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Smart Sensing Based on DNA-metal Interaction Enables A Label-free and Resettable Security Model of Electrochemical Molecular Keypad Lock Yan Du, Xu Han, Chenxu Wang, Yunhui Li, Bingling Li, and Hongwei Duan ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00735 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 18, 2017
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ACS Sensors
Smart Sensing Based on DNA-metal Interaction Enables A Label-free and Resettable Security Model of Electrochemical Molecular Keypad Lock Yan Du,*,† Xu Han, # Chenxu Wang,‡ Yunhui Li,# Bingling Li,*,† and Hongwei Duan*,‡ † State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, P.R. China. # School of chemistry and environmental engineering, Changchun university of science and technology, Changchun, Jilin 130022, P.R. China. ‡ School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, Singapore 637457.
Supporting Information Placeholder ABSTRACT: Recently, molecular keypad locks have received increasing attention. As a new sub-group of smart biosensors, they show great potential for protecting information as a molecular security data processor, rather than merely molecular recognition and quantitation. Herein, label-free electrochemically transduced Ag+ and cysteine (Cys) sensors were developed. Molecular keypad lock model with reset function was successfully realized based on the balanced interaction of metal ion with its nucleic acid and chemical ligands. The correct input of “1-2-3” (i.e. “Ag+Cys-cDNA”) is the only password of such molecular keypad lock. Moreover, the resetting process of either correct or wrong input order could be easily made by Cys, buffer and DI water treatment. Therefore, our system provides an even smarter system of molecular keypad lock, which could inhibit illegal access of unauthorized users, holding great promise in information protection at molecular level.
and smarter functions for these “games”.19-21 In particular, one still needs to solve common shortcomings of most molecular keypad locks reported previously, for example, difficulty to reset and requirement of multiple labelling steps. Without a resetting function, the password may be cracked by any of those unauthorized or wrong inputs. And using many labeling or synthesis steps will further delay and limit the implementation of molecular keypad lock.
KEYWORDS: keypad lock, resettable, label-free, electrochemical, logic sensor In the past decades, new methods for biosensing have been extensively explored to achieve more advanced functions such as multiplex assay, smarter assay, and even intelligent assay.1 Among these new methods, molecular logic gates that can perform Boolean operations have raised considerable interest because they can carry out molecular computational functions besides merely molecular recognition and quantitation.1,2 Recently, one such molecular logic device can even be set up responding only to proper combination of molecular inputs with defined input order.3,4 In other words, with a unique input order as a password, the device behaves just like an electronic keypad lock, but at a very magical molecular level.5,6 Different from those simple logic gates, the socalled molecular keypad locks protect information as a molecular security data processor, and thus received increasing attention.3,7,8 Major progress were made in converting input combinations of gases,9 metals,7 DNAs,10-13 enzymes14 or antibodies15-17 to fluorescence, colorimetric, or electrochemical outputs. However, up to now existing processes are on a ‘proof-of-concept’ level, and hardly fulfils the needs of practical requirements. That is because a real molecular security function necessitates greater flexibility in controlling molecular interaction and molecule-signal relation.18 There are growing efforts on achieving more advanced
Scheme 1. Schematic illustration on the keypad lock system, Au/HS-P/MCH interface, presents ON signal only with right input sequence “1-2-3” (G1), and presents OFF signals with wrong input sequences “1-3-2” (G2), “2-1-3” (G3), “2-3-1” (G4), “3-12” (G5), and “3-2-1” (G6). To address these problems, herein we developed a label-free keypad lock model with reset function. Based on the highly specific base-pairing of nucleic acids and its specific interactions with metal ions such as Ag+ (through cytosine (C) -Ag+ pair22), a C-rich nucleic acid, a metal ion (Ag+), and a metal ligand (cysteine, Cys) were employed as “proof-of-concept” combination of three inputs to play the game. We used these inputs first because detection of Ag+ and Cys are both practically necessary. Ag+ has high toxicity to aquicolous organism, while abnormal Cys level is relevant to a number of syndromes such as edema, lethargy, hair depigmentation, liver damage, slow growth and skin lesion.23,24 Second, nucleic acid-metal interaction plays an important role in modulating biological systems. Thus, the three inputs have already been used in many other logic gates25 and molecular keypad locks.7
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Our keypad lock panel was built as follows: the thiol-modified C-rich linear single stranded DNA Probe (HS-P) was attached to a cleaned Au electrode surface through Au–S bond. 6-mercapto-lhexanol (MCH) was used to block uncovered surface of the electrode and reduce the nonspecific adsorption. Now, the modified Au electrode is ready to serve as the keypad lock panel and receive the three inputs, Ag+ (“1”), Cys (“2”) and the complementary DNA of HS-P (cDNA, “3”) for further signaling. The correct order (or password) of the keypad lock is designed to be “1-2-3” (grid 1, G1 in the Scheme 1A). The final output signal should be chronocoulometry (CC) signal generated by the absorbed [Ru(NH3)6]3+ (RuHex) onto electrode surface.26 In details, the first “1” input, Ag+, binds to C bases in HS-P on the electrode and forces the HS-P forming a hairpin conformation. The second “2” input, Cys, is a stronger Ag+ ligand than C base, and thus can remove Ag+ from C-Ag+-C base pairs.27 Hence, in the presence of “2”, the hairpin structured HS-P switches back to linear conformation again. The third “3” input, cDNA, is able to hybridize with HS-P to form a DNA duplex. Compared to either linear or hairpin HS-P itself, such duplex conformation increases negative charges on the keypad lock panel, which means increasing the amount of RuHex molecules absorbed onto the electrode. It thus increases the CC signal generated.28 Correspondingly, outputting signals with other input orders are shown in grid G2G6 of Scheme 1. When cDNA/HS-P binding is weaker than that of Ag+/HS-P, it could be derived that only when the inputs are added in the correct “1-2-3” order, can the duplex conformation been formed. Therefore, the input “1-2-3” is set to be the only correct password of the molecular keypad lock. The whole device does not require any probe labeling process. The resetting, for either correct or wrong input order, could be easily realized by treating the electrode surface with Cys, buffer and DI water. Therefore, our system provides an even smarter system during boosting the field of molecular keypad lock and logic world.
Ag+/HS-P and Ag+/Cys > Ag+/HS-P. The detection pathways are illustrated in Scheme 2. In Ag+ sensing platform, the HS-P modified electrode was saturated by 1 µM cDNA, after which the asformed cDNA/HS-P modified electrode was incubated with Ag+ for 90 min (Figure S2A, in SI). As shown in Figure 1A, the CC signal sharply decreased during the increase of Ag+ concentration, with high selectivity (Figure 1B). It verifies that the interaction between Ag+ and HS-P was indeed stronger than that of cDNA/HS-P. Therefore, Ag+ could draw the cDNA off the HS-P, leaving less negative charge on the electrode. Note that the CC signal no longer decreased when the concentration of Ag+ reached 8 µM. It indicates saturated binding of Ag+ with HS-P at this concentration. Once the concentration was higher (e.g. at 10 µM), the negatively charged HS-P might start non-specifically absorbing the positively charged Ag+. The accumulated Ag+ might show signifcant influences on the surface property of the electrode, possibly via (I) changing the double layer charge or conductivity near the electrode surface, or (II) getting mildly reduced under the potential pulse during CC scan.30 Either of the two processes will increase the the total charge Q that is the intercept at t=0 in CC curve).28,31 Based on the result of above Ag+ sensing platform, we performed the sensing of Cys. After the cDNA/HS-P modified electrode was saturated by 8 µM Ag+, the as-formed Ag+/HS-P modified electrode was immersed into Cys solution for 50 min (Figure S2B, in SI). Then the electrode was re-incubated with 1 µM cDNA. As shown in Figure 1C and 1D, the CC signal gradually and selectively recovered upon the increase of Cys concentration. This further verifies that the interaction between Ag+ and Cys was indeed stronger than that of Ag+/HS-P. Therefore Cys could competitively draw the Ag+ off the HS-P, leaving more positions for cDNA rebinding. Of note is that before serving as a keypad lock, the designed panel could at first serve as sensitive, selective and flexible sensors for both Ag+ and Cys, with a detection limit of 17.3 nM and 28.1 nM, respectively. Furthermore, the tests in 50% lake water samples showed high anti-interference ability of the panel (Table S1).
Scheme 2. The illustration of the sensing platforms for detecting of Ag+ and Cys. The fabrication of the interface of the biosensor and keypad lock panel was validated by electrochemical impedance spectroscopy (EIS). It was previously proved that for a DNA modified electrode, the natively charged surface repelled the negatively charged probe, [Fe(CN)6]4−/3− anions, blocking the interfacial electron-transfer of the redox probe.29 Such an effect gives rise to an increase of electron transfer impedance (Ret) and a larger diameter of the semicircle in EIS Nyquist plot, as shown in Figure S1 (in Supporting Information, SI). The bare Au electrode showed a very small Ret. After HS-P was assembled, the Ret was increased obviously due to the remarkably decreased electron-transfer efficiency. After MCH was assembled, the Ret increased further. All these results demonstrated the successful fabrication of the biosensor and keypad lock panel. Before the keypad lock function could be performed, several important parameters should be examined. At the beginning, Ag+ and Cys sensing platforms were designed to detect the two inputs and then the binding affinity between the components and inputs was confirmed to follow an expected order of cDNA/HS-P
