Biomacromolecular Logic Devices Based on Simultaneous

Aug 10, 2015 - A series of biomacromolecular logic gates and functional devices with cyclic voltammetric (CV) and electrochemiluminescence (ECL) respo...
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Biomacromolecular Logic Devices Based on Simultaneous Electrocatalytic and Electrochemiluminescence Responses of Ru(bpy)32+ at Molecularly Imprinted Polymer Film Electrodes Wenjing Lian,† Xue Yu,† Lei Wang,† and Hongyun Liu*,†,‡ †

College of Chemistry and ‡Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, PR China S Supporting Information *

ABSTRACT: A series of biomacromolecular logic gates and functional devices with cyclic voltammetric (CV) and electrochemiluminescence (ECL) responses as output signals were established on the basis of molecularly imprinted polymer (MIP) film electrodes. The MIP films were electropolymerized on the surface of Au electrodes with o-phenylenediamine (OPD) as the monomer and chloramphenicol (CP) as the template molecule. The simultaneous CV and ECL signals of Ru(bpy)32+ were significantly enhanced by the addition of natural DNA in solution at the MIP film electrodes after CP removal. The CV and ECL responses of the Ru(bpy)32+−DNA system at the MIP film electrodes were greatly influenced by CP removal and rebinding. Moreover, the addition of ferrocene methanol (FcMeOH) to the solution led to substantial quenching of the ECL signal and produced a new CV peak pair. On the basis of these results, 3-input/3-output and 3-input/5-output biomacromolecular logic gate systems were established with DNA, CP, and FcMeOH as inputs and the ECL responses at different levels or CV responses at different potentials as outputs. After an elaborate reconfiguration of inputs and outputs, the functional non-Boolean logic devices such as a 2-to-1 encoder, a 1-to-2 decoder, and a 1-to-2 demultiplexer were also constructed on the same platform.

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intensities with two thresholds.26 This obviously limits the further increase of the number of outputs. Famulok and coworkers reported a 3-output biomolecular logic gate system where the UV−vis absorbance at 652 nm and fluorescence intensities at 456 and 586 nm were used as the 3 outputs.27 However, the UV−vis and fluorescence signals had to be obtained separately using different instruments, which made the performance of the logic gate less convenient. Willner and coworkers developed a series of DNA logic gates with 3 outputs,28 where a specifically synthesized DNA was used and the same type of readout, i.e., the fluorescence responses at three different wavelengths, were defined as outputs. Thus, to implement more complex calculations and improve the functionality of biocomputing, the development of logic gates with different types of signals as multiple outputs obtained simultaneously is urgently needed. Electrochemiluminescence (ECL) is the process in which luminescence or light is emitted during electrochemical reactions in solutions, and it is usually accompanied by electron transfer reactions at electrodes.29 Thus, CV and ECL signals can be obtained simultaneously using the same instrument. Although ECL was already used as an output signal in the

INTRODUCTION Biomolecular computing or biocomputing is a type of chemical or molecular computing1,2 that usually employs biomacromolecules such as DNA and enzymes to process information through chemical or biochemical methods.3−8 In recent years, biocomputing has aroused great interest among researchers. Different biomacromolecular logic gate systems and various biomolecular non-Boolean devices such as encoder/decoders and multiplexers/demultiplexers have been developed.9−13 Biocomputing has also been applied in the fields of medical diagnostics,14,15 biosensing,16 drug delivery,17 and molecular recognition18 and has demonstrated great potential. In the research on biomacromolecular logic gates, one great challenge is to increase the complexity or dimensions of the systems so that more complicated tasks or functions can be accomplished. In this respect, the increase in the number of outputs is more difficult than that of inputs and thus becomes more challenging. Up to now, most of biochemical logic systems have only 1 or 2 outputs,19−25 and just a few reports have investigated logic gates with 3 or more outputs.26−28 For example, in our previous work, a 3-output logic gate system was constructed on the basis of the bioelectrocatalysis of glucose by glucose oxidase.26 However, these 3 output signals belonged to the same type of readout, i.e., cyclic voltammetric (CV) peak current, and their realization was just dependent on dividing the CV peak responses into 3 outputs according to their different © XXXX American Chemical Society

Received: July 6, 2015 Revised: August 10, 2015

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Figure 1. (A) Continuous CVs of 10 mM OPD at Au electrode at 0.05 V s−1 in a pH 5.2 buffer solution containing 10 mM CP. (B) FTIR spectra of OPD, POPD, CP, and POPD-CP MIP samples.

development of DNA logic gates,30 a logic gate system with simultaneous CV and ECL responses as two different types of output signals has been rarely reported.31 Recently, molecularly imprinted polymer (MIP) has received increasing interest among researchers in analytical chemistry and other areas, mainly because the specific recognition sites for the template molecule are formed in the MIP matrix, and excellent selectivity toward the target template is realized.32 Although the combination of MIP with CV and ECL in the realization of detection of various substances has been reported,33,34 to the best of our knowledge, no report has been made on the establishment of a logic gate system with MIP until now. In the present work, a series of biomacromolecular logic gates with multiple outputs based on simultaneous CV and ECL responses at MIP film electrodes were established. The MIP films were formed on the surface of Au electrodes by electropolymerization with o-phenylenediamine (OPD) as the monomer and chloramphenicol (CP) as the template. Herein, CP is a broad-spectrum antibiotic and is widely used for the treatment of infections in humans and animals such as salmonellosis and typhoid fever,35 and OPD is a common functional monomer in electropolymerization.36 Tris(2,2′bipyridine)ruthenium(II) (Ru(bpy)32+) in solution was used not only as an electroactive probe but also an ECL reagent.37 When double-stranded natural DNA was added to the Ru(bpy)32+ solution, enhanced CV and ECL peaks at approximately 1.15 V vs SCE were simultaneously observed at the MIP film electrodes after CP removal. Meanwhile, ferrocene methanol (FcMeOH) functioned as both the electroactive probe and the ECL quencher in the system.38−40 Thus, with DNA, CP, and FcMeOH as the three inputs and the corresponding CV and ECL peak responses as multiple outputs, the 3-input/3-output and 3-input/5-output logic gates were established. Furthermore, some non-Boolean logic devices, such as an encoder, a decoder, and a demultiplexer, were also constructed on the basis of the same system with elaborate designs. To the best of our knowledge, this work is the first report on a logic gate system with simultaneous CV and ECL signals as multiple outputs. Moreover, all of these logic gates and devices were constructed on the same platform on the basis of MIP film electrodes. This may open a new approach for designing and constructing more complicated biomacromolecular logic gate systems and devices.

ferrocenemethanol (FcMeOH) were obtained from SigmaAldrich. Chloramphenicol (CP) and o-phenylenediamine (OPD) were purchased from Aladdin. All other reagents were of analytical grade and used as received. Solutions were prepared with ultrapure water provided by a Millipore water purification system (18.2 MΩ cm). A 0.1 M phosphate buffer at pH 7.0 was used as the CV and ECL detection solution. Instruments. The simultaneous CV and ECL experiments were carried out using an MPI-E electrochemiluminescence analyzer system (Xi’An Remax Science & Technology) with the voltage of the photomultiplier tube set at 600 V. For some CV experiments such as electropolymerization, a three-electrode system was used with an Ag/AgCl electrode in a saturated KCl solution as the reference, a platinum wire as the counter, and an Au disk electrode (diameter 2 mm) with films as the working electrode. Fourier transform infrared spectra (FTIR) were monitored by a 380 FTIR spectrophotometer (Nicolet) at a resolution of 4 cm−1. Scanning electron microscopy (SEM) was carried out using an S-4800 scanning electron microscope (Hitachi) to characterize the surface morphology of POPD-CP MIP films. Thin platinum films were coated on the sample surface with an E-1045 sputtering coater (Hitachi) before SEM measurements. Preparation of POPD-CP MIP Film Electrodes. POPDCP MIP films were constructed on the electrode surfaces by electropolymerization. Prior to electrodeposition, the Au electrodes were sequentially polished with 1.0, 0.5, and 0.05 μm γ-alumina on chamois leather to obtain a mirrorlike surface, followed by cleaning in ethanol and water for 5 min respectively in an ultrasonic bath. After consulting previous research41,42 and further optimization, the electropolymerization of MIP films was carried out by 30 cycles of CV at Au electrodes between 0 and 0.8 V at 50 mV s−1 in 0.1 M acetate buffer solutions at pH 5.2 containing 10 mM CP template and 10 mM OPD monomer. After being dried at room temperature for 10 min, CP-free MIP films were prepared by placing the MIP film electrodes in 20 mL of methanol/acetic acid (9:1, v/v) solutions for 20 min with magnetic stirring so that the CP molecules previously entrapped in the MIP matrix could be removed. After removal of CP, the CP-free MIP film electrodes were rinsed with water and dried at room temperature. The CP-rebinding MIP film electrodes were formed by incubation of the CP-free MIP film electrodes in the CP solution at different concentrations for 15 min, followed by water rinsing and room temperature drying. The nonmolecularly imprinted polymer (NIP) films were prepared with the same method as the MIP films except for the absence of the CP template in the electropolymerization solution.

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EXPERIMENTAL SECTION Chemicals. Tris(2,2′-bipyridine)ruthenium(II) chloride hexahydrate (Ru(bpy)3Cl2·6H2O), salmon testes doublestranded DNA (41.2% GC content, ∼ 2000 bp), and B

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RESULTS AND DISCUSSION Electropolymerization of POPD-CP MIP Films on Electrode Surface. The electrodeposition of poly(o-phenylenediamine) (POPD)-containing CP (POPD-CP) was carried out by CV in the presence of CP template and OPD monomer in pH 5.2 buffer solutions. A typical CV record is shown in Figure 1A. The anodic peak current at approximately 0.55 V for the irreversible OPD oxidation43 was observed in the first CV cycle. The peak current then decreased gradually during subsequent cycles until its complete disappearance after 30 cycles. These results suggest that the POPD polymer films with entrapped CP are formed on the Au electrode surface and the compact and insulated films block the electroactive OPD monomer from accessing the electrode surface. To further confirm the formation of MIP films, FTIR analyses of OPD, POPD, CP, and POPD-CP MIP samples were performed (Figure 1B), and the attributions of the characteristic peaks of these samples are summarized in Table S1 according to the literature.44,45 The characteristic IR vibration bands of the phenazine rings (νphenazine) at 1409 cm−1 and those of the C−N−C stretching (νC−N−C) in the benzenoid units at 1111 cm−1 for POPD46 were also observed for POPD-CP MIP but not for OPD samples, indicating that the OPD monomer is electropolymerized into the POPD polymer in electrodeposition. In addition, the characteristic IR vibration bands of the CO stretching (νCO) at 1687 cm−1 and the −NO2 symmetrical stretching (νs −NO2)44 at 1347 cm−1 for the CP sample were also observed in the MIP samples, illustrating that the template CP is entrapped into the polymer films. SEM top-view images showed that the surface morphology of POPD-CP MIP films was obviously different from that of bare Au electrodes (Figure S1). All of these results indicate that the POPD-CP MIP films are successfully electrodeposited on the electrode surface. Recognition of POPD-CP MIP Films toward CP. Ru(bpy)32+ was used as the electroactive probe to investigate the recognition ability of POPD-CP MIP films toward CP by simultaneous CV and ECL. With the bare Au electrodes, Ru(bpy)32+ showed a pair of CV redox peaks at approximately 1.15 V (Figure 2A, curve a), characteristic of the Ru(III)/ Ru(II) redox couple.37,47,48 With the POPD-CP MIP film electrodes, however, the peaks of Ru(bpy)32+ could hardly be

observed (Figure 2A, curve b) because the diffusion of Ru(bpy)32+ to the electrode surface is seriously hindered by the compact MIP films. After the POPD-CP MIP film electrodes were placed in methanol/acetic acid solutions for 20 min with magnetic stirring, the CP molecules previously entrapped in the MIP films were removed into the solution, forming CP-free MIP films. When the CP-free film electrodes were transferred into the Ru(bpy)32+ solutions, the CV redox peaks at 1.15 V were observed again (Figure 2A, curve c). This is because the removal of the CP molecules from MIP films results in many cavities or holes in the films, and the more porous films allow the probe to pass through the films and then transfer electrons with the electrode more easily. After the CPfree MIP films were incubated in 50 μM CP solution for 15 min, the CP-rebinding MIP films were formed. At the CPrebinding MIP film electrodes, the CV peaks of Ru(bpy)32+ at 1.15 V were significantly suppressed (Figure 2A, curve d) because of the refilling of the recognition cavities by CP molecules. The decrease in CV oxidation peak current (Ipa) at 1.15 V for the probe at CP-rebinding MIP film electrodes in comparison with that at CP-free MIP film electrodes was dependent on the concentration of CP (CCP) in the rebinding solution (Figure S2). These results indicate that the CP-free MIP films have a specific recognition function toward CP molecules, similar to the other MIP films reported previously.49,50 As the most frequently used ECL reagent, Ru(bpy)32+ showed an obvious ECL peak at approximately 1.15 V when the potential was scanned at the bare Au electrodes (Figure 2B, curve a). This phenomenon was observed previously and was attributed to the interaction of Ru(bpy)33+ with the hydroxyl species that had formed on the electrode surface.51,52 The ECL peak signals at POPD-CP MIP, CP-free MIP, and CP-rebinding MIP film electrodes as well as at bare Au electrodes (Figure 2B) demonstrated the same trend as the CV peaks at 1.15 V with the same reason (Figure 2A). In contrast, at the NIP film electrodes, Ru(bpy) 3 2+ demonstrated very small CV and ECL responses with the same removal and rebinding steps, much smaller than those at bare Au electrodes (Figure S3). This is understandable because there is no template CP molecule within the NIP films, and the removal and rebinding steps will not affect the structure of the NIP films. DNA-Sensitive ECL-Switching Behavior for CP-free MIP Films in Ru(bpy)32+ Solution. For CP-free MIP film electrodes, after the addition of 0.3 mg mL −1 DNA (optimization experiments, Figure S4) to the pH 7.0 buffer containing Ru(bpy)32+, the CV oxidation peak at approximately 1.15 V increased slightly (Figure 3A, curves a and b), whereas the corresponding ECL peak increased significantly by more than five times (Figure 3B, curves a and b). All of these results indicate that DNA in the solution can enhance both the CV oxidation peak current and the ECL peak signal of Ru(bpy)32+, and the mechanism may be expressed by the following equations:53,54

Figure 2. Simultaneous (A) CV and (B) ECL response curves of 0.5 mM Ru(bpy)32+ at 0.05 V s−1 in pH 7.0 buffers at (a) bare Au electrodes, (b) POPD-CP MIP film electrodes, (c) CP-free MIP film electrodes, and (d) CP-rebinding MIP film electrodes after 50 μM CP rebinding.

Ru(bpy)32 + ⇄ Ru(bpy)33 + + e− at electrode

(1)

Ru(bpy)33 + + DNA‐G → Ru(bpy)32 + + DNA‐GOx

(2)

Ru(bpy)33 + + DNA‐GOx → Ru(bpy)32 +* + DNA‐G2Ox (3) C

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and the CV Ipa at CP-rebinding MIP as the off state, the CPsensitive CV on−off behavior of the system was observed and could be reversibly repeated at least 6 cycles without obvious change of Ipa at the on state by switching the MIP film electrode between CP removal and rebinding (Figure S6A). The ECL peak signals of the system were also sensitive to the CP rebinding for the CP-free MIP films and showed similar on−off behavior to CV (Figure 3B, curves b and c). The CPsensitive ECL-switching behavior of the system could be reversibly repeated many times with good reproducibility (relative standard deviation of ECL intensity at the on state < 4%) by switching the MIP film electrode between CP removal and rebinding (Figure S6B). Quenching Effect of FcMeOH on ECL Signals of Ru(bpy)32+−DNA System. FcMeOH is an electroactive probe and also an efficient quencher for the ECL signal of Ru(bpy)32+ in solution38−40 and was thus used in the present work. After the addition of 1 mM FcMeOH to the buffer solution containing Ru(bpy)32+ and DNA, a new pair of CV quasi-reversible peaks appeared at approximately 0.35 V at the CP-free MIP film electrodes (Figure 4A, curve b), which is

Figure 3. Simultaneous (A) CV and (B) ECL response curves of 0.5 mM Ru(bpy)32+ at 0.05 V s−1 at CP-free MIP film electrode in pH 7.0 buffer solutions containing (a) 0 and (b) 0.3 mg mL−1 DNA, and (c) CP-rebinding MIP film electrode after 50 μM CP rebinding.

Ru(bpy)32 + * ⇄ Ru(bpy)32 + + hν (610 nm)

(4)

First, the electrochemical oxidation of Ru(bpy)32+ occurs at 1.15 V at the electrode, producing Ru(bpy)33+ (eq 1). Ru(bpy)33+ is then reduced back to Ru(bpy)32+ by the guanine groups in the DNA (DNA-G, eq 2), thus forming an electrocatalytic cycle. Ru(bpy)33+ may also be further reduced by the single-electron oxidation product of DNA guanine groups (DNA-GOx) and become its excited state Ru(bpy)32+* (eq 3). When the Ru(bpy)32+* decays to its ground-state Ru(bpy)32+, it simultaneously luminesces at 610 nm (eq 4), leading to an ECL catalytic cycle. Thus, the addition of DNA into the Ru(bpy)32+ solution increases not only the CV oxidation peak but also the ECL peak at the CP-free MIP film electrodes. Considering that the magnification effect of DNA for ECL was much more significant than that for CV, the ECL peak response of the Ru(bpy)32+-MIP system was used to investigate the DNA-sensitive switching behavior. By defining the ECL peak intensity of Ru(bpy)32+ at the CPfree MIP film electrodes in the presence of 0.3 mg mL−1 DNA as the “on” state and the peak intensity in the absence of DNA as the “off” state, the DNA-sensitive on−off behavior of the system was observed and could be repeated many times by switching the CP-free MIP film electrode in the Ru(bpy)32+ solutions from with to without DNA (Figure S5). This suggests that the ECL-switching property of the system is reversible. The system was also very stable. Whenever the system was at the on state during the repeated tests, the ECL peak intensity remained at almost the same high level. CP-Sensitive CV and ECL-Switching Behaviors for MIP Films in Ru(bpy)32+−DNA Solutions. Considering that the CP concentration in CP-rebinding solutions had a great effect on the CV signals of Ru(bpy)32+ at the CP-rebinding MIP film electrodes (Figure S2), 50 μM CP was used herein as a stimulus to switch the CV oxidation peak signals for the Ru(bpy)32+−DNA system. After rebinding 50 μM CP, the CVIpa of the system at the CP-rebinding MIP film electrodes was much smaller than that at the CP-free MIP film electrodes (Figure 3A, curves b and c). This is because most of the vacant recognition sites in the CP-free MIP films are occupied again by CP molecules after the CP rebinding, making the diffusion of Ru(bpy)32+ through the films and its approach to the electrode surface very difficult. By defining the CV Ipa of Ru(bpy)32+− DNA system at the CP-free MIP film electrodes as the on state

Figure 4. Simultaneous (A) CV and (B) ECL response curves of 0.5 mM Ru(bpy)32+ in pH 7.0 buffers containing 0.3 mg mL−1 DNA at 0.05 V s−1 at CP-free MIP film electrode (a) without and (b) with 1 mM FcMeOH and (c) CP-rebinding MIP film electrode after 50 μM CP rebinding in the solution of b.

attributed to the ferricinum/ferrocene redox couple (eq 5).55 However, no obvious change was observed for the CV oxidation peak of Ru(bpy)32+ at 1.15 V (Figure 4A, curves a and b). Meanwhile, the ECL peak intensity decreased dramatically (Figure 4B, curve b) because of the ECL quenching effect of FcMeOH eq 6.38−40 FcMeOH ⇄ FcMeOH+ + e− at electrode

(5)

FcMeOH+ + Ru(bpy)32 +* → FcMeOH + Ru(bpy)33 + (6)

FcMeOH was first oxidized to FcMeOH at the electrodes (eq 5), and FcMeOH+ was then reacted with Ru(bpy)32+* (eq 6). In this case, the excited state Ru(bpy)32+* produced in eq 3 was consumed and could not follow eq 4 to give ECL signal. After optimization (Figure S7), 1 mM FcMeOH was used as a stimulus in the FcMeOH-sensitive CV and ECL on−off experiments for the system. By switching the CP-free MIP film electrode in the Ru(bpy)32+−DNA solutions between with and without FcMeOH, the reversible CV and ECL on−off behaviors were observed (Figure S8). +

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Figure 5. (A) Truth table and (B) logic circuit for the 3-input/3-output logic gate system.

3-Input/3-Output Logic Gate Based on CV and ECL Responses Controlled by DNA, CP, and FcMeOH. According to the above results, a 3-input/3-output logic gate was constructed by defining DNA, CP, and FcMeOH as inputs A, B, and C, respectively. For Input A, the presence and absence of 0.3 mg mL−1 DNA in solution were defined as the 1 and 0 states, respectively. For Input B, CP-free and 50 μM CPrebinding MIP films were considered to be the 1 and 0 states, respectively. For Input C, the presence of 1 mM FcMeOH was considered the 1 state, and the absence of FcMeOH was the 0 state. The ECL peak signal and CV Ipa at 1.15 and 0.35 V for the system were defined as outputs E1, I1, and I2, respectively. For output E1, an ECL peak intensity greater than or equal to 300 was considered the 1 state, and that less than 300 was considered the 0 state, with 300 as the threshold. For output I1, a CV Ipa at 1.15 V greater than or equal to 2 μA was defined as 1; otherwise, it was 0 with 2 μA as the threshold. For output I2, a CV Ipa at 1.15 V greater than or equal to 1 μA was considered to be 1 state; otherwise, it was 0 with 1 μA as the threshold. Eight different combinations of the 3 inputs and the corresponding outputs are shown in Figure S9. For output E1, only the input combination (1,1,0) led to the 1 state (Figure S9A) because the presence of DNA significantly enhanced the ECL peak intensity of the system at the CP-free MIP film electrodes in the absence of FcMeOH. For output I1, input B (CP) played a key role, but input A (DNA) and input C (FcMeOH) had very small influences on the CV Ipa of Ru(bpy)32+ at 1.15 V. Whenever input B was at the 0 state (CPrebinding MIP), output I1 would be at the 0 state (Figure S9B). For output I2, only two input combinations, (0,1,1) and (1,1,1), resulted in the 1 state (Figure S9C) because the presence of FcMeOH and use of the CP-free MIP film electrodes are the two prerequisites of the observation of the FcMeOH redox peaks at 0.35 V, and these peaks have nothing to do with DNA. According to these results, a truth table was established (Figure 5A), and the corresponding 3-input/3-output logic gate system was constructed (Figure 5B), which is a combination of AND, INHIBIT, and YES gates.56 2-to-1 Encoder, 1-to-2 Decoder, and 1-to-2 Demultiplexer Based on ECL and CV Signals for the System. On the basis of the same system, a simple encoder and decoder could be established. Herein, an encoder is considered to be a non-Boolean operation or device that converts data from one format to another for the purpose of compression.57 For example, a 2-to-1 encoder can compress 2 input data into 1 output datum. A decoder is a device that does the reverse operation of an encoder.57 Thus, a 1-to-2 decoder can convert 1 input datum to 2 output data. In the present system, to construct the 2-to-1 encoder, CP (input B) and FcMeOH (input C) were chosen as the two inputs, and the ECL peak signal was used as the single output

(output E1). When the input combination was (1,0), output E1 was 1 because of the enhanced ECL peak intensity for the Ru(bpy)32+−DNA system at the CP-free MIP film electrodes in the absence of FcMeOH. However, if the input combination was (0,1), then output E1 was at the 0 state. Namely, with the CP-rebinding MIP films and by the addition of FcMeOH to Ru(bpy)32+−DNA solution, the corresponding ECL peak of the system became very small because of both the inhibition effect of the CP rebinding and the quenching effect of FcMeOH. This simple 2-to-1 encoder was thus constructed and defined by the truth table (Figure 6A). Herein, it must be understood that for all of the nonexplicitly defined input combinations such as (0,0) and (1,1) the corresponding outputs are treated as irrelevant.57

Figure 6. (a) Truth tables and (b) schematic representations of (A) 2to-1 encoder with CP and FcMeOH as two inputs and ECL peak intensity as the output and (B) 1-to-2 decoder with FcMeOH as the input and ECL peak intensity and CV Ipa at 0.35 V as two outputs.

To construct the 1-to-2 decoder, FcMeOH (input C) was selected as the sole input, and the ECL peak intensity (output E1) and the CV Ipa at 0.35 V (output I2) were chosen as the two outputs. When input C was at the 1 state, output E1 was 0 because the ECL signal was greatly quenched by FcMeOH. In the meantime, output I2 was 1 because of the CV response of FcMeOH at 0.35 V. If input C was at the 0 state, then output E1 was 1 because the ECL peak signal remained at the high level in the absence of FcMeOH. Meanwhile, output I2 was 0 because with no addition of FcMeOH in the testing solution there was no CV response at 0.35 V. The truth table of the 1to-2 decoder and the corresponding schematic representation are presented in Figure 6B. In electronics, a demultiplexer is a non-Boolean device that takes a single or small number of input signals and switches them between multiple output signals.58,59 For example, the operation of forwarding 1 input signal into 2 different output channels can be accomplished by a 1-to-2 demultiplexer. Unlike the decoder, however, the demultiplexer needs the additional address input that selects either output channel to be used to take the input signal.58,59 The present Ru(bpy)32+−DNA system could also be used to develop a 1-to-2 demultiplexer using CP as the input (input B) and FcMeOH as address input E

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FcMeOH. All other six input combinations resulted in the 1 state of output E3 (ECL ≤ 100). This is understandable because either the recognition cavities in CP-free MIP films are filled by CP molecules after CP rebinding, which inhibits the Ru(bpy)32+ in solution from diffusing to the electrode surface, or the ECL signals of Ru(bpy)32+ are quenched by the addition of FcMeOH regardless of whether DNA exists in solution. The increase in the number of outputs from 3 to 5 greatly increased the function of the logic gate system, and the more complex logic gate network with 3-input/5-output was developed (Figure 8A), which is a combination of AND, INHIBIT, IMPLICATION, and YES gates.56 The truth table of the system is also presented in Figure 8B.

with output E1 and I2 as the two outputs (Figure 7). When address input was set at the 0 state (without FcMeOH), the

Figure 7. (A) Truth table, (B) logic circuit, and (C) equivalent switching device for the 1-to-2 demultiplexer with CP as the input, FcMeOH as the address input, and ECL peak intensity and CV Ipa at 0.35 V as two outputs.

binary state of input B was transmitted directly to output E1. That is, the logic value 1 of input B (CP-free MIP) would lead to the 1 state of output E1 because the high ECL peak intensity for the Ru(bpy)32+−DNA system could be observed at the CPfree MIP film electrodes in the absence of FcMeOH, and input B 0 (CP-rebinding MIP) would result in output E1 0 because of the inhibiting effect of CP rebinding. Conversely, if the address input was switched to the 1 state (with FcMeOH), then the binary data of input B were directed to output I2. Namely, the logic value 1 of input B (CP-free MIP) would lead to the 1 state of output I2 because of the large CV response of FcMeOH at 0.35 V, and the 0 state of input B (CP-rebinding MIP) would result in output I2 0 because of the inhibiting effect of CP rebinding. The truth table, the logic circuit, and the diagram of the equivalent switching device for the 1-to-2 demultiplexer are shown in Figure 7. 3-Input/5-Output Logic Gate Based on CV and ECL Responses Controlled by DNA, CP, and FcMeOH. To further increase the dimension and complexity of the 3-input/ 3-output logic gate system (Figure 5), the ECL peak responses of the system could be divided into 3 outputs according to their different magnitudes with 2 thresholds (100 and 300) (Figure S10). When the ECL response was greater than or equal to 300, output E1 was defined as the 1 state; otherwise, it was at the 0 state. When the ECL response was between 100 and 300, output E2 was considered to be 1; otherwise, it was 0. If the ECL response was less than or equal to 100, then output E3 was at the 1 state; otherwise, it was 0. Other inputs and outputs were defined as before. Thus, a 3-input/5-output logic gate system was established on the basis of the same platform. Eight possible combinations of the three inputs and the corresponding outputs are shown in Figure S10. Only input combination (1,1,0) led to the 1 state of output E1 (ECL ≥ 300) because the addition of DNA to the Ru(bpy)32+ solution could greatly increase the ECL peak intensity at the CP-free MIP film electrodes in the absence of FcMeOH. Input combination (0,1,0) exclusively resulted in the 1 state of output E2 (100 < ECL < 300) because although the absence of DNA in the solution would not lead to the increase in the ECL response of Ru(bpy)32+ the ECL peak could still be kept at a relatively high level at the CP-free MIP film electrodes in the absence of

Figure 8. (A) Equivalent electronic circuit and (B) truth table for the 3-input/5-output logic gate with DNA, CP, and FcMeOH as inputs and ECL peak intensities, CV Ipa at 1.15 V, and CV Ipa at 0.35 V as outputs.

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CONCLUSIONS In this work, POPD-CP MIP films were electropolymerized on Au electrodes, and the addition of natural DNA to a Ru(bpy)32+ solution greatly increased the CV and ECL peak responses at 1.15 V at CP-free MIP electrodes. Meanwhile, the addition of FcMeOH to the testing solution would substantially quench the ECL signal and demonstrate a new CV peak pair at 0.35 V. On the basis of this system, the biomacromolecular logic gates of 3-input/3-output and 3-input/5-output were constructed. Because the ECL and CV responses of the system could be obtained simultaneously with one simple instrument, the number of outputs and the corresponding complexity of the logic gates were unprecedentedly increased. In addition, some non-Boolean logic devices such as a 2-to-1 encoder, a 1-to-2 decoder, and a 1-to-2 demultiplexer were also established on the basis of the same system, illustrating that by careful and elaborate design different logic functions could be implemented on the same platform with a single interface. Another F

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The Journal of Physical Chemistry C distinguishing feature of the work was the first introduction of MIP into the construction of biomacromolecular logic gates and devices. Although the current work is just at the proof-ofprinciple stage, it does reflect important progress in biocomputing. This study not only provides a new strategy for the development of biomolecular logic gates and devices but also may become a foundation for the establishment of more complicated biomacromolecular computing devices. In addition, the present system can be easily extended. For example, other monomers rather than OPD and other template molecules instead of CP may be used for the construction of MIP films. Moreover, other ECL quenchers rather than FcMeOH, coreactants of Ru(bpy)32+ such as tripropylamine, and DNA damage reagents such as methylmethanesulfonate may also be selected as the inputs. All these demonstrate the generality and flexibility of the present system.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.5b06456. Assignment of characteristic IR absorption peaks of OPD, POPD, MIP and CP samples, SEM images of the surface of bare Au electrode and MIP film electrode, CV Ipa of Ru(bpy)32+ at CP-free MIP film electrode after rebinding CP at different concentrations, simultaneous CV and ECL response of Ru(bpy)32+ at various modified electrodes, dependence of the ECL response of Ru(bpy)32+ at CP-free MIP film electrode on the solutions with and without DNA, dependence of CV Ipa at 1.15 V and ECL peak intensity of Ru(bpy)32+ on the DNA concentration, dependence of CV Ipa at 1.15 V and ECL peak intensity of Ru(bpy)32+−DNA on the CP-sensitive switching behavior between CP removal and CP rebinding, ECL peak intensity of Ru(bpy)32+−DNA at CP-free MIP film electrodes with different concentrations of FcMeOH, dependence of CV Ipa at 0.35 V and ECL peak response of Ru(bpy)32+−DNA at CP-free MIP film electrodes on the FcMeOH-sensitive switching behavior, ECL peak intensity, CV Ipa at 1.15 and 0.35 V for different combinations of the three inputs of DNA, CP, and FcMeOH, and ECL peak intensity for different combinations of DNA, CP, and FcMeOH inputs with two thresholds. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 10 5880 7843. Fax: +86 10 5880 2075. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support from the National Natural Science Foundation of China (NSFC 21105004), the Major Research Plan of NSFC (21233003), and the Fundamental Research Funds for the Central Universities is gratefully acknowledged.

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