1 and 2 shown in the truth table of the ternary OR logic gate in Figure 4. As shown in Figure 4, the four designed single-stranded DNAs used in ternary OR logic gate were acted as the two inputs, input 1 and input 2 (IN1 and IN2), respectively. For IN1, the value of


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Figure 4. (A) The working scheme of the GO/P-DNA based system for the reaction mechanism of the ternary OR logic gate. (B) The NMM fluorescence response of the ternary OR logic systems. (C) The truth table of the ternary OR logic gate. [1] and [2] represented the addition of a1 and the mixture of a1 and a2, respectively. For IN2, the value of [1] and [2] represented the introduction of b1 and the mixture of b1 and b2, respectively. To be sure beforehand, all the ssDNAs can hybridize with P-DNA to form the structure of G-quadruplex. When the addition of a1 and b1 to the system, they had the priority to hybridize and form the duplex of a1/b1 and then form the G-quadruplex structure with P-DNA. The feasibility of the ternary OR logic gate was further demonstrated by introducing various combinations of inputs to the fluorescence outputs as shown in Figure 5. To establish optimal conditions, the concentrations of a1 and the mixture of a1 and a2 were explored (see Figure S3). At the initial state of GO/P-DNA that represented the inputs of “0/0” in logic gate, the



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P-DNA anchors on the GO, producing a low NMM fluorescence signal with the output of “0” in logic gate, (Figure 5A curve a). When the addition of a1, the input of “1/0” in logic gate, it could hybridize with P-DNA and form the duplex of P-DNA/a1 with the G-quadruplex structure, performing the medium NMM fluorescence marked as “1”, (curve b), and so did the addition of b1 (input of “0/1” in logic gate), which formed the duplex of P-DNA/b1 and produced the NMM fluorescence marked as “1”, (curve d). When the addition of the mixture of a1 and a2 (input of “2/0” in logic gate), they all could hybridize with P-DNA, performing the strongest fluorescence of NMM marked as “2”, (curve c). For the same reason, the existence of b1 and b2 (input of “0/2” in logic gate) could also perform the strongest fluorescence of NMM marked as “2”, (curve e). When the existence of both a1 and b1 (input of “1/1” in logic gate), they had the priority to hybridization, leaving the GGGT sequence of a1 to create G-quadruplex structure with P-DNA and forming the NMM signal marked as “1”, (curve f). However, when the addition of b1 and the mixture of a1 and a2 (input of “2/1” in logic gate), a1 could hybridize with b1 and then forming the G-quadruplex structure with P-DNA, a2 could also hybridize with P-DNA and form the G-quadruplex structure, performing the strongest fluorescent of NMM marked as “2”, (curve h). For the same reason, the addition of a1 and the mixture of b1 and b2 (input of “1/2” in logic gate) could also perform the strongest fluorescent of NMM marked as “2”, (curve g). When the coexistence of the mixture of a1 and a2 and the mixture of b1 and b2, the input of “2/2” in logic gate, forming the highest NMM fluorescence marked as “2”, (curve i). The fluorescence responses of NMM at 607 nm were plotted


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as column bar in Figure 4B to define the output threshold values with an undefined range. The output threshold value was defined as “0” when the normalized fluorescence intensity was lower than 0.25. The threshold value “1” was defined when the normalized fluorescence intensity was higher than 0.3 and lower than 0.7. And then the output threshold value can be defined as “2” when the normalized fluorescence intensity is higher than 0.75.

Figure 5. (A) The ternary OR logic gate fluorescence emission response profiles of the curve a: no inputs, curve b: a1, curve c: a1+a2, curve d: b1, curve e: b1+b2, curve f: a1+b1, curve g: a1+(b1/b2), curve h: b1+(a1/a2), curve i: (a1/a2)+(b1/b2). (B) The electronic equivalent circuitry of the ternary OR logic systems. We have also applied PAGE and CD experiments to demonstrate the mechanism of the ternary OR logic system. The native PAGE was used to investigate the hybridization reaction of these input DNA strands of the ternary OR logic gate shown in Figure 6A. From Lane 1 to Lane 5, the belts showed the individual DNA strands of P-DNA, a1, the mixture of a1 and a2, b1, the mixture of b1 and b2 in sequence as control. Because of the similarity in a1 and a2, the belts locations were almost the same in Lane 3, and so did b1 and b2 in Lane 5. When the addition of P-DNA and a1



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to the system of ternary OR logic, they could hybridize with each other and form the duplex of P-DNA/a1, generating a new belt in Lane 6. It was much the same with the addition of P-DNA and b1 to the system and form the duplex of P-DNA/b1 in Lane 8. When the addition of P-DNA and the mixture of a1 and a2, a1 and a2 all could hybridize with P-DNA and form the duplex of P-DNA/a1 and P-DNA/a2 with two

Figure 6. (A) Polyacrylamide gel analysis of the ternary OR logic gate. Different DNA samples were added into lanes 1-13. Lane 1: P-DNA, (P) Lane 2: a1, Lane 3: a1+a2, Lane 4: b1, Lane 5: b1+b2, Lane 6: P+a1, Lane 7: P+a1+a2, Lane 8: P+b1, Lane 9: P+b1+b2, Lane 10: P+a1+b1, Lane 11: P+a1+b1+b2, Lane 12: P+ a1+a2+b1, Lane 13: P+a1+a2+b1+b2. (B) The circular dichroism of different DNA inputs. new belts locations in Lane 7. For this reason, two duplexes of P-DNA/b1 and P-DNA/b2 were also formed when the addition of P-DNA and the mixture of b1 and b2 to the system, forming two new belts in Lane 9. When the addition of P-DNA, a1 and b1 to the system, a1 and b1 had the priority to hybridization, leaving the GGGT sequence of a1 to create G-quadruplex structure with P-DNA and generating a new belt in Lane 10. Based on this reason, when the addition of P-DNA, a1 and the


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mixture of b1 and b2 to the system, another new belt was produced by the duplex of P-DNA/b2 in Lane 11. However, when the addition of P-DNA, b1 and the mixture of a1 and a2 to the system, another new belt was produced by the duplex of P-DNA/a2 in Lane 12. When the coexistence of P-DNA, the mixture of a1 and a2 and the mixture of b1 and b2 in the system, three new belts were produced as mentioned above in Lane 13. Moreover, the CD experiments were also performed to further confirm the formation of G-quadruplex structure in the implementation of ternary OR logic operation (Figure 6B). As shown in Figure 4B, the CD spectrum of P-DNA, a1, the mixture of a1 and a2, b1, the mixture of b1 and b2 were of relatively low amplitude (curve 1-5) in Tris-HCl buffer with GO, indicating random DNA structures. When adding a1 in P-DNA, two obvious peaks were found for the formation of G-quadruplex structure between a1 and P-DNA (curve 6). The addition of a1 and a2 into P-DNA could also form G-quadruplex structure (curve 7). When the addition of b1 or the mixture of b1 and b2 into P-DNA, the G-quadruplex structure could be also formed (curve 8 and 9). So when the addition of a1 and b1, a1 and the mixture of b1 and b2, b1 and the mixture of a1 and a2 or all the inputs DNA sequence to P-DNA, they all could form the G-quadruplex structure (curve 10, 11, 12 and 13). Each curve in Figure 6B was corresponding to PAGE in Figure 6A, which further demonstrated the feasibility of the ternary OR logic gate.

4. CONCLUSIONS In conclusion, a ternary INHIBIT logic gate and a ternary OR logic gate in an



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enzyme-free condition by integration of graphene oxide and DNA as a platform were successfully presented for the first time. By conjugate GO with P-DNA, combing with the hybridization of input DNA sequences, the construction of advanced ternary logic gates were simply realized in cost effective and highly repeatable way. Different from binary logic gate, the developed ternary logic gate has great potential to increase the number of meaningful states to bring about the higher information processing capability. It can be preconceived that such functions endow the multi-valued logic gate as a promising candidate for the development of biosensing and biomedical systems.

ASSOCIATED CONTENT Supporting Information Materials including sequences of the oligonucleotides used in this work were listed in the table. The optimization conditions of reacting materials and inputs were listed in figures.

AUTHOR INFORMATION Corresponding Authors *E.K.W.: e-mail, [email protected]. *S.J.D.: e-mail, [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes


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The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 211900040 and No. 21427811), the State Key Project of Ministry of Science and Technology of China (No. 2016YFA02032000 and No. 2013YQ170585).

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A

Unimolecular,

Emission-Mode,


Molecular


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