Entropy-driven DNA Research Articles

Self-reinforced D-MOF and autocatalytic AuPd@SnS2 dual-wavelength electrochemiluminescence biosensor for detection of ctDNA and CEA

Trace detection of circulating tumor DNA (ctDNA) and CEA is of great significance for early prevention and diagnosis of cancer. In this work, the well electrochemiluminescence (ECL) of the dual-ligand metal-organic frameworks (D-MOFs) and AuPd@SnS2 QDs nuclear-satellite conjugate was newly explored, and a dual-wavelength biosensing platform based on ECL energy transfer between D-MOFs and AuPd NPs coupled with catalytic effect of AuPd NPs on ECL of SnS2 QDs was developed for detection of trace ctDNA and CEA. Here, thanks to the ordered heterogeneous arrangement and network charge transfer of the luminescent ligand, the prepared D-MOF exhibits highly enhanced ECL without exogenous co-reactants. Target ctDNA was exponentially amplified using an entropy-driven DNA cycle amplification strategy to obtain bandage DNA, which triggered the DNA walker to connect the AuPd@SnS2 core-satellite probe to the electrode. Therefore, the ECL ratio of D-MOF and sensitized SnS2 at 535 nm and 650 nm was used to achieve ultrasensitive detection of trace ctDNA. Moreover, the specific recognition of target CEA to T1 DNA induced changes in dual-wavelength signals to achieve further detection of CEA, and ensure the accuracy of cancer analysis. The work opened up two novel ECL materials and developed a novel ECL biosensor for rapid detection of double tumor markers, providing a new way for early prevention and diagnosis of actual cancer.

A self-propelled DNAzyme-mediated cascading entropy-driven catalysis DNA nanodevice for high-efficiency imaging in living cells and mouse bodies under near-infrared light initiation

Despite the promising application prospects exhibited by entropy-driven catalysis (EDC) DNA nanodevice-founded fluorescence biosensors for imaging disease biomarkers in living bio-samples, they still face challenges of inadequate sensitivity and uncontrollable initiation. In response, we devise a self-propelled DNAzyme-mediated cascading EDC DNA nanodevice initiated by near-infrared light. Firstly, the wide-ranging interface of MnO2 nanosheets is utilized to co-load all essential nucleic acid components required for EDC through physisorption. Following reduction by the prevalent existence of glutathione in biological media, the resultant Mn2+ serves as the self-propelling force for a Mn2+-reliant DNAzyme, which further combines with EDC to carry out a cascading dual-cycle signal amplification. Subsequently, the analyte binding unit is locked by inserting one DNA modulation with a photon-triggered cleavage connector, thereby implementing an upconverting luminescence-enabled NIR light initiation concept to prevent the biosensor from being prematurely activated during bio-delivery. Upon opting for a non-coding microRNA (miRNA-21, commonly overexpressed in diverse cancers) as the model low-abundance analyte, our newly-developed DNA nanodevice showcases ultra-high sensitivity and satisfactory specificity in detection performance. Looking ahead, this biosensing system can function as a high-efficiency imaging toolbox for analyte analysis in living cells and even mouse bodies, making a considerable contribution to employing DNA nanodevices for disease diagnosis.

New quasi-Co/Cu-MOF nanozyme coupled with entropy-driven DNA amplification cascades for highly sensitive electrochemical aptamer growth differentiation factor 15 assay

BackgroundThe monitoring of concentration variation of the newly developed growth differentiation factor 15 (GDF15) biomarker in human serum is of great significance for diagnosing cardiovascular diseases. Current methods for the detection of the GDF15 protein mainly are based on antibody-assisted immunoassays, which encounter the limitations in terms of sensitivity, complexity and costs. The development of simple and sensitive biosensors for GDF15 can therefore facilitate the diagnosis of cardiovascular diseases. ResultsA new bimetallic quasi-Cu/Co-MOF nanozyme with high catalytic performance for electrochemical reduction of H2O2 is synthesized via simple one-step precipitation and low-temperature calcination method. Such nanozymes are further employed as amplification tags and coupled with cyclic entropy-driven DNA signal enhancement strategies to construct ultrasensitive aptamer-based biosensor for detecting GDF15 in human serums. GDF15 molecules associate with two aptamers and release the ssDNA trigger sequences via target-binding induced displacement reaction. These ssDNAs subsequently initiate cyclic DNA-fueled strand displacement and catalytic hairpin assembly (CHA) reaction cascades for confining many quasi-Cu/Co-MOF nanozymes on sensor electrode, which yield drastically amplified H2O2 reduction current for detecting GDF15 down to 0.12 pg mL−1 with a dynamic range of 0.5 pg mL−1 to 20 ng mL−1. The electrochemical aptasensor also presents good reproducibility and selectivity and exhibits the capability to detect GDF15 in diluent serums. SignificanceOur aptamer-based GDF15 protein electrochemical assay clearly outperforms current existing antibody-based methods and the quasi-Cu/Co-MOF nanozyme/entropy-driven cascaded signal amplification means can be used as a universal strategy for sensitive monitoring of different biomolecular markers for diverse applications.

A GSH/APE1-controlled framework nucleic acids based entropy-driven DNA circuit for high-contrast miRNAs imaging

Catalytic DNA circuits show great potential for precise intracellular imaging. However, their selectivity and efficiency are often hindered by low anti-interference performance in the cytoplasm’s complex environment. Thus, designing DNA circuits that exhibit enhanced stability and specific activation is crucial for the accurate intracellular biomolecule imaging. Herein, we proposed a framework nucleic acids based endogenously activated entropy-driven catalytic (EDC) circuit, FEED, for in vivo microRNA imaging with enhanced selectivity and efficiency. To mitigate undesired signal leakage before reaching the target cells, the dominating EDC circuitry fuel strand was initially blocked with a disulfide bond modified DNA strand, and the target recognition site of sensing module was also closed by apurinic/apyrimidinic (AP) sites. This configuration allowed selective activation of the EDC by endogenous GSH and human apurinic/apyrimidinic endonuclease 1 (APE1) in cancer cells, facilitating high-contrast miRNA imaging. Additionally, the integrating a distinctive DNA tetrahedral structure into the EDC circuit enhances both biostability and cellular reaction efficacy. This in-site activation FEED circuit offers a programmable and modular amplification strategy for biomarker detection in live cells and mice, providing a potentially valuable molecular tool for living systems.

An ultra-sensitive CRISPR-Cas12a and aptamer-based biosensor utilizing Entropy-driven catalytic DNA networks for precise detection of DNA Methyltransferase 1

DNA Methyltransferase 1 (DNMT1) serves as a crucial biomarker associated with various diseases and is essential for evaluating DNA methylation levels, diagnosing diseases, and evaluating prognosis. As a result, a convenient, quantitative, and sensitive assay for detecting DNMT1 is in high demand. However, current techniques for DNMT1 detection struggle to balance accuracy, low cost, and high sensitivity, limiting their clinical usefulness. To address this challenge, we have developed a DNMT1 detection method (CAED), which combines aptamer-specific recognition with a highly programmable Entropy-driven catalysis DNA network and is further integrated with the CRISPR-Cas12a system. This innovative approach achieves a detection limit as low as 90.9 fmol/L. To demonstrate the clinical applicability and significance of our CAED method, we successfully measured DNMT1 levels in 10 plasma samples 10 cervical tissue samples. These results underscore the potential of our method as an accurate, affordable, and ultra-sensitive tool for evaluating DNMT1 levels. This innovative method offers a potent means for assessing DNMT1 levels and significantly advances disease diagnosis and health risk prediction. Plus, it establishes an innovative design framework for CRISPR-Cas12a-based biosensors, tailored explicitly for enzyme content quantification.

Homogeneous electrochemical ratiometric biosensor for MircoRNA detection based on UiO-66-NH2 signal probe and waste-free entropy-driven DNA machine

The construction of efficient methods for highly sensitive and rapid detection of disease markers is essential for the early diagnosis of serious diseases. In this paper, taking advantage of the UiO-66-NH2 signal molecule in combination with a waste-free entropy-driven DNA machine, a novel homogeneous electrochemical ratiometric platform is developed to detect MircoRNA (miRNA). Metal-organic framework materials (UiO-66-NH2 MOF) and ferrocene were utilized as electrochemical signal tags and reference probes, respectively. The target-initiated waste-free three-dimensional (3D) entropy-driven DNA nanomachine is activated in the presence of miRNA, resulting in DNA-labeled-UiO-66-NH2 falling off from the electrode, leading to a decrease in the signal of UiO-66-NH2 at 0.83V. Our strategy can mitigate false positive responses induced by the DNA probes immobilized on electrodes in traditional distance-dependent signal adjustment ratiometric strategies. The proposed ratiometric platform demonstrates superior sensitivity (a detection limit of 9.8 fM), simplified operation, high selectivity, and high repeatability. The ratiometric biosensor is also applied to detect miRNA content in spiked serum samples.

Ultrasensitive serological digoxin fluorescence sensor based on cascade amplification of entropy-driven DNA nanomachine and proximity ligation assay

A nanobiosensor was prepared by DNA nanomachine based on proximity ligation assay (PLA) for the detection of digoxin (DIG) in serum. In this sensor the DNA nanomachine releases a large number of signal probes (SP) after addition of DIG, which participate in PLA as the connector strand, initiating real-time quantitative polymerase chain reaction (qPCR) and producing fluorescence signals. For the benefit of clinical therapeutics, we determined the detection range of 0.1 ∼ 5 ng/mL. Under the optimal experimental conditions, the cycle threshold (Ct) value showed a good linear relationship with DIG concentrations, and the detection limit was as low as 0.06 ng/mL (3σ). This method achieved the ultrasensitive detection of DIG by signal amplification and was successfully used for the detection of DIG in real serum, which has a good potential for application in the field of therapeutic drug monitoring.

Electrochemical Monitoring of Sphingosine-1-phosphate-Induced ATP Release Using a Microsensor Based on an Entropy-Driven Bipedal DNA Walker.

Neuropathic pain is a chronic and severe syndrome for which effective therapy is insufficient and the release of ATP from microglia induced by sphingosine-1-phosphate (S1P) plays a vital role in neuropathic pain. Therefore, there is an urgent demand to develop highly sensitive and selective ATP biosensors for quantitative monitoring of low-concentration ATP in the complex nervous system, which helps in understanding the mechanism involved in neuropathic pain. Herein, we developed an electrochemical microsensor based on an entropy-driven bipedal DNA walker. First, the microsensor specifically recognized ATP via ATP aptamers, initiating the entropy-driven bipedal DNA walker. Subsequently, the bipedal DNA walker autonomously traversed the microelectrode interface, introducing methylene blue to the electrode surface and achieving cascade signal amplification. This microsensor showed excellent selectivity, stability, and a low limit of detection at 1.13 nM. The S1P-induced ATP release from BV2 cells was successfully monitored, and it was observed that dicumarol could inhibit this release, suggesting dicumarol as a potential treatment for neuropathic pain. The microsensor's small size exhibited significant potential for monitoring ATP level changes in neuropathic pain in vivo, which provides a new strategy for in situ and quantitative monitoring of nonelectroactive biomolecules associated with neurological diseases.

Latent Toehold-Mediated DNA Circuits Based on a Bulge-Loop Structure for Leakage Reduction and Its Application to Signal-Amplifying DNA Logic Gates.

DNA circuits based on successive toehold-mediated DNA displacement reactions, particularly entropy-driven DNA circuit (EDC) systems, have attracted considerable attention as powerful enzyme-free tools for dynamic DNA nanotechnology. However, background leakage (noise signal) often occurs when the circuit is executed nonspecifically, even in the absence of the appropriate catalyst DNA (input). This study designed and developed a new latent toehold-mediated DNA circuit (LDC) system that relies on a bulge-loop structure as a latent toehold toward leakage reduction. Furthermore, the number (size) of nucleotides (nt) in the bulge-loop is found to play a significant role in the performance (i.e., leakage, signal, and kinetics) of LDC systems. In fact, the signal rate for the LDC systems increased as the number of nt in the bulge-loop increased from 4 to 8, whereas the leakage rate of the LDC systems with bulge-loops of 7 nt or less was low, but the leakage rate of the LDC system with a bulge-loop of 8 nt increased significantly. Note that the LDC system with the optimal bulge-loop (7 nt) was capable of not only reducing the leakage but also accelerating the circuit speed without any signal loss, unlike methods of reducing the leakage by reducing the signal reported previously for the conventional EDC systems. These facts indicate that the 7 nt bulge-loop acts as a "latent" toehold for the DNA circuit system. By using the amplification function of output signals with an accelerated circuit and reduced leakage, our LDC system with a 7 nt bulge-loop could be applied directly and successfully to signal-amplifying DNA logic gates such as OR and AND gates, and thus, sufficient output signals could be obtained even with a small amount of input. These findings reveal that our LDC systems with a bulge-loop structure can replace the conventional EDC system and have enormous potential in the field of DNA nanotechnology.

An electrochemical biosensor designed with entropy-driven autocatalytic DNA circuits for sensitive detection of ovarian cancer-derived exosomes

Intelligent artificial DNA circuits have emerged as a promising approach for modulating signaling pathways and signal transduction through rational design, which may contribute to comprehensively realizing biomolecular sensing of organisms. In this work, we have fabricated an electrochemical biosensor for the sensitive and accurate detection of ovarian cancer-derived exosomes by constructing an entropy-driven autocatalytic DNA circuit (EADC). Specifically, the robust EADC is prepared by the self-assembly of well-designed DNA probes, and upon stimulation of the presence of ovarian cancer cells-derived exosomes, numerous inputs can be produced to feedback and accelerate the reaction. The catalytic abilities of the generated input sequences play a pivotal role in EADC and dramatically enhance the signal amplification capability. Through the combination of the autocatalytic circuit and circular cleavage reactions, significantly changed electrochemical signals can be recorded for sensitive analysis of the exosomes with a remarkably low detection limit of 30 particles/μL. Moreover, the proposed enzyme-free biosensor shows exceptional performance in distinguishing patient samples from healthy samples, which exhibits promising prospects for the clinical diagnosis of ovarian cancer.

NIR light-controlled DNA nanodevice for amplified mRNA imaging and precise gene therapy

Accurately delivering antisense oligonucleotides (ASOs) to tumor cells for gene therapy poses a significant challenge. To address this issue, we develop a NIR light-activable DNA nanodevice to target survivin mRNA and simultaneously release ASOs in tumor cells, which allows high-precision spatiotemporal imaging and efficient gene therapy. The concept is based on upconversion nanoparticles, which can convert NIR light into UV emissions to activate an entropy-driven DNA walking system for mRNA recognition in tumor cells. The intramolecular toehold-mediated entropy-driven catalytic reaction generates a large amount of Bcl-2 ASOs, which allows accurate delivery of ASOs for gene therapy due to the specific targeting of mRNA in tumor cells. The results from in vitro and in vivo experiments show that the NIR light-activated DNA nanodevice can detect mRNA with high sensitivity by DNA walking amplification and high precision in gene delivery. Moreover, the released ASOs from DNA walking system down-regulate the expression of Bcl-2 anti-apoptosis protein and induce tumor cell apoptosis, thereby suppressing tumor growth without a transfection reagent. The NIR-light-controlled DNA nanodevice holds great potential for precise gene therapy in clinical applications.

Nickel Single-Atom Catalyst Boosts Electrochemiluminescence of Graphitic Carbon Nitride for Sensitive Detection of HBV DNA.

Owing to excellent catalytic activity, single-atom catalysts (SACs) have recently attracted considerable research interest in the electrochemiluminescence (ECL) field. However, the applications of SACs are mostly limited to conventional luminol ECL system. Hence, it is necessary to explore the application of SACs in more ECL systems. In this work, nickel single-atom catalysts (Ni SACs) were successfully applied in the graphitic carbon nitride (g-C3N4)-H2O2 ECL system to significantly enhance its cathodic emission. Notably, g-C3N4 acted not only as an ECL luminophore but also as a support to anchor Ni SACs. Ni SACs can significantly activate H2O2 to produce abundant OH• radicals for enhancing the cathodic ECL emission of g-C3N4. Ni SACs-anchored g-C3N4 (Ni SACs@g-C3N4) had a 10-fold enhanced ECL intensity as compared to g-C3N4. Finally, the Ni SACs@g-C3N4-H2O2 ECL system was developed to detect hepatitis B virus (HBV) DNA by incorporating an entropy-driven DNA walking machine-assisted CRISPR-Cas12a amplification strategy. The constructed biosensor exhibited excellent detection performance for HBV DNA with a limit of detection as low as 17 aM. This work puts forward a new idea for enhancing the cathodic ECL of g-C3N4-H2O2 and expands the application of SACs in the ECL system.

Entropy-driven multicolor DNA nanoflowers for simultaneous and rapid detection of multiple heavy metal ions in water

Heavy metal ions (HMIs), including Pb2+, Cu2+, and Hg2+, has shown great threat to the environment and public health worldwide owing to their high toxic at trace levels. However, conventional laboratory-based methods for analyzing HMIs require expensive instrumentation and complex operations limit their further application. In this study, we present a novel “two-step” approach based on entropy-driven multicolor DNA nanoflowers (DNFL) for the simultaneous ultrasensitive detection of three typical HMIs (Pb2+, Cu2+, and Hg2+). Through specific cleavage/competition reactions initiated by the target metal ions, a large number of single-stranded DNA (ssDNA) are produced, triggering the blooming of DNFL. Subsequently, with the assistance of fuel strands, an entropy-driven strand displacement reaction (ESDR) will further accelerate the conformational change of DNFL within one hour. Different fluorophores (FAM, ROX, and Cy5) attached to the opened DNFL serve as indicator molecules for the respective HMIs. After reasonable design and optimization, the developed fluorescence analysis method demonstrates excellent analytical performance (with limit of detection, 0.13 nM, 3.5 pM, 4.6 pM for Pb2+, Cu2+, and Hg2+, respectively) and can be applied to the simultaneous and rapid detection of multiple HMIs in water environment.

A novel isothermal amplification strategy for rapid and sensitive detection of Matrix Metalloproteinase 2 using a bipedal DNA walker in anti-aging research

In the field of anti-aging research, a groundbreaking study introduces a novel isothermal amplification strategy for the rapid and sensitive detection of Matrix Metalloproteinase 2 (MMP-2) utilizing a bipedal DNA walker. MMP-2 plays a crucial role in skin aging due to its involvement in the degradation of collagen and elastin, key components of the extracellular matrix. The dysregulation of MMP-2 activity contributes to the breakdown of the extracellular matrix, leading to manifestations of skin aging such as wrinkles, sagging, and loss of elasticity. This research presents a novel method for detecting MMP-2 using a bipedal DNA walker. The method uses peptide nucleic acid (PNA) as a substrate for MMP-2, which can release a bandage strand that forms the bipedal walker “leg” with two DNA strands. The bipedal walker “leg” initiates the bipedal walker isothermal amplification strategy that involves entropy-driven DNA walking and catalytic hairpin assembly on a PEI@Ru(bpy)32+-Ti3C2 @AuNPs-modified electrode. The walker’s motion produces an electrochemiluminescence (ECL) signal that can be rapidly analyzed by a biosensor. The detection limit of this method is 80.6 fM, which is significantly lower than other methods. Overall, this study represents an important step towards the development of effective anti-aging research methods.

A dual amplification-based CRISPR/Cas12a biosensor for sensitive detection of miRNA in prostate cancer

MicroRNA (miRNA) has gained significant attention as a potential biomarker for cancer clinics, and there is an urgent need for developing sensing strategies with high selectivity, sensitivity, and low background. In vitro diagnosis based on Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated protein (CRISPR/Cas) technology could simplify the detection procedure, improve sensitivity and selectivity, and has broad application prospects as the next-generation molecular diagnosis technology. We propose a novel dual signal amplification strategy, called CENTER, which integrates the CRISPR/Cas12a system, an entropy-driven DNA signaling network, and strand displacement amplification to achieve ultrasensitive detection of miR-141, a potential marker for prostate cancer. The experimental results demonstrate that CENTER can distinguish single nucleotide mutations, and the strategy exhibits a good linear calibration curve ranging from 100 aM to 1 pM. Due to dual signal amplification, the detection limit is as low as 34 aM. We proposed a method for identifying miR-141 expressed in human serum and successfully distinguished between prostate cancer patients (n = 20) and healthy individuals (n = 15) with an impressive accuracy of 94%. Overall, CENTER shows great promise for the detection of miRNA.

Inverting the DNA tetrahedron: A novel strategy for sensitive and stable detection of aging-related enzyme MMP2

In the current study, a novel electrochemiluminescence biosensor based on the entropy-driven DNA tetrahedron for the detection of matrix metalloproteinase 2 (MMP2), an enzyme that regulates extracellular matrix remodeling and affects aging was reported. The biosensor utilizes an inverted DNA tetrahedron structure, which exposes three vertices to the solution, as molecular recognition units for capturing specific biomolecules. The biosensor also employs a ratiometric method and an entropy-driven reaction, which enhance the response rate and sensitivity of the detection. The biosensor can detect MMP2 with a detection limit of 55.2 fM, which is lower than that of conventional sensors. The biosensor also exhibits excellent stability and reproducibility, and can accurately measure MMP2 levels in complex samples, such as human serum. The paper demonstrates the feasibility and effectiveness of using the “inverted” DNA tetrahedron structure and the entropy-driven process to construct interfacial biosensors. The paper also discusses the potential applications of the biosensor in clinical diagnosis and anti-aging research, where MMP2 plays a crucial role in tissue damage and repair. The paper provides a valuable contribution to the field of biosensor development, and opens up new possibilities for using DNA nanotechnology for sensitive and reliable detection of various biomolecules.

Butanol accelerated entropy-driven DNA walking machine for rapid and ultrasensitive determination of alkaline phosphatase activity

Alkaline phosphatase (ALP) as an important biomarker as well as an index for the pasteurization degree of dairy food. However, there is a dilemma between the sensitivity and time-cost of ALP determination based on nucleic acid amplification approach. Herein, an ultrasensitive and rapid detection method for the ALP assay was developed based on entropy-driven DNA machine. In our design, the ALP catalyzed dephosphorylation of detection probe, which inhibited the digestion effect of lambda exonuclease. The remaining probe as a linker to tether the walking strand proximity to the surface of track strand modified gold nanoparticle, activating entropy-driven DNA machine. Accompany with walking strand moving, a large amount of assembled dye-labelled strand dissociated from gold nanoparticle with fluorescence recovery. More importantly, to further improve the walking efficiency, butanol was introduced to accelerated the signal amplification at interface, which short the incubation time from several hours to 5 min. Under the optimum condition, the change of fluorescence intensity was proportion to the concentration of ALP in the range from 0.05 U L−1 to 5 U L−1 with an ultralow limit of detection of 2.07 × 10−3 U L−1 was achieved, which is superior to other reported methods. Furthermore, the proposed method also successfully applied for the spiked milk sample assay with satisfactory recovery in the range of 98.83%–103.00%. This work proposed a new strategy for the application of entropy-driven DNA machine in the field of rapid and ultrasensitive detection.

N/S-Co-doped carbon dot-based FRET ratiometric fluorescence aptasensing platform modulated with entropy-driven DNA amplifier for ochratoxin A detection.

This study proposes a nitrogen and sulfur co-doped carbon dot (N/S-CD)-based FRET ratiometric fluorescence aptasensing strategy modulated with entropy-driven DNA amplifier for sensitive and accurate detection of ochratoxin A (OTA). In the strategy, a duplex DNA probe containing OTA aptamer and complementary DNA (cDNA) is designed as arecognition and transformation element. Upon sensing of target OTA, the cDNA was liberated, and triggered a three-chain DNA composite-based entropy-driven DNA circuit amplification, making CuO probes anchor on amagnetic bead (MB). The CuO-encoded MB complex probe is finally turned into abundant Cu2+, which oxidizes o-phenylenediamine (oPD) to generate 2,3-diaminophenazine (DAP) with yellow fluorescence and further triggers FRET between the blue fluorescent N/S-CDs and DAP. The changes in ratiometric fluorescence are related to the OTA concentration. Originating from the synergistic amplifications from the entropy-driven DNA circuits and Cu2+ amplification, the strategy dramatically enhanced detection performance. A limit of detection as low as 0.006 pg/mL of OTA was achieved. Significantly, the aptasensor can visually evaluate the OTA via on-site visual screening. Moreover, the high-confidence quantification of the OTA in real samples with results consistent with that of the LC-MS method indicated that the proposed strategy has practical application prospects for sensitive and accurate quantification in food safety.

Entropy-driven DNA circuit with two-stage strand displacement for elegant and robust detection of miRNA let-7a

MicroRNAs (miRNAs) research in cancer diagnosis is expanding, on account of miRNAs were demonstrated to be key indicator of gene expression and hopeful candidates for biomarkers. In this study, a stable miRNA-let-7a fluorescent biosensor was successfully designed based on an exonuclease Ⅲ-assisted two-stage strand displacement reaction (SDR). First, an entropy-driven SDR containing a three-chain structure of the substrate is used in our designed biosensor, leading to reduce the reversibility of the target recycling process in each step. The target acts on the first stage to start the entropy-driven SDR, which generates the trigger used to stimulate the exonuclease Ⅲ-assisted SDR in the second stage. At the same time, we design a SDR one-step amplification strategy as a comparison. Expectly, this developed two-stage strand displacement system has a low detection limit of 25.0 pM as well as a broad detection range of 4 orders of magnitude, making it more sensitive than the SDR one-step sensor, whose detection limit is 0.8 nM. In addition, this sensor has high specificity across members of the miRNA family. Therefore, we can take advantage of this biosensor to promote miRNA research in cancer diagnosis sensing systems.

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.