Boolean Logic Tree of Label-Free Dual-Signal Electrochemical

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Boolean Logic Tree of Label-Free Dual-Signal Electrochemical Aptasensor System for Biosensing, Three-State Logic Computation, and Keypad Lock Security Operation Jiao Yang Lu, Xin Xing Zhang, Wei Tao Huang, Qiu Yan Zhu, Xuezhi Ding, Liqiu Xia, Hong Qun Luo, and Nian Bing Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01498 • Publication Date (Web): 15 Aug 2017 Downloaded from http://pubs.acs.org on August 16, 2017

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Analytical Chemistry

Boolean Logic Tree of Label-Free Dual-Signal Electrochemical Aptasensor System for Biosensing, Three-State Logic Computation, and Keypad Lock Security Operation Jiao Yang Lua,#, Xin Xing Zhanga,#, Wei Tao Huanga,*, Qiu Yan Zhua, Xue Zhi Dinga, Li Qiu Xiaa, Hong Qun Luob, and Nian Bing Lib,* a

State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science,

Hunan Normal University, Changsha 410081, P. R. China b

Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education),

School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R. China *Corresponding author: E-mail: [email protected]. Fax: (+86)731-8887-2905; Tel: (+86)731-8887-2905 E-mail: [email protected]. Fax: (+86)23-6825-3237; Tel: (+86)23-6825-3237

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ABSTRACT The most serious and yet unsolved problems of molecular logic computing, consist in how to connect molecular events in complex systems into a usable device with specific functions and how to selectively control branchy logic processes from the cascading logic systems. This report demonstrates that Boolean logic tree is utilized to organize and connect “plug and play” chemical events DNA, nanomaterials, organic dye, biomolecule, and denaturant for developing the dual-signal electrochemical evolution aptasensor system with good resettability for amplification detection of thrombin, controllable and selectable three-state logic computation and keypad lock security operation. The aptasensor system combines the merits of DNA-functionalized nano-amplification architecture and simple dual-signal electroactive dye brilliant cresyl blue for sensitive and selective detection of thrombin with a wide linear response range of 0.02–100 nM and a detection limit of 1.92 pM. By using these aforementioned chemical events as inputs and the differential pulse voltammetry current changes at different voltages as dual outputs, a resettable 3-input biomolecular keypad lock based on sequential logic is established. Moreover, the first example of controllable and selectable three-state molecular logic computation with active-high and active-low logic functions can be implemented and allows the output ports to assume a high impediment or nothing (Z) state in addition to the 0 and 1 logic levels, effectively controlling subsequent branchy logic computation processes. Our approach is helpful in developing the advanced controllable and selectable logic computing and sensing system in large-scale integration circuits for application in biomedical engineering, intelligent sensing and control.


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INTRODUCTION Life is the highest and most complex expression of chemistry which is the science of matter and of its transformations. The power and mystery of life itself is founded on a much longer-term evolution of the matter in a natural logic manner. By learning and using this natural logic, molecular information processing1 in molecular interactions and complex biological processes have been developed for Boolean logic computations,2,3 neural network computation,4 and artificial intelligence systems.5-7 By utilizing biochemical molecules (nucleic acids,8,9 enzymes,3 and organic molecules10-12) and engineered biological units (such as living cell)13-15 in complex chemical or life system as building blocks, ongoing efforts within molecular information technology have been directed toward the constructing and designing of novel artificial intelligent sensing and computing devices6,16 with information encoding,17 encryption,18 even fuzzy search19 functions. To a certain extent, the complexity of computing circuitries in biochemical systems was achieved by the self-organizing interactions of the inputs with libraries of molecular events that provide “plug and play” computing modules, by cascading logic gates,20 and by branching logic computation processes and adapting thresholds.4 Nonetheless, such efforts are limited by the difficulties arising from lack of effective strategies for connecting molecular events in complex molecular systems into an organic whole with some specific functions.19 Furthermore, it is very challenging for the cascading molecular logic computing systems to selectively control and/or effectively remove the outputs of branchy logic computation processes from the circuits. In recent years, an intense interest has grown in combinations based on interactions between versatile biomolecules (nucleic acids, antibodies, enzymes) and nanomaterials in solution.21 These 3 ACS Paragon Plus Environment


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combinations provide excellent background for various multifunctional logic programmable devices application and development via organizing and connecting various molecular events on nanomaterials surface. The solution-based systems are favorable for mixing and interacting of biochemical events, but easy to result in the accumulation of inputs/outputs and inaccuracy of logic operations. With the help of strengths of solid-phase substrate in easy solid-liquid separation and eliminating invalid inputs and verbose outputs, the versatility and universality of solid-based platform gives a favorable opportunity to realize sensing and computing integration for effectively organizing and regulating logic functions and processes.22 Many solid-based electrochemical sensing systems combined biomolecules (such as DNA, RNA, peptides, enzymes) as functional connecting “wires” with nanomaterials (such as gold nanoparticles (AuNPs), graphene, quantum dots) as nano-amplification23 to modify and expand surface of the electrodes for applications in biosensing,24 biofuel cell,25 and logic gate operations. Because the labeled biomolecules were expensive, and one redox label (ferrocene or methylene blue) could only be tagged to one biomolecule, leading to a small electrochemical signal and bioaffinity reduction.26 Label-free electrochemical sensing systems based on various amplification strategies27 have attracted increasing attention from researchers. However, most of them have possessed just a single response. The increase of the number of the signal outputs, as well as inputs, can not only increase sensitivity, but also give them significant potential for enhancing computational complexity and anti-interference ability of logic computing. Up to now, only a few works have reported the solid-based label-free electrochemical sensing systems with dual outputs.28,29 4 ACS Paragon Plus Environment

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Event tree analysis using Boolean logic to combine a series of lower-level events30 may be an effective strategy for connecting complex molecular systems into an organic whole. Moreover, it was suggested that three-state logic computation31 assuming a high impediment or nothing (Z) state in addition to the 0 and 1 logic levels could be a potential and efficient paradigm for selectively controlling and/or removing the inputs or outputs of logic computing systems. In the present study, the Boolean logic tree19 of complex chemical evolution systems on solid-based platform is demonstrated and applied to organizing and connecting “plug and play” chemical events (DNA “wires”, nanoparticle amplification supports, dual-output electrochemical dye, biomolecule “winder”, and denaturant) for developing the label-free dual-signal electrochemical evolution aptasensor system. Solid-based reactions based on these aforementioned chemical events are programmed for dual-signal amplification detection of thrombin, controllable and selectable three-state logic computation and resettable keypad lock security operation. As far as we are aware, the three-state molecular logic computation has never been demonstrated, and this study adds a new dimension to expanding molecular logic computing and designing controllable and selectable logic computing. The present report will provide more opportunities for output enable of the complex biochemical system for the development and design of intelligent molecular computing and sensing applications, such as biosensing, medical diagnosis, intelligent sensing and control.

EXPERIMENTAL SECTION

Reagents. A 15-mer thrombin-binding aptamer (TBA) with a –SH group labeled at its 5’ end (5’-SH-(CH2)6-GGTTGGTGTGGTTGG-3’) and different length ranges of aptamer-complementary 5 ACS Paragon Plus Environment


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DNA oligonucleotide

(cTBA)

with

a

–SH

group

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labeled

at

its

5’ end

(5-mer:

5’-SH-(CH2)6-CCAAC-3’, 7-mer: 5’-SH-(CH2)6-CCAACCA-3’, 9-mer: 5’-SH-(CH2)6-CCAACC ACA-3’, 12-mer: 5’-SH-(CH2)6-CCAACCACACCA-3’) were purchased from Songon Inc. (Shanghai, China) and purified by high performance liquid chromatography. Thrombin (TB), bovine serum albumin (BSA), lysozyme (Lys), hemoglobin (Hb), guanidine hydrochloride denaturant (GH), 6-mercapto-1-hexanol (MCH) were purchased from Sigma. Tris-(2-carboxyethyl) phosphine hydrochloride (TCEP) were obtained from Alfa Aesar (Tianjing, China). Tris(hydroxymethyl)aminomethane (Tris), hydrogen tetrachloroaurate (III) hydrate (HAuCl4), brilliant cresyl blue (BCB), sodium citrate was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Unless otherwise noted, all solutions were prepared with ultrapure water (18.2 MΩ cm). All other chemicals not mentioned here were of analytical reagent grade and were used as received. Apparatus. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were carried out with a CHI 660B electrochemical workstation (Shanghai Chenhua Instrument, China). Differential pulse voltammetry (DPV) was performed with an Autolab PGSTAT302 electrochemical workstation (Eco Chemie BV, Utrecht, The Netherlands). A conventional three-electrode cell was employed, which involved a modified gold electrode (2 mm in diameter) as a working electrode, a platinum wire as an auxiliary electrode and an Ag/AgCl (3 M KCl) reference electrode. All the potentials in this paper are given with respect to SCE. The electrolyte buffer was thoroughly purged with nitrogen before experiments. The pH measurements were made by PHS-3C pH meter (Shanghai Leici Apparatus Manufactory, Shanghai, China). The UV–vis absorption 6 ACS Paragon Plus Environment

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spectra are taken on a Shimadzu UV-2450 UV–vis spectrophotometer. Quartz cuvettes with a 10 mm path length were used for the spectral measurements. Transmission electron microscopy (TEM) images were taken using a Hitachi 600 (Hitachi Ltd., Tokyo, Japan). Preparation of AuNPs and DNA-Functionalized AuNPs. AuNPs were prepared by citrate reduction of HAuCl4 according to the literature.32 Briefly, 10 mL of 38.8 mM sodium citrate was immediately added to 100 mL of 1 mM HAuCl4 refluxing solution under stirring, and the mixture was kept boiling for another 20 min. The solution color turned to wine red and was cooled to room temperature with continuous stirring. The sizes of the AuNPs were characterized by TEM photographs and UV–visible spectrophotometry. As shown in Supporting Information Figure S1A, average particle diameters were consistently in the range of ∼13 nm. Supporting Information Figure S1B shows the characteristic absorption peak at approx. 520 nm. The extinction value of the 520-nm plasmon peak is ∼1.4, and the nanoparticle concentration is ∼6 nM.33 DNA-functionalized AuNPs was carried out as follows according to the literature.32 Briefly, 5 µL cTBA (50 µM) was activated with 10 µL acetate buffer (10 mM, pH 5.2) and 1.5 µL TCEP (20 mM) at room temperature for 1 h and then added into 200 µL of freshly prepared AuNPs for 16 h in darkness at room temperature. Then, the DNA–AuNPs conjugates were ‘‘aged’’ in salts (0.1 M NaCl, 10 mM Tris acetate, pH 7.4) for 24 h. Excess reagents were removed by centrifuging at 15000 rpm for 30 min. The red precipitate was washed, recentrifuged, dispersed in 100 µL Tris acetate buffer (10 mM, pH 7.4 containing 0.1 M NaCl), and stored at 4 ℃. Evolution and Regeneration of the Electrochemical Aptasensor System. The electrochemical aptasensor system with the thrombin-binding aptamer as the model recognition unit was fabricated 7 ACS Paragon Plus Environment


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on gold electrodes (2 mm diameter). The electrodes were polished carefully with 0.3- and 0.05-µm alumina powder, then sonicated with Milli-Q water, ethanol, and Milli-Q water (each for 3–5 min), and finally electrochemically cleaned by consecutive cycling of the electrodes in 0.5 M H2SO4 solution in the potential range from −0.2 to +1.6 V until a reproducible cyclic voltammogram was obtained. A 50 µL of 20 mM Tris–HCl buffer (pH 7.4) containing 5 µM SH-labeled TBA and 8 mM TCEP was dropped onto the clean Au electrodes and the electrodes were covered with a plastic cap to avoid evaporation of the solution. After being kept overnight at room temperature, the modified electrodes were then immersed in 1 mM MCH for 1 h to cover aptamer-unbound surface and obtain a well-aligned aptamer monolayer. After that, the modified working electrodes were further immersed in a cTBA–AuNPs solution for 6 h at room temperature. Because different length ranges of cTBA affected the hybridization and disassociation with TBA, led to a much effect on the sensitivity of the aptasensor. According to optimization result of the number of bases of cTBA on DPV peak currents of (BCB•(cTBA•AuNPs)•(TBA•GE)) before and after incubating 10 nM TB (Figure S2 in Supporting Information), 7-mer cTBA was chosen in following experiments. Finally, the modified gold electrodes were then immersed in 200 µM BCB solution without applying any potential to the electrode at room temperature for 24 min (the optimization results of BCB concentration and accumulation time in Figure S3 in Supporting Information). The electrode surfaces were rinsed with 20 mM Tris–HCl buffer (pH 7.4) containing 150 mM NaCl and Milli-Q water after each step of the fabrication process to replace any un-adsorbed species. The regeneration of the electrochemical aptasensor system was performed by dipping the 8 ACS Paragon Plus Environment

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electrodes previously exposed to thrombin solution into 6 M guanidine hydrochloride denaturant (pH 7.4) for 20 min and then removing the electrodes from the solution and rinsing them with Milli-Q water. After that, the electrodes were re-immersed into a DNA–AuNPs solution and BCB solution. The electrodes were finally rinsed with Tris–HCl buffer and Milli-Q water before re-use. Measurement Procedure. The electrochemical measurements including CV and EIS were carried out in a PBS solution (200 mM, pH 7.4) containing 100 mM KCl and 10 mM K3[Fe(CN)6]/K4[Fe(CN)6]. CV curves were scanned from −0.3 to 0.7 V at a scan rate of 100 mV/s. EIS was performed under an amplitude of 5 mV over the frequency range of 105 Hz to 0.1 Hz. DPV was performed in 20 mM Tris–HCl buffer (containing 150 mM NaCl, pH 7.4). DPV parameters applied were: 50 mV pulse amplitude, 200 ms pulse width, 0.5 s pulse period, and voltage range from −0.1 to −0.5 V. All the electrochemical measurements were carried out at room temperature. After accumulation of BCB, the modified electrode was rinsed with water thoroughly. Then, 20 µL of thrombin solution (a series of concentrations from 0.02 nM to 100 nM) was placed onto the modified electrode for 30 min. At last, DPV signal was evaluated in 20 mM Tris–HCl buffer solution (pH 7.4). As for the controlled experiment, the sensing interface was separately treated with 1 µM BSA, Lys, and Hb solutions under similar experimental conditions.

RESULTS AND DISCUSSION

Boolean Logic Tree of Solid-Based Electrochemical Evolution Aptasensor System. There are two types of symbols which appear in the Boolean logic tree structure: gates and events. Typical 9 ACS Paragon Plus Environment


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examples of basic, intermediate, and top event symbols are illustrated in green box of Scheme 1. The events linked using “gate” symbols. Common gates are shown in red box of Scheme 1. There are two fundamental logic gates “AND” and “NOT” in this Boolean logic tree (“INHIBIT (INH)” gate is formed by combining “AND” and “NOT”). For an AND gate, the output event occurrence requires the simultaneous existence of all of the input events. The output to a NOT gate happens as long as the input event does not. Firstly, thiol-labeled TBA “wires” (basic event) were self-assembled on the surface of a solid-phase bare gold electrode (GE, basic event) to form an AND-gate DNA monolayer-modified electrode (TBA•GE, intermediate event, the dot “•” is used to represent AND Boolean algebra, Scheme 1a). Thiol-modified cTBA “wires” (basic event) were covalently coupled to AuNPs (basic event) which implemented AND gate operation and exported cTBA-functionalized AuNPs (cTBA•AuNPs, intermediate event, Scheme 1b). The cTBA•AuNPs (intermediate event) were further connected to a TBA-modified gold electrode (intermediate event) via hybridization of TBA “wires” with cTBA “wires” in which the DNA “wires” were responsible for introducing other chemical events into the solid-phase electrode, resulting in superimposed AND-gates nanoamplifier (cTBA•AuNPs)•(TBA•GE) (intermediate event, Scheme 1c). The cTBA•AuNPs localized at the electrode surface could adsorb abundant positively charged molecules of BCB (basic event), which served as the dual-signal electroactive signaling molecules, due to their electrostatic attraction and intercalation effects with DNA “wires”, resulting in a more complex combinatorial AND-gates electrochemical aptasensor (BCB•(cTBA•AuNPs)•(TBA•GE)) (intermediate event, Scheme 1d). Upon the introduction of TB “winder” (basic event), the aptamer “wires” preferred to bind with 10 ACS Paragon Plus Environment

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target molecule TB rather than with the complementary DNA. As a result, the BCB•(cTBA•AuNPs) (top event) was replaced by the target TB to form the TB–G-quadruplex aptamer complex (TB•TBA)•GE (intermediate event, Scheme 1e), with the extrication of the abundant BCB molecules from the electrode into the solution, leading to a significant decrease in the current signal that can be used for sensitive and selective sensing of thrombin. Importantly, the used solid-based electrochemical aptasensor was easily re-usable by denaturing TB in GH solution (basic event), corresponding to the INH gate (TB•TBA)•GE ANDNOT GH gate (Scheme 1f), and then re-hybridizing the aptamer “wires” assembled onto the electrode with the cTBA•AuNPs. Thus, we can represent the whole evolution system as TB•(BCB•(cTBA•AuNPs)•(TBA•GE))• GH .

Scheme 1. Schematic illustration of Boolean logic tree of a solid-based electrochemical evolution



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aptasensor system based on interactions among DNA “wires” (TBA and cTBA), AuNPs (amplification supports), gold electrode (solid-based substrate), electroactive dye BCB (a dual-signal probe), TB (a “winder”), GH for the amplification detection of TB and molecular logic computation.

The

Boolean

(cTBA•AuNPs)•(TBA•GE)

logic

tree (c),

consists

of

TBA•GE

(a),

cTBA•AuNPs

BCB•(cTBA•AuNPs)•(TBA•GE)

(b), (d),

TB•(BCB•(cTBA•AuNPs)•(TBA•GE)) (e), (TB•TBA)•GE ANDNOT GH (f) gates. The dot “•” represents AND Boolean algebra. Three typical event symbols (basic, intermediate, and top events) and three common “gate” symbols (AND, NOT, INH gates) are illustrated in green and red boxes, respectively. The orange glow represents electroactive signal.

Characterization of the Electrochemical Evolution Aptasensor System. In order to clarify the electrochemical properties of the resulting aptasensor system, CV and EIS were used for monitoring and characterizing the logic evolution process of the aptasensor system in each step. As expected, the behavior of K3[Fe(CN)6]/K4[Fe(CN)6] on a bare GE was reversible with a peak-to-peak separation ∆Ep of 107 mV (Figure 1Aa). Since the TBA could block electron transfer, the redox peak (Figure 1Ab) decreased after immobilizing TBA “wires” on the electrode surface (Scheme 1a). After treatment with MCH, the redox peak decreased (Figure 1Ac), implying a remarkable increase in the resistance to electron transfer. After self-assembly of cTBA•AuNPs and BCB (Scheme 1c,d), an increase in the ∆Ep of the electrode was observed (Figure 1Ad). It was largely attributed to the fact that the surface of AuNPs was covered with DNA “wires” and the bulk negative charges could further repel the probes of [Fe(CN)6]3−/4− anions. After incubating TB 12 ACS Paragon Plus Environment

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“winder” on the electrode surface (Scheme 1e), the reversibility of the resulting electrode was partly restored (Figure 1Ae). After treated with GH solution on the (TB•TBA)•GE electrode surface for disassociating TB, the CV curve of the regenerable MCH•TBA•GE was similar to that of initial MCH•TBA•GE (Figure 1Ac,f). Furthermore, after re-assembled BCB•(cTBA•AuNPs) on the regenerable

MCH•TBA•GE

electrode

surface,

the

CV

curve

of

the

regenerable

(BCB•(cTBA•AuNPs)•(TBA•GE)) was similar to that of initial (BCB•(cTBA•AuNPs)•(TBA•GE)) (Figure 1Ad,g). A consistent result was also provided in Figure 1B. The electron-transfer resistance (Ret) increased in the order of the bare GE (0.114 kΩ), TBA•GE (9.597 kΩ), MCH•TBA•GE (12.938 kΩ), and (BCB•(cTBA•AuNPs)•(TBA•GE)) (20.501 kΩ, Figure 1Ba-d). The increase in Ret indicated that the TBA “wires” and cTBA•AuNPs were successfully immobilized on the electrode surface. After incubating TB on the electrode surface, Ret decreased obviously from 20.501 to 19.002 kΩ (d to e in Figure 1B), proving that cTBA•AuNPs molecules were dissociated from the electrode as anticipated (Scheme 1e). After adding GH solution on the electrode surface, Ret further decreased significantly from 19.002 to 13.014 kΩ and was close to that (12.938 kΩ) of MCH•TBA•GE (Figure 1Bc,f), implying disassociating TB from the electrode (Scheme 1f). Subsequently, the electrodes were re-immersed into a cTBA•AuNPs solution and BCB solution, Ret re-increased significantly from 13.014 to 21.006 kΩ (Figure 1Bg), suggesting that the prepared electrochemical aptasensor system was re-generable (Scheme 1c,d).



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Figure 1. Cyclic voltammograms (A) and Nyquist plots of impedance spectra (B) of the bare GE (a), TBA•GE (b), MCH•TBA•GE (c), and (BCB•(cTBA•AuNPs)•(TBA•GE)) (d) in 10 mM K4[Fe(CN)6]/K3[Fe(CN)6] solution. (e) is (d) treated with 10 nM TB for 30 min. (f) is (e) treated with 6 M GH solution for 20 min. (g) is (f) re-assembled with BCB•(cTBA•AuNPs). Points are experimental data and lines are fitting data. The inset shows the equivalent circuit applied to fit the experimental impedance data. Rs is solution resistance; Ret charge transfer resistance, ZW is Warburg impedance and CPE is constant phase element.34 The scan rate of cyclic voltammetry is 100 mV/s. The frequency range is from 0.1 to 105 Hz and the amplitude of the alternate voltage is 5 mV.

Dual-Signal Amplification Detection of Thrombin Based on Electrochemical Evolution Aptasensor.

Figure

2A

shows

the

DPVs

of

the

fabricated

aptasensor

(BCB•(cTBA•AuNPs)•(TBA•GE)) for amplification detection of TB with different concentrations under the same experimental conditions (comparison of DPV currents between nano-amplification and no amplification in Supporting Information Figure S4). It can be seen that BCB due to its two-electron redox reaction process35 had two peak currents at −0.35 V (Ip1) and at −0.21 V (Ip2), 14 ACS Paragon Plus Environment

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moreover Ip1 and Ip2 decreased gradually with increasing concentration of TB from 0 nM to 100 nM (Figure 2A). The linear relationship between the Ip1, Ip2, Ip1+p2 of BCB and the logarithm to the base 10 of TB concentration (lgCTB) over a range of 0.02–100 nM is shown in the Figure 2B-C, which could be expressed as Ip1 = 20.259 lgCTB − 62.481 (r = 0.993) with a detection limit of 2.74 pM, Ip2 = 16.423 lgCTB− 47.008 (r = 0.994) with a detection limit of 2.89 pM, Ip1+p2 = 36.638 lgCTB − 109.862 (r = 0.996) with a detection limit of 1.92 pM (defined as 3σ, where σ is the standard deviation of the blank solution, n = 11; I in µA, CTB in nM). TB is present in blood, a complex sample matrix, with hormones, lipids, blood cells and other proteins. Serum albumin is the main protein in plasma. The blood cells are mainly red blood cells, white blood cells and platelets. The most abundant cells are red blood cells, which contain Hb. Moreover, the most abundant type of white blood cells in most mammals is neutrophils, which have abundant immunoreactive Lys. Moreover, molecular weight of the TB (70 kDa) is similar with that of BSA (66.5 kDa) and Hb (64.5 kDa).36 Therefore, to investigate the selectivity of the fabricated aptasensor for detection of TB, we conducted the control experiments using these three physiologically and clinical diagnostic relevant proteins typically present in blood, including BSA 1 µM, Lys 1 µM, Hb 1 µM, as negative controls.37,38 As shown in Figure 2D, despite the concentration of analog molecules are 100-fold than the TB (10 nM), there were no apparent changes in DPV peak currents (∆I = I(p1+p2)/sample − I(p1+p2)/blank). However, the presence of TB resulted in the dramatic increase of ∆I. The results demonstrated that the fabricated aptasensor showed a high specificity for the sensing of TB.



(A) 0

(B) 0 I = 16.423 lgC − 47.008 P2 TB

100 nM

-25 r = 0.994 I (µA)

I (µA)

-30 -60

-50

p1 p2

-75

p2 -90

0 nM

p1 -0.5

-0.4

-0.3 -0.2 E (V)

-60

Ip1 = 20.259 lgCTB − 62.481 r = 0.993

-100

-0.1

-1.6

-0.8

0.0 0.8 lgCTB (nM)

(D)100

(C) -30 Ip1+p2 = 36.638 lgCTB − 109.862

1.6

2.4

p2 p1

r = 0.996

75

-90

∆I (µA)

Ip1+p2 (µA)

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-120 -150

50 25

-180 -1.6

-0.8

0.0 0.8 lgCTB (nM)

1.6

2.4

0 Thrombin

BSA

Lys

Hb

Figure 2. (A) DPV responses of the fabricated aptasensor with Tris-HCl buffer (20 mM, pH 7.4) containing 150 mM NaCl after incubation with different concentrations of TB (from lower to upper): 0, 0.02, 0.05, 0.1, 0.2, 0.5, 2, 5, 10, 40 and 100 nM. The linear relationships between lgCTB and Ip1, Ip2 (B), and Ip1+p2 (C) in a range of 0.02 to 100 nM. (D) DPV responses of the aptasensor towards 10 nM TB, 1 µM BSA, 1 µM Lys and 1 µM Hb.

Controllable and Selectable Three-State Logic Computation Based on the Aforementioned System. In digital electronics, three-state logic (or three-state buffer), which plays important roles in many registers, bus drivers, flip-flops, and microprocessors, allows an output port to assume a


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high impediment or nothing (Z) state in addition to the 0 and 1 logic levels, effectively controlling when an input signal passes through the device, and when it doesn't.31 It should be noted that three-state logic should not be confused with three-valued logic. In logic, a three-valued logic (also trinary logic) is any of several many-valued logic systems in which there are three truth values indicating true, false and some indeterminate third value. Molecular ternary computing has already been implemented in a ternary DNA computing device39 and a ternary photoelectrochemical logic device.40 A three-state logic has two inputs: a data input X and a control input C. The control input C acts like a “wire”. When the control input C is active (1), the "wire" is connected (Figure 3Ab), the output enables and its logical value is equal to that of the data input X. When the control input C is inactive (0), the "wire" is open (Figure 3Ab), even if the data input X is 0 or 1, the output is high impediment or nothing (Z, this means that it is neither 0 nor 1). The aforementioned processes in Scheme 1 show that DNA “wires”, nanomaterial supports, dual-signal electrochemical probe and biomolecule “winder” can self-assemble, compete, and connect with each other in solid-based electrochemical GE platform. We used the DPV current changes at −0.35 V (Ip1) and at −0.21 V (Ip2) in the different combinations of these aforementioned chemical events for implementing controllable and selectable three-state molecular logic computation with two electrochemical outputs (Ip1 and Ip2) and two different kinds of inputs for active-high (Figure 3A) and active-low (Figure 3B) three-state logic functions. Logical outputs which indicated the accumulative amount of BCB on the electrode platform in response to input combinations, were examined by DPV and defined when 0.1 µA < Ip1 or Ip2 < 20 µA for logical 0, Ip1 or Ip2 > 20 µA for logical 1, and Ip1 or Ip2 < 17 ACS Paragon Plus Environment


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0.1 µA for Z state, respectively. The logic responses of the system were studied by adding different input combinations of chemical events (Figure 3Aa and Ba). If the control input C = TBA was inactive or absent (0) in GE, whatever the data input X = cTBA•AuNPs was absent (0) or present (1), the GE could not further bind the electroactive signaling molecules BCB, and even capture TB due to lack of TBA linker, resulting in nothing on the surface of GE and inability to execute logic evolution process in scheme 1a,c-f (Figure 3Aa). That is, the output Y of GE was nothing (Z), and the control input C = TBA can selectively control or effectively remove the outputs of subsequent branchy logic computation processes from electrochemical evolution aptasensor system. Once the control input C = TBA was active or present (1) in GE, when the data input X = cTBA•AuNPs was absent (0), the GE just covalently coupled to TBA “wires” which could capture a few BCB, thus the output Y of GE was a low DPV current signal (0). When the data input X = cTBA•AuNPs was present (1), the GE could bind cTBA•AuNPs nanoamplifier via hybridization of TBA “wires” with cTBA “wires” and further capture abundant BCB, thus the output Y of GE was a high DPV current signal (1). Therefore, by combining the two inputs TBA and cTBA•AuNPs in accordance with the truth table (Figure 3Aa), the solid-based electrochemical GE platform controllably and selectively gave the DPV current signal and implement subsequent branchy logic computation processes in scheme 1c-f, only when the control input C = TBA was active or present (1), corresponding to active-high three-state logic function (Figure 3A). Besides, some three-state logic are active low. In an active-low three-state logic, C = 0 makes the “wire” close, while C = 1 turns it open. By combining the two inputs TB and cTBA•AuNPs in different TBA•GE platform in accordance with the truth table (Figure 3Ba), the TBA•GE platform 18 ACS Paragon Plus Environment

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controllably and selectively gave the DPV current signal only when the control input C = TB was inactive, corresponding to active-low three-state logic function (Figure 3B). When the control input TB was active, TBA “wires” in the electrochemical platform were constrained and specifically induced to form G-quadruplex structures by target TB, resulting in that the electrochemical platform could not connect with other chemical input events (such as BCB), equivalently, high impediment (Z). Thus, the constructed three-state molecular logic computation implementing active-high and active-low logic functions, allow the output ports to assume a high passivation or nothing (Z) state in addition to the 0 and 1 logic levels, effectively controlling when an input signal passes through the circuits.



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Figure 3. Controllable and selectable three-state logic computation in solid-based electrochemical platform. Processing performance and the truth table (a), logical symbol (b,c) of the active-high three-state logic (A) and the active-low three-state logic (B) with two chemical inputs and two electrochemical outputs (Ip1 and Ip2). TB, 20 µl/10 µM; TBA, 50 µl/5 µM; cTBA•AuNPs, 5 µl/50 µM•200 µl/6 nM; BCB, 200 µM.

Keypad Lock Security Operation Based on the Aforementioned System. The combinatorial logic circuits produce output signals as a result of processing input signals; however, they do not have no memory.1 One challenging step forward in the development of controllable molecular digital systems will be the design of memory systems41 for mimicking sequential logic operations, which depend on both the input combination and the input sequence. Application of sequential logic operations allows the construction of memory elements, including flip-flops,42 latches,43 and registers. Herein, our proposed electrochemical evolution aptasensor system can perform sequential logic 20 ACS Paragon Plus Environment

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operation and function as a molecular keypad lock44 with three inputs and two electrochemical outputs Ip1 and Ip2 (Figure 4). The AND-gate TBA monolayer-modified electrode (TBA•GE) was used as an initial system which involved stepwise treatment with cTBA (designated as the character “a”), AuNPs (designated as the character “b”) and BCB (designated as the character “c”) defined as the inputs. The high and low DPV current values (Ip1 and Ip2) of the outputs were considered as 1 and 0, respectively, with a threshold value of 20 µA. The logic responses of the system were studied by adding different input combinations of chemical events (Figure 4B,C). Because only cTBA “wires” were covalently self-assembled on the surface of AuNPs to form an AND-gate nanoamplifier cTBA•AuNPs, the initial system TBA•GE could further bind cTBA•AuNPs nanoamplifier via hybridization of TBA “wires” with cTBA “wires” to construct signal amplification architecture for adsorbing abundant BCB (Figure 4A). Thus, only the combination (a•b)c could trigger the system to adopt the “1” state (a high current response) (Figure 4B,C). In contrast, low current signals were detected in the presence of other input combinations, indicating failure of nano-amplification. Thus, the correct input sequence (a•b)c represented the password to open the keypad lock while all other input combinations failed to open the lock. Furthermore, due to the introduction of solid-phase substrate, the system could be separated easily from the current solution for removing invalid inputs and verbose outputs. Our developed keypad-lock can be facilely reset by TB and GH after operation (Figure 1Bf,g, Figure 4A,B, Figure S5), which is critical for multiple operations of the keypad-lock.44 Upon alternately assembling BCB•(cTBA•AuNPs) and incubating TB and GH, the DPV current signal of the electrochemical system alternately increased and decreased almost to its original value and the reset could be 21 ACS Paragon Plus Environment


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repeated at least several times (Figure 4B, cyan and green dots, Figure S5), suggesting that our developed molecular keypad-lock had good stability and resettability.

Figure 4. (A) Schematic representation of an electrochemical evolution system for sequential logic operating as a keypad lock with reset function, employing cTBA (a), AuNPs (b) and BCB (c) as the inputs, TB (d) and GH (e) for reset operation. (B) DPV responses (Ip1 and Ip2) of the electrochemical evolution system in the presence of the three input signals with different combination sequences and reset operations. Cyan (or green) and black represent logical values 1 and 0, respectively. (C) Truth table for the keypad lock system.

CONCLUSIONS

In summary, we utilized Boolean logic tree to organize and connect “plug and play” chemical 22 ACS Paragon Plus Environment

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events (DNA “wires”, nano-amplification supports, dual-signal electrochemical dye, biomolecule “winder”, and denaturant) in solid-based platform for developing the label-free dual-signal electrochemical evolution aptasensor system for amplification detection of thrombin, controllable and selectable three-state logic computation and resettable keypad lock security operation. By combining the merits of DNA-functionalized nano-signal amplification architecture and simple dual-signal electroactive dye, this system can sensitively and selectively determine thrombin with a wide linear response range of 0.02–100 nM and a detection limit of 1.92 pM. This solid-phase system with dual-signal output is not only easy solid-liquid separation and eliminating invalid inputs and verbose outputs, but also gives them significant potential for enhancing computational complexity and anti-interference ability of logic computing. Moreover, the first example of three-state molecular logic computation based on solid-based platform has been constructed, which is helpful to expand molecular logic computing and may be an attractive research interest to utilize biochemical events for controllable and selectable logic computing in large-scale integration circuits. Predictably, the three-state molecular logic computation will provide more opportunities for output enable of the complex biochemical system for applications in biomedical engineering, intelligent sensing and control.

Notes The authors declare no competing financial interest.

Author Contributions 23 ACS Paragon Plus Environment


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#

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These authors contributed equally to this work.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21505042), Natural Science Foundation of Hunan Province (No. 2016JJ3084), the Research Foundation of Education Bureau of Hunan Province (No. 15K084), the Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (No. 20134486), Youth Foundation of Hunan Normal University (No. 31403), Doctoral Scientific Research Foundation of Hunan Normal University (No. 150612), and Hunan Provincial Key Laboratory Open Foundation for Microbial Molecular Biology (No. 2014-03).

ASSOCIATED CONTENT

Supporting Information.

The Supporting Information is available free of charge on the ACS Publications website. TEM image and UV-vis absorption spectral data of AuNPs (Figure S1); optimization of different length ranges of cTBA (Figure S2), the BCB concentration and accumulation time (Figure S3); comparison of DPV currents between nano-amplification and no amplification (Figure S4); resettability of the electrochemical evolution system (Figure S5); and the corresponding discussion. (PDF).


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for TOC only


Boolean Logic Tree of Label-Free Dual-Signal Electrochemical

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