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. 2021 May 27;7(6):1036–1044. doi: 10.1021/acscentsci.1c00277

Cascade Strand Displacement and Bipedal Walking Based DNA Logic System for miRNA Diagnostics

Peng Miao †,‡,*, Yuguo Tang †
PMCID: PMC8228592  PMID: 34235264

Abstract

graphic file with name oc1c00277_0006.jpg

DNA logic gated operations empower the highly efficient analysis of multiplex nucleic acid inputs, which have attracted extensive attention. However, the integration of DNA logic gates with abundant computational functions and signal amplification for biomedical diagnosis is far from being fully achieved. Herein, we develop a bipedal DNA walker based amplified electrochemical method for miRNA detection, which is then used as the basic unit for the construction of various logic circuits, enabling the analysis of multiplex miRNAs. In the bipedal walking process, target triggered strand displacement polymerization is able to produce a large number of strands for the fabrication of three-way junction-structured bipedal walkers. The following catalytic hairpin assembly ensures the walking event and the immobilization of signal probes for output. Ultrahigh sensitivity is realized due to the integration of dual signal amplification. In addition, under logic function controls by input triggered cascade strand displacement reactions, NOT, AND, OR, NAND, NOR, XOR, and XNOR logic gates are successfully established. The as-developed DNA logic system can also be extended to multi-input modes, which holds great promise in the fields of DNA computing, multiplex analysis, and clinical diagnosis.

Short abstract

Cascade strand displacement reactions are developed for the construction of a series of miRNA responsive logic gates with bipedal walking for electrochemical signal outputs.

Introduction

miRNAs belong to noncoding RNAs, which act as critical gene regulators and are extensively involved in a range of biological processes.1−3 Numerous studies have revealed that abnormal expression levels of miRNAs are responsible for the occurrence of certain diseases including cancers.4,5 Selected miRNAs can thus serve as biomarkers for diagnosis and therapy purposes.6,7 Nevertheless, the inherent properties of miRNA such as short length, low abundance, and high sequence similarity between family members raise significant difficulties for rapid and convenient detection of miRNAs.8 Classic techniques including Northern blotting, microarray, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) suffer from certain limitations like tedious procedures, expensive instrumentation, and long reaction times. In recent years, researchers have developed various sensing platforms (e.g., electrochemistry, surface-enhanced Raman scattering, mass spectrum, fluorescence, and surface plasmon resonance), which partially resolve the above-mentioned limitations.9−12 For example, Gines et al. fabricated a background-free molecular circuit for the isothermal digital analysis of miRNA.13 Zhao et al. functionalized a DNA device on the surface of upconversion nanoparticles for NIR light gated miRNA sensing and imaging.14 Among the above techniques, electrochemistry based methods have attracted great attention, which permit an ultrasensitive and cost-effective analysis with a low background signal response.15,16 The interfacial properties can be accurately controlled by the engineering of immobilized biomolecules, which is essential for the reliable and stable analysis.17 To further improve the sensitivity of an electrochemical biosensor, amplification processes are always adopted, such as rolling circle amplification,18 hybridization chain reaction,19 strand displacement amplification,20 catalytic hairpin assembly,21 and so on. The DNA walker, as a typical DNA nanomachine, can also act as a powerful programmable amplification tool with excellent automaticity and controllability.22 By applying bipedal or multipedal walking, the efficiency can be further enhanced.23,24

However, most work can only accomplish a single target analysis. Actually, it is indicated that multiplex miRNAs always jointly regulate the function of target genes.25 Increasing demands have been raised to fabricate novel methodologies responding to multiplex miRNA targets for the purposes like precise screening of cancer types26 or signaling pathway investigation.27 DNA based mimicking Boolean logic gated operations provide great possibilities to achieve multifunctional information processing of miRNAs.28,29 DNA logic gates are a type of computational device with unique output patterns to process information and perform arithmetic operations.30,31 In addition, serial assembly of primary logic gates can be easily achieved so as to smartly respond to various analytes.32,33

The strand displacement reaction not only can be used to assist signal amplification34 but also provides toolboxes to implement DNA computing in most DNA logic operations.35 Although encouraging progress has been made in DNA logic gate based biomedical applications, most systems suffer from poor integration with cascade signal amplification, complicated designs, and limited computational functions. In this study, we first explore a highly sensitive electrochemical platform for an miRNA assay combining cascade strand displacement and bipedal DNA walking. A series of metastable DNA junction or duplex structures are then prepared. After undergoing a structure transformation by multiplex miRNA triggered strand displacement reactions, a series of DNA logic gates are established. This strategy not only reaches a low limit of detection but also distinguishes miRNAs with one base mutation. Moreover, the developed compact and efficient logic circuits give responses to multiplexed combinations of miRNA inputs, which holds intriguing potential for biological and clinical applications.

Results and Discussion

Architecture Design of Basic Bipedal Walking

The principle of the electrochemical sensing method for miRNA is illustrated in Figure 1a. First, miRNA hybridizes with single-stranded template1, which is extended with the existence of polymerase. The formed complete duplex is then recognized by nicking endonuclease, and a nick is created. Subsequently, polymerization continues, and TWJ1 is released. The upstream strand displacement amplification is responsible for the generation of a large number of TWJ1 strands. TWJ1, in turn, participates in the assembly of a three-way junction containing two “legs” in close proximity. The bipedal DNA walking can thus occur on the track modified electrode surface based on the criterion of the catalytic hairpin assembly. Generally, track and driver sequences are designed with two hairpins, which cannot interact with each other directly. In the presence of the bipedal walker “legs”, the simultaneous opening of two hairpins on neighboring track sequences is catalyzed. The released single-stranded domain in turn captures the driver sequence, which is able to displace one “leg” in order to form a complete duplex. The catalytic hairpin assembly is thus completed. Moreover, the freed “leg” of the bipedal walker is able to move to another hairpin-structured track for new cycles of immobilization. In the presence of sufficient driver sequences, the bipedal walking is executed by the catalytic hairpin assembly process, which brings abundant methylene blue (MB) close to the electrode. Combining upstream strand displacement amplification and downstream bipedal walking, a significantly increased signal response can be recorded to indicate trace levels of target miRNA.

Figure 1.

Figure 1

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Bipedal DNA walking based electrochemical detection of miRNA. (a) Scheme of the sensing principle. (b) Cyclic voltammograms, (c) Nyquist diagrams, and (d) square wave voltammograms of the bare electrode, track modified electrode, and those further treated with driver in the absence and presence of the bipedal walker. (e) Square wave voltammograms for the quantification of the target (from 10–16 to 10–11 M). (f) Calibration plot of SWV peak current variation versus the logarithmic miRNA level. (g) Selectivity performance of the electrochemical biosensor for the detection of target miRNA.

To ensure the feasibility of the DNA walker, we have first calculated and compared the melting temperature (Tm) of double-stranded regions during the process. Usually, higher Tm values are a reflection of a steadier state. In addition, with bigger Tm variation from the reactant to product, the reaction tends to be executed much faster. Tm values of the hairpin-structured track and driver are 45.6 and 30.2 °C, respectively, which are quite low. The duplex of the track and the walker sequence is increased to 50.8 °C. After the strand displacement reaction, the duplex of the track and driver is calculated to be 64 °C. Theoretically, stepwise walking reactions can be realized since the products possess higher Tm values. We have then carried out polyacrylamide gel electrophoresis (PAGE) experiments to confirm the feasibilities of the reactions of upstream strand displacement amplification (Figure S1a) and downstream walking (Figure S1b). The molecular weights of DNA probes before and after target triggered corresponding reactions appear in the expected positions in the gels, demonstrating the correct architecture design.

Ultrasensitive Detection of Target miRNA

We have conducted cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) to probe the electrochemical properties of the biosensor during stepwise reactions. The smaller CV peak current and larger semicircle diameter of the Nyquist diagram could reflect the increased degree of repellent between the molecule layer on the electrode and electrochemical species. With the DNA assembly on the electrode, the negatively charged phosphate backbone repels [Fe(CN)6]3–/4–, leading to a decrease of the current peak and increase of the semicircle domain of EIS. After the completion of bipedal walking, the formed double stranded DNA leads to the smallest CV peak currents and largest electron transfer resistance (Figure 1b,c). Furthermore, since MB is labeled on the end of the driver, after effective DNA walking, the immobilized MB contributes to a significant square wave voltammetry (SWV) current peak, which can be applied for quantification purposes (Figure 1d). Some important parameters are optimized including the 0.5 μM track, 0.8 nM template, 0.1 unit μL–1 endonuclease, 0.4 μM TWJ2/3, 60 min strand displacement amplification, and 60 min walking reaction (Figure S2). Under these experimental parameters, miRNA with different concentration triggered reactions are investigated by SWV. The curves are displayed in Figure 1e. With the increase of target level, more bipedal walkers are produced, which help the immobilization of more electrochemical probes on the electrode interface. The peak intensity becomes larger as expected. In addition, the calibration curve is reflected in Figure 1f, and a wide linear concentration range is from 100 aM to 0.1 pM with the equation of ip = 18.965 + 1.117 log(c) (n = 3, R2 = 0.995). The detection limit is estimated to be 10 aM (S/N = 3), which is lower than or at least comparable with most reported methods (Table S1).

The specificity performance is one of the most critical considerations for a biosensor. To verify the high specificity of this method, we have introduced a series of mismatched miRNAs to replace the target miRNA. After the standard experimental protocol, the obtained peak current is much lower than that of target miRNA. In addition, after further spiking with target miRNA, the peak currents grow significantly and are comparable with the original value, verifying the ability to distinguish the target from potential interfering sequences (Figure 1g). Next, cellular miRNA levels are determined by the proposed method. MCF-7, HeLa, A549, and MCF-10A cells are cultured, and miR-21 extracted from these cells is analyzed by the electrochemical method and a qRT-PCR assay. According to the standard curve established in Figure 1f, tumorigenic cells show much higher miR-21 expression levels than nontumorigenic MCF-10A, which is as reported. In addition, the calculated concentrations are in good accordance with the qRT-PCR assay, indicating the excellent accuracy of this method (Figure S3a). We have further challenged this miRNA assay with tissues from human beings. After analyzing the samples from healthy individuals and breast cancer patients, respectively, miR-21 concentrations are calculated, and a scattered plot is plotted. Much higher expressions of miR-21 are observed for the disease group, demonstrating the potential utility of this method in clinical applications (Figure S3b).

Construction of the NOT Logic Gate

YES and NOT logic gates are the foundations of the architecture of logic systems. A high (logic “1”) or low (logic “0”) concentration of target miRNA is the single input; meanwhile, a high (logic “1”) or low (logic “0”) SWV peak current is the output. The biosensor performed to detect target miR-21 can be regarded as the YES logic gate, and a threshold of 2 μA is selected. Having established the YES gate, we then demonstrate a NOT gate by modifying the template of strand displacement and employing the signal transducer (TWJ1NOT). As illustrated in Figure 2a, in the absence of target, TWJ1NOT is directly added in the system for the fabrication of a three-way junction, and the following bipedal walking is able to locate MB on the electrode. The remarkable electrochemical response provides the logic “1” result (larger than 2 μA). On the contrary, a hairpin-structured templateNOT is introduced to recognize target miRNA, and the subsequent strand displacement amplification produces numerous P1NOT probes, which block TWJ1NOT by forming a complete duplex. With insufficient TWJ1NOT, the electrochemical response is supposed to be declined. By applying 0.1 pM miR-21, a peak current lower than 2 μA is observed (Figure 2b). These results fulfill the truth table for the NOT gate (Figure 2c).

Figure 2.

Figure 2

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Construction of the NOT gate. (a) Scheme of the reaction process. (b) Square wave voltammogram peak currents obtained in various input modes. (c) Truth table for the NOT logic gate.

Construction of Two-Input Logic Gates

miR-21 and miR-20a are utilized as the target examples for two-input logic gates. AND and OR logic gates are developed by introducing another three-way junction and a longer template, respectively. In addition, NAND and NOR gates are fabricated by coupling corresponding AND and OR gates with a NOT gate. The working mechanisms are depicted in Figure 3a. First, miR-21 and miR-20a initiate strand displacement amplifications to produce P1X and P1Y using template2X and template2Y, respectively.

Figure 3.

Figure 3

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Two-input logic gate construction. (a) Scheme of reaction processes for AND, OR, NAND, NOR, XOR, and XNOR gates. The inserted table lists the sequences involved in the sub logic gates for XOR and XNOR logic gates. (b) Truth table for the above two-input logic gates.

For the AND gate, a DNA three-way junction is constructed by the hybridization between P1AND, P2AND, TWJ1, and P1X. P1X acts as the first primer, which can be extended along the template of P1AND. After that, the duplex of P2AND and TWJ1 are released, the 3′ end single-stranded region of which captures P1Y for the secondary strand displacement reaction. The subsequently released TWJ1 undergoes the following bipedal walker formation and reaction processes like the YES gate. High electrochemical response can be obtained only in the presence of both of the two target miRNAs. For the OR gate, a long single-stranded template2OR is introduced that locks TWJ1 at its 3′ end by hybridization. There are another two domains that are complementary with P1X and P1Y, respectively. Either one of the two sequences can initiate polymerization, which meanwhile releases TWJ1 for the following reactions. NAND and NOR gates are based on the combination of AND and OR gates with the NOT gate. P1AND, P1NAND, P1NOT, and P1X are assembled as the three-way junction for the NAND gate. Template2NOR is used to block P1NOT instead of TWJ1 for the NOR logic gate. In addition, TWJ1NOT is spiked for both of the two gates. XOR and XNOR logic gates can be regarded as the combination of three sub logic gates. For the XOR gate, after inputting miR-21 and miR-20a generated P1X and P2Y, the outputs of a NAND gate and an OR gate are in turn applied as the secondary inputs of another AND gate. For the XNOR gate, the last logic gate of the three sub logic gates is changed to another NAND gate. The DNA strands involved in these sub gates are listed in Figure 3a and form corresponding three-way junctions and long templates for strand displacement reactions. Detailed operations can be found in the Experimental Section.

We have conducted electrochemical measurements, and the collected SWV peak currents are compared with the threshold of 2 μA. As summarized in Figure 4, “true” or “1” results are achieved in the input modes of (1,1) for the AND gate; (0,1), (1,0), and (1,1) for the OR gate; (0,0), (0,1), and (1,0) for the NAND gate; (0,0) for the NOR gate; (0,1) and (1,0) for the XOR gate; and (0,0) and (1,1) for the XNOR gate. These results are fully consistent with the expected truth table as listed in Figure 3b, demonstrating the successful fabrication of these two-input logic gates.

Figure 4.

Figure 4

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Square wave voltammogram peak currents obtained for two-input (a) AND, (b) OR, (c) NAND, (d) NOR, (e) XOR, and (f) XNOR logic gates in different input modes.

Construction of Three-Input Logic Gates

We have further tried to extend the logic gates responding to multi-inputs. miR-21, miR-20a, and miR-106a are applied as the three inputs. The working mechanisms are illustrated in Figure 5a. For the three-input AND gate, a four-way junction (P1AND, P5AND, P6AND, and TWJ1) is used instead of a three-way junction. The stand displacement reactions are conducted after the introduction of three inputs in succession, and the finally released TWJ1 strand is further applied to activate the electrochemical response. For the three-input OR gate, a long single-stranded DNA (template3OR) is prepared to capture TWJ1 by hybridization. The remaining single-stranded sequence contains three domains that are complementary with the P1X, P1Y, and P1Z. Either one of the three inputs can be extended in the presence of polymerase. The displaced TWJ1 strand is used for the bipedal walking reaction on the surface of the electrode. Various input modes are challenged with the two logic gates, and SWV peak currents are analyzed. As depicted in Figure 5b, only in the presence of the three inputs, a peak current larger than 2 μA is detected for the AND logic gate. For the OR logic gate, either one, two, or three inputs can trigger the walking event, and the peak currents are over the threshold (Figure 5c). The results demonstrate the successful construction of the three-input AND and OR gates, which are consistent with the expected truth table (Figure 5d).

Figure 5.

Figure 5

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(a) Scheme of reaction processes for three-input AND and OR gates. Square wave voltammogram peak currents obtained in various input modes for (b) the three-input AND gate and (c) the three-input OR gate. (d) Truth table for the three-input logic gates.

Conclusion

Usually, signal amplification is a critical process for ultrasensitive assays and an important mechanism for biological communications. However, in complicated logic circuits, the integration of signal amplification elements is always difficult. Previous studies might take advantage of DNAzyme for cyclical activation. However, most amplifiers have not yet achieved high sensitivity. Herein, we have carefully designed different structures and programmable DNA intermediates between inputs and outputs. Cascade strand displacement reactions and downstream bipedal walking by a catalytic hairpin assembly are coupled to enhance the output signal intensity. The background of the electrochemical signal is limited compared with that of the commonly applied fluorescence signal. In addition, the whole process does not involve strand cleavage at the sensing interface, and regeneration is possible, which is one key capability for further DNA computation investigation.

As a proof-of-concept, an ultrasensitive electrochemical biosensor for single miRNA detection is first constructed, which can be directly used for miRNA diagnosis. Through selective inhibition of bipedal walking, a NOT gate is successfully engineered. AND and NAND logic gates fabricated in this study give expected outputs only in the presence of all inputs. The feature is especially favorable in the applications of signaling pathway investigations which require that all elements participate. OR and NOR logic gates are able to respond to several inputs at the same time, which makes them useful tools for the analysis of multiple analytes. In addition, XOR and XNOR gates answer whether only one input exists in the system. The combinatorial logic gates can be applied to make judgments about detailed information on inputs. The serialized logic gates provide great prospects in the analysis of multiplex analysts and interaction networks, which can also be easily extended to more types or a greater number of targets by the modification of the architecture design.

In summary, we have engineered a DNA logic gate system with succinct molecular computing designs for the electrochemical diagnosis of multiplex miRNA inputs. By coupling cascade strand displacement reactions and bipedal walking on the electrode surface, this system performs a series of Boolean logic operations correctly. The developed logic gates effectively eliminate conventional limitations of DNA circuitry with low sensitivity. Ultrahigh sensitivity is achieved with improved processing density compared with traditional DNA logic gates. The robustness and accuracy are demonstrated by challenging biological samples. In light of these results, we envision that the logic system brings smart tools to biomedical research (e.g., in vivo controlled drug delivery) and clinical diagnosis. More broadly, the strategy can be extended to more types or greater numbers of targets and is highly appropriate for a range of biomedical applications.

Experimental Section

Materials and Chemicals

Tris(2-carboxyethyl)phosphine (TCEP), ethylenediaminetetraacetic acid (EDTA), diethylpyrocarbonate (DEPC), mercaptohexanol (MCH), and Tri reagent were ordered from Sigma. Polymerase and endonuclease were purchased from New England Biolabs (Beijing, China). Tumorigenic MCF-7, HeLa, and A549 and nontumorigenic MCF-10A cells were ordered from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The qRT-PCR kit was from Tiangen Biotech Co., Ltd. (Beijing, China). All oligonucleotides and 20 bp DNA Ladder were ordered from Takara Biotechnology Co., Ltd. (Dalian, China). The sequences are shown in Table S2. Human tissue samples were collected from a local hospital (Suzhou, China). Water was purified by a Millipore water purification system and then treated with DEPC (0.1%) before use. In all experiments, no unexpected or unusually high safety hazards were encountered.

Preparation of Track Modified Gold Electrode

The substrate gold electrode was first incubated with piranha solution for 5 min. The electrode was then rinsed. After that, it was carefully polished with silicon carbide paper (P3000) and alumina powders (diameter: 1.0, 0.3, 0.05 μm), successively. The electrode was further incubated with nitric acid (50%) for 30 min and then electrochemically cleaned with 0.5 M H2SO4. The pretreated electrode was dried with nitrogen before DNA modification. All DNA strands were heated to 94 °C and cooled to 20 °C slowly. The track probe was dissolved in 10 mM Tris-HCl (0.5 μM, 10 mM TCEP, 0.1 M NaCl, 1 mM EDTA, pH 7.5). The electrode was incubated with track for 8 h and then MCH (1 mM) for 30 min to achieve a well-aligned DNA monolayer on the electrode surface.

Bipedal DNA Walking

Standard miRNA solutions were prepared with a series of concentrations. 0.8 nM template1 was prepared with 20 mM Tris-acetate (10 mM magnesium acetate, 100 μg mL–1 BSA, 50 mM potassium acetate, pH 7.8), 120 μM dNTPs, 0.04 unit μL–1 Klenow fragment polymerase, and 0.1 unit μL–1 Nb.BbvCI. Samples were spiked with the above solution. Polymerization and nicking reactions lasted for 1 h at 30 °C to produce TWJ1 strands. In order to construct and purify the desired three-way junction nanostructure, the obtained solution was further blended with 0.4 μM TWJ2, TWJ3, and driver probes. After 1 h, 15 μL of the mixture was dropped on the track immobilized electrode for another 1 h at room temperature. The electrode was then rinsed and used for electrochemical experiments.

Logic Gates Operations

NOT, two-input, and three inputs DNA gates were operated previously before mixing with TWJ2/3 and driver for bipedal walking around the electrode surface. First, 1.5 nM DNA probes were mixed for 1 h to form the three-way junctions: briefly, P1AND, P2AND, and TWJ1 for TWJAND; P1AND, P1NAND, and P1NOT for TWJNAND; P1AND, P2NAND, and P2NOT for TWJXOR; P3AND, P4AND, and TWJ1 for TWJXOR′; and P3AND, P3NAND, and P1NOT for TWJXNOR. Two nM P1AND, P5AND, P6AND, and TWJ1 were mixed for 1 h to form the four-way junction FWJAND. For the NOT gate, 0.8 nM template1NOT was used replacing template1 to produce P1NOT via miR-21 triggered strand displacement amplification. After that, the solution was blended with 0.5 nM TWJ1NOT for 30 min. For the AND gate, 0.8 nM template2X and template2Y were used replacing template1 to produce P1X and P1Y in the presence of miR-21 and miR-20a, respectively. The resulting two strands were mixed with 0.5 nM TWJAND in the buffer solution containing 120 μM dNTPs, 0.04 unit μL–1 Klenow fragment polymerase, and 0.1 unit μL–1 Nb.BbvCI for 1 h. For the OR gate, 1 nM TWJ1 and template2OR strands were first blended for 1 h, which then interacted with P1X and P1Y under strand displacement amplification buffer conditions. Similarly, to achieve the NAND gate and NOR gate, the operations were first carried out by replacing TWJAND with TWJNAND and template2OR with template2NOR. Subsequently, 0.5 nM TWJ1NOT was spiked before following the bipedal walking process. XOR and XNOR gates could be divided into three sub logic gates. 0.8 nM template2X and template2Y were applied for strand displacement reactions to produce the primary inputs of P1X and P1Y. For the XOR gate, the above solution was mixed with 0.5 nM TWJXOR, P2X, template2OR′/P2Y duplex, and TWJXOR′ before the strand displacement reaction. For the XNOR gate, the above solution was mixed with 0.5 nM TWJXOR, P2X, template2OR′/P2Y duplex, and TWJXNOR before the strand displacement reaction. For the three-input AND gate, 0.8 nM template3Z was utilized to produce P1Z in the presence of miR-106a, which was mixed with 0.5 nM FWJAND under strand displacement amplification buffer conditions. For the three-input OR gate, the template3OR/TWJ1 duplex with the concentration of 0.5 nM was formed and then blended with P1X, P1Y, and P1Z under strand displacement amplification buffer conditions.

PAGE

The sizes of reactants and products after reactions were characterized by polyacrylamide gel electrophoresis. 10 μL of the DNA sample was blended with 2 μL of loading buffer. Later, the mixtures were added into a nondenaturing polyacrylamide hydrogel. Electrophoresis was then carried out at 110 V in 0.5× tris-borate-EDTA buffer for 90 min. After staining with diluted GelRed, various DNA bands in the gels appeared under UV light.

Cell Culture and RNA Extraction

All cells were cultured in DMEM with 10% FBS (37 °C, 5% CO2). After reaching the exponential growth period, the cells were washed using PBS. Subsequently, these cells were treated with Tri reagent, and the solutions were passed several times through a pipet to form homogeneous lysates. After chloroform extraction, total RNAs in the colorless upper phase were removed to new tubes and precipitated using isopropyl alcohol. The formed pellets were then washed twice in 70% ethanol. Subsequently, it was dried out under a vacuum and dissolved with RNase-free water.

Electrochemical Detection

A CHI 660D workstation was applied for all electrochemical measurements (CH Instruments). A saturated calomel electrode, a platinum wire electrode, as well as a gold electrode were assembled, forming a three-electrode system. CV and EIS experiments were performed in the 5 mM [Fe(CN)6]3–/4– with 1 M KNO3. The scan range and scan rate for CV were from +0.7 to 0 V and 50 mV s–1, respectively. The parameters for EIS include 5 mV amplitude, 0.207 V biasing potential, and the frequency range 0.1–100 000 Hz. SWV was performed in the electrolyte of 20 mM Tris-HCl buffer containing 100 mM NaCl, 50 μM TCEP, and 50 mM MgCl2. The parameters include the scan range from 0.2 to −0.6 V, 50 mV s–1 scan rate, 4 mV step potential, and 70 Hz frequency.

Acknowledgments

The authors gratefully acknowledge financial support from the Science and Technology Cooperation Project between the Chinese and Australian Governments (Grant 2017YFE0132300), China Scholarship Council (Grant 201904910056), and the National Natural Science Foundation of China (Grant 81771929). The authors also wish to acknowledge Prof. Nadrian C. Seeman and Prof. Ruojie Sha for their helpful discussions.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.1c00277.

  • PAGE characterization of strand displacements, DNA walking and logical operations, optimization of experimental conditions, cell sample and human tissue sample analysis, comparison of miRNA assays, and DNA/RNA sequences (PDF)

The authors declare no competing financial interest.

Supplementary Material

oc1c00277_si_001.pdf (769KB, pdf)

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