DNA Circuits Research Articles

Triplex-structure based DNA circuits with ultra-low leakage and high signal-to-noise ratio

DNA circuits are powerful tools in various applications such as logical computation, molecular diagnosis and synthetic biology. Leakage is a major problem in constructing complex DNA circuits. It directly affects the output signal and harms the circuit's performance significantly. In the traditional DNA circuits, the gate complex is a duplex structure. There are insufficient energy barriers to prevent spontaneous detachment of strands, resulting in a leak prone. Herein, we have developed triplex-structure based DNA circuit with ultra-low leakage and high signal-to-noise ratio (SNR). The triplex structure improves the stability in the absence of input. At the same time, the driving force of the strand displacement cascades reduces the influence of the triplex structure on the desired reaction. The SNR of the DNA circuit was increased to 695, while the desired reaction rate remained 90% of the conventional translator circuit. The triplex-structure mediated leakage prevention strategy was further tested at different temperatures and in DNA translator and seesaw circuits. We also constructed modular basic logic gates with a high efficiency and low leakage. On this basis, we further constructed triplex-structure based tertiary DNA logic circuits, and the SNR reached 295, which, to the best of our knowledge, was among the highest of the field. We believe that our scheme provides a novel, valid, and general tool for reducing leakages, and we anticipate that it will be widely adopted in DNA nanotechnology.

“Repaired and initiated” intramolecular DNA circuit enables the amplified imaging of DNA repair enzyme activity in live cells

Intramolecular catalytic hairpin assembly (intraCHA) has recently been developed. However, non-nucleic acid-initiated intraCHA is rarely explored. The only protein-initiated intraCHA depends on the unwarrantable binding affinity between protein and its aptamer, which generally results in limited initiation efficiency. Herein, a “repaired and initiated” intraCHA (RI-intraCHA) nanosystem was designed and its operation was reported for amplified imaging of DNA repair enzyme activity in live cells. 8-oxoguanine (8-OG) DNA glycosylase was selected as the model DNA repair enzyme. The RI-intraCHA nanosystem was constructed by linking a dsDNA (containing 8-OG sites and trigger strand), two hairpins to different vertexes of DNA tetrahedron. The nanosystem could independently enter into live cells in virtue of cell permeability of DNA tetrahedron. Upon repair action of endogenous 8-OG DNA glycosylase, intraCHA reaction in the nanosystem was initiated efficiently, generating amplified fluorescence signal. The nanosystem provided a detection limit of 0.2443 U/mL for 8-OG DNA glycosylase, which was lower than that of the reported imaging approaches. Furthermore, the nanosystem exhibited satisfactory biosafety and biostability, achieving amplified imaging of intracellular 8-OG DNA glycosylase activity. The designed RI-intraCHA nanosystem provided a promising tool for the research of basic biology of intracellular DNA repair enzymes as well as their clinic correlations.

An unique DNA junction scaffold-based electrochemical sensing platform achieving multilevel signal responses for amplified multiplexed nucleic acid analysis and logic operation

Herein, a DNA junction scaffold structure was exploited as the only immobilization probe to construct a simple and multifunctional electrochemical sensing platform. The programmable operation of a catalytic DNA circuit reaction on it afforded the amplified and well-controllable signal responses for the individual and simultaneous analysis of two nucleic acid targets. Also, it could be flexibly harnessed for each target assay based on multilevel signal response behaviours including single-signal, dual-signal and ratiometric signal responses. Then, its application for the operation of two logic devices of “AND” and “OR” gates toward different target inputs was demonstrated. Interestingly, the “OR” gate could be in-situ evolved from the “AND” status by simply controlling signal output. The low detection limit could reach 10 fM whether for the simultaneous or individual analysis of two targets in buffer or serum. Current sensing platform by only a DNA junction scaffold probe features in facile biosensor fabrication and operation, sensitive and selective analysis of multiplexed targets, well-controllable and multilevel signal response behaviours, and expandable logic operation. It thus may open a promising avenue toward the development of advanced biosensors or logic devices applied for molecular diagnosis or information processing.

Rational Design of Cascade DNA System for Signal Amplification.

System leakage critically confines the development of cascade DNA systems that need to be implemented in a strict order-by-order manner. In principle, ternary DNA reactants, composed of three single-strand DNA (ssDNA) with a strict equimolar ratio (1:1:1), have been indispensable for successfully cascading upstream entropy-driven DNA circuit (EDC) with downstream circuits, and system leakage will occur with any unbalance of the molar ratio. In this work, we proposed "splitting-reconstruction" and "protection-release" strategies on the potential downstream circuit initiator derived from upstream EDC to guide the construction of EDC-involved cascade systems independent of system leakage derived from unpurified reactants. Both the reconstructed and released downstream circuit initiators were in compliance with the principle of the cascade AND logic gate. Using these two strategies, two cascade systems─EDC2-4WJ-TMSDR and EDC3-HCR─were developed to carry out the designed order, which did not require that the ratio of 1:1:1 be maintained. Furthermore, the inherent property of the upstream EDC could transfer into the downstream circuit, endowing the developed cascade systems with a more powerful signal amplification ability for the sensitive detection of the corresponding initiator strand. These two strategies may provide new insights into the process of constructing EDC-like circuit-involved high-order DNA networks.

A Smart Nano-Theranostic Platform Based on Dual-microRNAs Guided Self-Feedback Tetrahedral Entropy-Driven DNA Circuit.

MicroRNAs (miRNAs) can act as oncogenes or tumor suppressors, capable of up or down-regulating gene expression during tumorigenesis; they are diagnostic biomarkers or therapeutic targets for tumors. To detect low abundance of intracellular oncogenic miRNAs (onco-miRNAs) and realize synergistic gene therapy of onco-miRNAs and tumor suppressors, a smart nano-theranostic platform based on dual-miRNAs guided self-feedback tetrahedral entropy-driven DNA circuit is created. The platform as a delivery vehicle is a DNA tetrahedral framework, in which the entropy-driven DNA circuit achieves a dual-miRNAs guided self-feedback, between an in situ amplification of the onco-miRNAs and activation of suppressor miRNAs release. To test this platform, dual-miRNAs are selected, miRNA-155, an up-regulated miRNA, as cancer indicators, and miRNA-122, a down-regulated miRNA as therapy targets in hepatocellular carcinoma, respectively. Through the circuit, the platform to detect onco-miRNAs at femtomolar level as well as visualized miRNAs inside cells, fixed tissues, and mice is programmed. Furthermore, triggered by miRNA-155, preloaded miRNA-122 is amplified via the self-feedback and released into target cells; the sudden increase of miRNA-122 and simultaneous decrease of miRNA-155 synergistically served as therapeutic drugs for gene regulation with enhanced antitumor efficacy and superior biosafety. It is envisioned that this nano-theranostic platform will initiate an essential step toward tumor theranostics in personalized/precise medicine.

An all-in-one assay based on CRISPR/Cas13a and a DNA circuit for rapid and ultrasensitive detection of Echovirus 11

Echovirus 11 (ECHO 11) is a positive-stranded RNA virus. The infections of ECHO 11 are associated with many clinical syndromes, thus the deployment of rapid point-of-care diagnostics is necessary. This study reported a new diagnostic assay based on All-In-One CRISPR/Cas13a and a DNA circuit (termed AIOC) for ECHO 11 detection. By combination of CRISPR/Cas13a and catalytic hairpin assembly (CHA) circuit in one-pot reaction, multiple handling steps and uncapping contamination could be effectively avoided. After recognition of target RNA, the activation of CRISPR/Cas13a induced the RNase activity of Cas13a to cleave hairpin probes, resulting in subsequent CHA amplification. This simplified assay was able to detect ECHO 11 RNA in the range of 500 × 10-18 M to 5 × 10-9 M for VP1 gene and 100 × 10-18 M to 5 × 10-9 M for RdRP gene. And the limit of detection was 152 × 10-18 M and 24 × 10-18 M, respectively. More importantly, the practicability of AIOC assay was validated by clinical ECHO samples. Taking together, the AIOC assay is rapid (∼30 min), isothermal (37 ℃), ultrasensitive, specific, user-friendly and does not need the step of RNA reverse-transcription, making it an excellent detection performance in one-pot point-of-care assay.

Programmable DNA Circuit-Facilitated Determination of Circulating Extracellular Vesicle PD-L1 for Lung Cancer Diagnosis and Immunotherapy Response Prediction.

Circulating extracellular vesicle (EV) PD-L1 is correlated with the occurrence and progression of lung cancer and has great potential as a valuable diagnostic and immunotherapy predictive biomarker. In this work, we propose a fluorescent biosensing method for the sensitive and accurate determination of circulating EV PD-L1. Specifically, after the phosphatidylserine-targeting peptide-assisted magnetic enrichment, a programmable DNA circuit is designed to translate the presence of PD-L1 to the appearance of numerous duplex DNA probes on the circulating EV surface. Upon fructose treatment, these newly formed duplex DNA probes are released from the EV surface to activate the trans-cleavage activity of CRISPR/Cas12a system, which finally produces a significant fluorescence signal. Experimental results reveal that the method not only enables sensitive determination of EV PD-L1 with a detection limit of 67 particles/mL but also demonstrates the potential use in the diagnosis and immunotherapy response prediction of lung cancer in a principle-of-proof study. Therefore, the method may provide a useful tool for EV PD-L1 determination, which may provide valuable information for the precise diagnosis and personalized treatment of lung cancer patients.

Construction of a self-sufficient DNA circuit for amplified detection of kanamycin

Improper use of kanamycin can lead to trace kanamycin residues in animal-derived foods, which can pose a potential threat to public health. Isothermal enzyme-free DNA circuits have provided a versatile toolbox for detecting kanamycin residues in complicated food samples, yet they are always limited by low amplification efficiency and intricate design. Herein, we present a simple-yet-robust nonenzymatic self-driven hybridization chain reaction (SHCR) amplifier for kanamycin determination with 5800-fold sensitivity over that of the conventional HCR circuit. The analyte kanamycin-activated SHCR circuitry can generate numerous new initiators to promote the reaction and improve the amplification efficiency, thus achieving an exponential signal gain. With precise target recognition and multilayer amplification capability, our self-sustainable SHCR aptasensor facilitated the highly sensitive and reliable analysis of kanamycin in buffer, milk, and honey samples, thus holding great potential for the amplified detection of trace contaminants in liquid food matrices.

Stimuli‐Responsive DNA Circuits for High‐Performance Bioimaging Application

AbstractProbing endogenous molecules in living entities is significant to help decipher biological functions and exploit novel theranostics. DNA circuits that can recognize molecular inputs of interest and transduce them into readable signal outputs in an isothermal and autonomous manner have been actively pursued as versatile toolkits for intracellular biosensing research. Tremendous efforts are being devoted to developing integrated DNA circuits with high sensitivity, while spatiotemporal selectivity is often overlooked in the construction of functional DNA circuitry systems. This requires the development of stimuli‐responsive DNA circuits that can be activated on‐demand from the initial sensing‐blunt state to the sensing‐ready state under a programmable manner, for achieving precise bioimaging with high spatiotemporal control. In this review, an overview of recent advances in the construction of stimuli‐responsive DNA circuits that respond to particular triggers, including external physical stimuli and endogenous biological cues, and their spatiotemporally controllable molecular bioimaging applications is provided. The current challenges and potential solutions of these stimuli‐responsive DNA circuits for their future developments in this emerging field are also discussed.

High-throughput, modular and logic analysis platform based on binary photonic crystals array and entropy-driven amplifier for multiplex biomarkers detection

The development of high-throughput and modular assay of multiplex biomarkers has great significance for disease diagnosis but it still faces some difficulty. Herein, a high-throughput, modular and logic analysis platform combining binary photonic crystals (PCs) array and entropy-driven DNA amplifier (EDA) was developed to achieve simple, high-sensitive, and nonenzymic detection of multiplex biomarkers. In this platform, three modules including logic-recognition, EDA and binary PCs array were integrated, and each module had independent and universal design but connected functions. The binary PCs array was fabricated conveniently through stacking self-assembly of two kinds of colloidal nanospheres, and could specifically enhance two kinds of fluorescence based on the match between emission peak and photonic band gap. Functionally, the input of two mRNA biomarkers initiated the logical operation of logic-recognition module, and the released DNA strand triggered corresponding EDA module for amplified fluorescence signal output (FAM or Cy5). The whole DNA circuit was performed in one step. Then, binary PCs array module further enhanced both two fluorescence signals, and got direct fluorescence signal readout by microplate reader (scanning rate of 96 samples/ 7 min). It is the first time that the binary PCs array-based high-throughput platform has achieved logical and sensitive detection of multi-biomarkers by employing logic gate and EDA. Therefore, the proposed platform has merits of logical analysis, high sensitivity, programmable design, high throughput and multiplex detection, showing potential application in bioanalysis and clinical diagnostics.

Miniature Hierarchical DNA Hybridization Circuit for Amplified Multiplexed MicroRNA Imaging.

Accurate diagnosis requires the development of multiple-guaranteed DNA circuits. Nevertheless, for reliable multiplexed molecular imaging, existing DNA circuits are limited by poor cell-delivering homogeneity due to their cumbersome and dispersive reactants. Herein, we developed a compact-yet-efficient hierarchical DNA hybridization (HDH) circuit for in situ simultaneous analysis of multiple miRNAs, which could be further exploited for specifically discriminating cancer cells from normal ones. By integrating the traditional hybridization chain reaction and catalytic hairpin assembly reactants into two highly organized hairpins, the HDH circuit is fitted with condensed components and multiple response domains, thus permitting the programmable multiple microRNA-guaranteed sequential activations and the localized cascaded signal amplification. The synergistic multi-recognition and amplification features of the HDH circuit facilitate the magnified detection of multiplex endogenous miRNAs in living cells. The in vitro and cellular imaging experimental results revealed that the HDH circuit displayed a reliable sensing performance with high selective cell-identification capacity. We anticipate that this compact design can provide a powerful toolkit for accurate diagnostics and pathological evolution.

Colorimetric aptasensor for fumonisin B1 detection based on the DNA tetrahedra-functionalized magnetic beads and DNA hydrogel-coated bimetallic MOFzyme.

The toxicity and incidence of fumonisin B1 (FB1) pose a major challenge to public health and the environment, prompting the development of alternative quantitative strategies for FB1. Herein, a colorimetric aptasensor was constructed based on DNA tetrahedra-functionalized magnetic beads (MBs) and DNA hydrogel-coated Mn-Zr bimetallic metal-organic frameworks-based nanozyme (MOFzyme). Initially, MBs functionalized by DNA tetrahedra demonstrated excellent capturing capability for FB1. Along with the capture of FB1, catalyst DNA (C) was released into the supernatant. Aided by fuel DNA (F), C can trigger continuous cleavage of the main chains and cross-linking points of the DNA hydrogel through an entropy-driven DNA circuit integrated into the hydrogel coating. Subsequently, the bimetallic MOFzyme encapsulated inside the DNA hydrogel was exposed and exerted its superb peroxidase-like activity, producing a colorimetric signal whose intensity was positively dependent on the amount of FB1. The developed aptasensor exhibited good linearity in the range of 5×10-4 to 50ngmL-1 with a limit of detection (LOD) of 0.38pgmL-1, and reasonable specificity in different matrices. Furthermore, the aptasensor was successfully applied to quantify FB1 in actual samples with recoveries fell within 92.25 %-108.00 %, showing its great potential in environmental monitoring and food safety.

Tailoring a Minimal Self-Replicate DNA Circuit for Highly Efficient Intracellular Imaging of microRNA.

Trace analyte detection in complex intracellular environment requires the development of simple yet robust self-sufficient molecular circuits with high signal-gain and anti-interference features. Herein, a minimal non-enzymatic self-replicate DNA circuitry (SDC) system is proposed with high-signal-gain for highly efficient biosensing in living cells. It is facilely engineered through the self-stacking of only one elementary cascade hybridization reaction (CHR), thus is encoding with more economic yet effective amplification pathways and reactants. Trigger (T) stimulates the activation of CHR for producing numerous T replica that reversely motivate new CHR reaction cycles, thus achieving the successive self-replication of CHR system with an exponentially magnified readout signal. The intrinsic self-replicate circuity design and the self-accelerated reaction format of SDC system is experimentally demonstrated and theoretically simulated. With simple circuitry configuration and low reactant complexity, the SDC amplifier enables the high-contrast and accurate visualization of microRNA (miRNA), ascribing to its robust molecular recognition and self-sufficient signal amplification, thus offering a promising strategy for monitoring these clinically significant analytes.

Self-Stacking Autocatalytic Molecular Circuit with Minimal Catalytic DNA Assembly.

Isothermal autocatalytic DNA circuits have been proven to be versatile and powerful biocomputing platforms by virtue of their self-sustainable and self-accelerating reaction profiles, yet they are currently constrained by their complicated designs, severe signal leakages, and unclear reaction mechanisms. Herein, we developed a simpler-yet-efficient autocatalytic assembly circuit (AAC) for highly robust bioimaging in live cells and mice. The scalable and sustainable AAC system was composed of a mere catalytic DNA assembly reaction with minimal strand complexity and, upon specific stimulation, could reproduce numerous new triggers to expedite the whole reaction. Through in-depth theoretical simulations and systematic experimental demonstrations, the catalytic efficiency of these reproduced triggers was found to play a vital role in the autocatalytic profile and thus could be facilely improved to achieve more efficient and characteristic autocatalytic signal amplification. Due to its exponentially high signal amplification and minimal reaction components, our self-stacking AAC facilitated the efficient detection of trace biomolecules with low signal leakage, thus providing great clinical diagnosis and therapeutic assessment potential.

Catalyst-Accelerated Circular Cascaded DNA Circuits: Simpler Design, Faster Speed, Higher Gain.

DNA cascaded circuits have great potential in detecting low abundance molecules in complex biological environment due to their powerful signal amplification capability and nonenzymatic feature. However, the problem of the cascaded circuits is that the design is relatively complex and the kinetics is slow. Herein, a new design paradigm called catalyst-accelerated circular cascaded circuits is proposed, where the catalyst inlet is implanted and the reaction speed can be adjusted by the catalyst concentration. This new design is very simple and only requires three hairpin probes. Meanwhile, the results of a series of studies demonstrate that the reaction speed can be accelerated and the sensitivity can be also improved. Moreover, endogenous mRNA can also be used as a catalyst to drive the circuits to amplify the detection of target miRNA in live cells and in mice. These catalyst-accelerated circular cascaded circuits can substantially expand the toolbox for intracellular low abundance molecular detection.

Chemiluminescence resonance energy transfer-based multistage nucleic acid amplification circuits for MiRNA detection with low background.

Chemiluminescence resonance energy transfer (CRET)-based assays have shown great potential in biosensing due to their negligible background autofluorescence, yet are still limited by their low sensitivity and short half-life luminescence. Herein, a multistage CRET-based DNA circuit was constructed with amplified luminescence signals for accurate miRNA detection and fixed reactive oxygen species (ROS) signals for cell imaging. The DNA circuit is designed through an ingenious programmable catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), and the use of DNAzyme to realize target-triggered precise regulation of distance between the donor and acceptor for CRET-mediated excitation of photosensitizers. In detail, the analyte catalyzes the hybridization of CHA reactants, which leads to the assembly of multiple HCR-mediated DNAzyme nanowires. Subsequently, DNAzymes catalyze the oxidation of luminol by H2O2, and the adjacent photosensitizer chlorin e6 (Ce6) anchored on the DNA nanostructure is stimulated by the CRET process, resulting in the amplified long-wavelength luminescence and the generation of single oxygen signals through further energy transfer to oxygen. The biomarker miRNA can be detected with great sensitivity by integrating the recognition module into a universal platform. Furthermore, the DNA circuit enables CRET-mediated intracellular miRNA imaging, by detecting singlet oxygen signals through a ROS probe. The significant amplification effect is attributed to the robust multiple recognition of the target and the guaranteed transduction of the CRET signal through programmable engineering of DNA nanostructures. The CRET-based DNA circuit achieves amplified long-wavelength luminescence for accurate miRNA detection with low background and ROS-mediated signal fixation for cell imaging, making it a promising candidate for early diagnosis and theranostics.

Temporal logic circuits implementation using a dual cross-inhibition mechanism based on DNA strand displacement.

Molecular circuits crafted from DNA molecules harness the inherent programmability and biocompatibility of DNA to intelligently steer molecular machines in the execution of microscopic tasks. In comparison to combinational circuits, DNA-based temporal circuits boast supplementary capabilities, allowing them to proficiently handle the omnipresent temporal information within biochemical systems and life sciences. However, the lack of temporal mechanisms and components proficient in comprehending and processing temporal information presents challenges in advancing DNA circuits that excel in complex tasks requiring temporal control and time perception. In this study, we engineered temporal logic circuits through the design and implementation of a dual cross-inhibition mechanism, which enables the acceptance and processing of temporal information, serving as a fundamental building block for constructing temporal circuits. By incorporating the dual cross-inhibition mechanism, the temporal logic gates are endowed with cascading capabilities, significantly enhancing the inhibitory effect compared to a cross-inhibitor. Furthermore, we have introduced the annihilation mechanism into the circuit to further augment the inhibition effect. As a result, the circuit demonstrates sensitive time response characteristics, leading to a fundamental improvement in circuit performance. This architecture provides a means to efficiently process temporal signals in DNA strand displacement circuits. We anticipate that our findings will contribute to the design of complex temporal logic circuits and the advancement of molecular programming.

An allosteric DNA switch-mediated catalytic DNA circuit for ratiometric and sensitive nucleic acid detection.

Herein, a new allosteric DNA switch-mediated catalytic DNA circuit reaction strategy has been proposed for ratiometric and sensitive nucleic acid detection. The sensing system was based on two DNA hybrid probes, each of which was constructed by annealing a reconfigurable DNA hairpin with single-stranded DNA. Upon target recognition by the first DNA hybrid probe, a reconfigurable DNA switch was liberated, triggering a toehold-mediated strand displacement reaction (TSDR) with the second DNA hybrid probe, which was accompanied by the release of another reconfigurable DNA switch. This released allosteric DNA switch could further interact with the first hybrid DNA probe via the TSDR strategy to form a reciprocal strand displacement network between the two DNA hybrid probes. Theoretically, this reciprocal strand displacement reaction would continue till the complete consumption of the reaction substrates. Thus, it provides a new signal amplification method leading toward target recognition. More interestingly, it creates a ratiometric signal response mode for target recognition, which involves the fluorescence increment of one fluorophore (Cy5) and concurrent decrement of another fluorophore (Cy3) accompanied by the target-triggered reciprocal strand displacement reaction. This process could achieve a low detection limit of about 0.1 pM toward the target nucleic acid and selective discrimination toward different mismatched targets. It could also be applied for detection in a serum sample. Thus, the developed catalytic DNA circuit reaction strategy together with ratiometric signal readout provides a new avenue for programmable, reliable and sensitive detection of nucleic acids and might also pave the way for developing more advanced DNA circuits or biosensors.

A versatile and convenient tool for regulation of DNA strand displacement and post-modification on pre-fabricated DNA nanodevices.

Toehold-mediated strand displacement and its regulatory tools are fundamental for DNA nanotechnology. However, current regulatory tools all need to change the original sequence of reactants, making the regulation inconvenient and cumbersome. More importantly, the booming development of DNA nanotechnology will soon promote the production of packaged and batched devices or circuits with specified functions. Regarding standardized, packaged DNA nanodevices, access to personalized post-modification will greatly help users, whereas none of the current regulatory tools can provide such access, which has greatly constrained DNA nanodevices from becoming more powerful and practical. Herein, we developed a novel regulation tool named Cap which has two basic functions of subtle regulation of the reaction rate and erasability. Based on these functions, we further developed three advanced functions. Through integration of all functions of Cap and its distinct advantage of working independently, we finally realized personalized tailor-made post-modification on pre-fabricated DNA circuits. A pre-fabricated dual-output DNA circuit was successfully transformed into an equal-output circuit, a signal-antagonist circuit and a covariant circuit according to our requirements. Taken together, Cap is easy to design and generalizable for all strand displacement-based DNA nanodevices. We believe the Cap tool will be widely used in regulating reaction networks and personalized tailor-made post-modification of DNA nanodevices.

Colorimetric aptasensor for the sensitive detection of ochratoxin A based on a triple cascade amplification strategy

Ochratoxin A(OTA), a highly toxic mycotoxin commonly found in food, poses a serious threat to health even at low concentrations. Developing a sensitive, accurate, simple, and cost-effective detection method is of great significance for food safety and quality control. Herein, a triple cascade amplification strategy was used to construct the colorimetric assay for the detection of OTA, where the amplification process consists of an entropy-driven DNA circuit (EDC), a catalytic hairpin assembly (CHA), and Mg2+-assisted DNAzyme catalysis (MNAzyme). Through the specific binding of ochratoxin A (OTA) and its aptamer, an initiator strand is released to initiate upstream EDC and then produce a new trigger unit that motivates downstream CHA to generate MNAzyme, which further cleaves the substrate strand to induce the formation of G-quadruplex/hemin DNAzyme as a signal readout. The aptasensor was shown to detect OTA, with a low detection limit of 8.7 fM and good selectivity. The developed method could be used as a highly colorimetric aptasensor for the detection of OTA in spiked rice samples.