Enzyme-free and rapid colorimetric analysis of osteopontin via triple-helix aptamer probe coupled with catalytic hairpin assembly reaction.

BACKGROUND: Osteopontin (OPN) is closely associated with tumorigenesis, growth, invasion, and immune escape and it serves as a plasma biomarker for hepatocellular carcinoma (HCC). Nevertheless, the accurate and rapid detection of low-abundance OPN still poses significant challenges. Currently, the majority of protein detection methods rely heavily on large precision instruments or involve complex procedures. Therefore, developing a simple, enzyme-free, rapid colorimetric analysis method with high sensitivity is imperative. RESULTS: In this study, we have developed a portable colorimetric biosensor by integrating the triple-helix aptamer probe (THAP) and catalytic hairpin assembly (CHA) strategy, named as T-CHA. After binding to the OPN, the trigger probe can be released from THAP, then initiates the CHA reaction and outputs the signal through the formation of a G-quadruplex/Hemin DNAzyme with horseradish peroxidase-like activity. Consequently, this colorimetric sensor achieves visual free-labeled detection without additional fluorophore modification and allows for accurate quantification by measuring the optical density of the solution at 650 nm. Under optimal conditions, the logarithmic values of various OPN concentrations exhibit satisfactory linearity in the range of 5 pg mL-1 to 5 ng mL-1, with a detection limit of 2.04 pg mL-1. Compared with the widely used ELISA strategy, the proposed T-CHA strategy is rapid (∼105 min), highly sensitive, and cost-effective. SIGNIFICANCE: The T-CHA strategy, leveraging the low background leakage of THAP and the high catalytic efficiency of CHA, has been successfully applied to the detection of OPN in plasma, demonstrating significant promise for the early diagnosis of HCC in point-of-care testing. Given the programmability of DNA and the universality of T-CHA, it can be readily modified for analyzing other useful tumor biomarkers.

Development of Aptamer-DNAzyme based metal-nucleic acid frameworks for gastric cancer therapy.

The metal-nucleic acid nanocomposites, first termed metal-nucleic acid frameworks (MNFs) in this work, show extraordinary potential as functional nanomaterials. However, thus far, realized MNFs face limitations including harsh synthesis conditions, instability, and non-targeting. Herein, we discover that longer oligonucleotides can enhance the synthesis efficiency and stability of MNFs by increasing oligonucleotide folding and entanglement probabilities during the reaction. Besides, longer oligonucleotides provide upgraded metal ions binding conditions, facilitating MNFs to load macromolecular protein drugs at room temperature. Furthermore, longer oligonucleotides facilitate functional expansion of nucleotide sequences, enabling disease-targeted MNFs. As a proof-of-concept, we build an interferon regulatory factor-1(IRF-1) loaded Ca2+/(aptamer-deoxyribozyme) MNF to target regulate glucose transporter (GLUT-1) expression in human epidermal growth factor receptor-2 (HER-2) positive gastric cancer cells. This MNF nanodevice disrupts GSH/ROS homeostasis, suppresses DNA repair, and augments ROS-mediated DNA damage therapy, with tumor inhibition rate up to 90%. Our work signifies a significant advancement towards an era of universal MNF application.

Photoactivated Controlled Dnazyme Platform for on-Demand Activation Sensitive Electrochemiluminescence mRNA Analysis.

Programming ultrasensitive and stimuli-responsive DNAzyme-based probes holds great potential for on-demand biomarker detection. Here, an optically triggered DNAzyme platform was reported for on-demand activation-sensitive electrochemiluminescence (ECL) c-myc mRNA analysis. In this design, the sensing and recognition function of the split DNAzyme (SDz) probe was silent by engineering a blocking sequence containing a photocleavable linker (PC-linker) group at a defined site that could be indirectly cleaved by 302 nm ultraviolet (UV) light. When the SDz probes were assembled on the Au nanoparticles and potassium (K) element doped graphitic carbon nitride nanosheet (K-doped g-C3N4) covered electrode, UV light activation induces the configurational switching and consequently the formation of an active DNAzyme probe with the help of target c-myc mRNA, allowing the cleavage of the substrate strand by magnesium ions (Mg2+). Thus, the release of a ferrocene (Fc)-labeled DNAzyme 2 strand contributed to an extreme ECL signal recovery. In the meantime, the released target c-myc mRNA combined another inactive SDz motif to form active DNAzyme and repeat the cyclic cleavage reaction, resulting in the signal amplification. Furthermore, according to the responses toward two other designed nPC-SDz and m-SDz probes, we demonstrated that controlled UV light mediated photoactivation of the DNAzyme biosensor "on demand" effectively constrained the ECL signal to the mRNA of interest. Moreover, false positive signals could also be avoided due to such a photoactivation design with UV light. Therefore, this study provided a simple methodology that may be broadly applicable for investigating the mRNA-associated physiological events that were difficult to access using traditional DNAzyme probes.

Multiarmed DNA jumper and metal-organic frameworks-functionalized paper-based bioplatform for small extracellular vesicle-derived miRNAs assay.

Small extracellular vesicle-derived microRNAs (sEV-miRNAs) have emerged as promising noninvasive biomarkers for early cancer diagnosis. Herein, we developed a molecular probe based on three-dimensional (3D) multiarmed DNA tetrahedral jumpers (mDNA-Js)-assisted DNAzyme activated by Na+, combined with a disposable paper-based electrode modified with a Zr-MOF-rGO-Au NP nanocomplex (ZrGA) to fabricate a novel biosensor for sEV-miRNAs Assay. Zr-MOF tightly wrapped by rGO was prepared via a one-step method, and it effectively aids electron transfer and maximizes the effective reaction area. In addition, the mechanically rigid, and nanoscale-addressable mDNA-Js assembled from the bottom up ensure the distance and orientation between fixed biological probes as well as avoid probe entanglement, considerably improving the efficiency of molecular hybridization. The fabricated bioplatform achieved the sensitive detection of sEV-miR-21 with a detection limit of 34.6 aM and a dynamic range from100 aM to 0.2 µM. In clinical blood sample tests, the proposed bioplatform showed results highly consistent with those of qRT-PCRs and the signal increased proportionally with the NSCLC staging. The proposed biosensor with a portable wireless USB-type analyzer is promising for the fast, easy, low-cost, and highly sensitive detection of various nucleic acids and their mutation derivatives, making it ideal for POC biosensing.

The 8-17 DNAzyme can operate in a single active structure regardless of metal ion cofactor.

DNAzymes - synthetic enzymes made of DNA - have long attracted attention as RNA-targeting therapeutic agents. Yet, as of now, no DNAzyme-based drug has been approved, partially due to our lacking understanding of their molecular mode of action. In this work we report the solution structure of 8-17 DNAzyme bound to a Zn2+ ion solved through NMR spectroscopy. Surprisingly, it turned out to be very similar to the previously solved Pb2+-bound form (catalytic domain RMSD = 1.28 Å), despite a long-standing literature consensus that Pb2+ recruits a different DNAzyme fold than other metal ion cofactors. Our follow-up NMR investigations in the presence of other ions - Mg2+, Na+, and Pb2+ - suggest that at DNAzyme concentrations used in NMR all these ions induce a similar tertiary fold. Based on these findings, we propose a model for 8-17 DNAzyme interactions with metal ions postulating the existence of only a single catalytically-active structure, yet populated to a different extent depending on the metal ion cofactor. Our results provide structural information on the 8-17 DNAzyme in presence of non-Pb2+ cofactors, including the biologically relevant Mg2+ ion.

Target-driven cascade amplified assembly of covalent organic frameworks on tetrahedral DNA nanostructure with multiplex recognition domains for ultrasensitive detection of microRNA.

BACKGROUND: MicroRNA (miRNA) emerges as important cancer biomarker, accurate detection of miRNA plays an essential role in clinical sample analysis and disease diagnosis. However, it remains challenging to realize highly sensitive detection of low-abundance miRNA. Traditional detection methods including northern blot and real-time PCR have realized quantitative miRNA detection. However, these detection methods are involved in sophisticated operation and expensive instruments. Therefore, the development of novel sensing platform with high sensitivity and specificity for miRNA detection is urgently demanded for disease diagnosis. RESULTS: In this work, a novel electrochemical biosensor was constructed for miRNA detection based on target-driven cascade amplified assembly of electroactive covalent organic frameworks (COFs) on tetrahedral DNA nanostructure with multiplex recognition domains (m-TDN). COFs were employed as nanocarriers of electroactive prussian blue (PB) molecules by the "freeze-drying-reduction" method without the use of DNA as gatekeeper, which was simple, mild and efficient. The target-triggered catalytic hairpin assembly (CHA) and glutathione reduction could convert low-abundance miRNA into a large amount of Mn2+. Without the addition of exogenous Mn2+, the dynamically-generated Mn2+-powered DNAzyme cleavage process induced abundant PB-COFs probe assembled on the four recognition domains of m-TDN, resulting in significantly signal output. Using miRNA-182-5p as the model target, the proposed electrochemical biosensor achieved ultrasensitive detection of miRNA-182-5p in the range of 10 fM-100 nM with a detection limit of 2.5 fM. SIGNIFICANCE AND NOVELTY: Taking advantages of PB-COFs probe as the enhanced signal labels, the integration of CHA, Mn2+-powered DNAzyme and m-TDN amplification strategy significantly improved the sensitivity and specificity of the biosensor. The designed sensing platform was capable of miRNA detection in complex samples, which provided a new idea for biomarker detection, holding promising potential in clinical diagnosis and disease screening.

A colorimetric tandem combination of CRISPR/Cas12a with dual functional hybridization chain reaction for ultra-sensitive detection of Mycobacterium bovis.

Tuberculosis caused by Mycobacterium bovis poses a global infectious threat to humans and animals. Therefore, there is an urgent need to develop a sensitive, precise, and easy-to-readout strategy. Here, a novel tandem combination of a CRISPR/Cas12a system with dual HCR (denoted as CRISPR/Cas12a-D-HCR) was constructed for detecting Mycobacterium bovis. Based on the efficient trans-cleavage activity of the active CRISPR/Cas12a system, tandem-dsDNA with PAM sites was established using two flexible hairpins, providing multiple binding sites with CRISPR/Cas12a for further amplification. Furthermore, the activation of Cas12a initiated the second hybridization chain reaction (HCR), which integrated complete G-quadruplex sequences to assemble the hemin/G-quadruplex DNAzyme. With the addition of H2O2 and ABTS, a colorimetric signal readout strategy was achieved. Consequently, CRISPR/Cas12a-D-HCR achieved a satisfactory detection linear range from 20 aM to 50 fM, and the limit of detection was as low as 2.75 aM with single mismatched recognition capability, demonstrating good discrimination of different bacterial species. Notably, the practical application performance was verified via the standard addition method, with the recovery ranging from 96.0% to 105.2% and the relative standard deviations (RSD) ranging from 0.95% to 6.45%. The proposed CRISPR/Cas12a-D-HCR sensing system served as a promising application for accurate detection in food safety and agricultural fields.

Construction of a streptavidin-based dual-localized DNAzyme walker for disease biomarker detection.

A dual-localized DNAzyme walker (dlDW) was constructed by utilizing multiple split DNAzymes with probes, and their substrates are separately localized on streptavidin and AuNPs, serving as walking pedals and tracks, respectively. Based on dlDW, biosensing platform was successfully constructed and showed great potential application in clinical disease diagnosis.

Autonomous Feedback-Driven Engineered DNAzyme-Coated Trojan Horse-like Nanocapsules for On-Demand CRISPR/Cas9 Delivery.

Manipulating the expression of cellular genes through efficient CRISPR/Cas9 delivery is rapidly evolving into a desirable tumor therapeutics. The exposure of CRISPR/Cas9 to a complex external environment poses challenges for conventional delivery carriers in achieving responsive and accurate release. Here, we report a Trojan horse-like nanocapsule for the on-demand delivery of CRISPR/Cas9 in a microRNA-responsive manner, enabling precise tumor therapy. The nanocapsule comprises a nanoassembled, engineered DNAzyme shell encasing a Cas9/sgRNA complex core. The DNAzyme, functioning as a catalytic unit, undergoes a conformational change in the presence of tumor-associated microRNA, followed by activating a positive feedback-driven autonomous catabolic cycle of the nanocapsule shell. This catabolic cycle is accomplished through chain reactions of DNAzyme "cleavage-hybridization-cleavage", which ensures sensitivity in microRNA recognition and effective release of Cas9/sgRNA. Utilizing this Trojan horse-like nanocapsule, as low as 1.7 pM microRNA-21 can trigger the on-demand release of Cas9/sgRNA, enabling the specific editing of the protumorigenic microRNA coding gene. The resulting upregulation of tumor suppressor genes induces apoptosis in tumor cells, leading to significant inhibition of tumor growth by up to 75.94%. The Trojan horse-like nanocapsule, with superior programmability and biocompatibility, is anticipated to serve as a promising carrier for tailoring responsive gene editing systems, achieving enhanced antitumor specificity and efficacy.

DNAzyme-activated CRISPR/Cas assay for sensitive and one-pot detection of lead contamination.

Hazardous lead ions (Pb2+) even at a minute level can pose side effects on human health, highlighting the need for tools for trace Pb2+ detection. Herein, we present a DNAzyme-activated CRISPR assay (termed DzCas12T) for sensitive and one-pot detection of lead contamination. Using an extension-bridged strategy eliminates the need for separation to couple the DNAzyme recognition and CRISPR reporting processes. The tandem design endowed the DzCas12T assay with high specificity and sensitivity down to the pM-level. This assay has been used to detect lead contamination in food and water samples, indicating the potential for monitoring lead-associated environmental and food safety.

Synchronously Sensitive Immunoassay and Efficient Inactivation of Living Zika Virus via DNAzyme Catalytic Amplification and In Situ Aggregation-Induced Emission Photosensitizer Generation.

Sensitive identification and effective inactivation of the virus are paramount for the early diagnosis and treatment of viral infections to prevent the risk of secondary transmission of viruses in the environment. Herein, we developed a novel two-step fluorescence immunoassay using antibody/streptavidin dual-labeled polystyrene nanobeads and biotin-labeled G-quadruplex/hemin DNAzymes with peroxidase-mimicking activity for sensitive quantitation and efficient inactivation of living Zika virus (ZIKV). The dual-labeled nanobeads can specifically bind ZIKV through E protein targeting and simultaneously accumulate DNAzymes, leading to the catalytic oxidation of Amplex Red indicators and generation of intensified aggregation-induced emission fluorescence signals, with a detection limit down to 66.3 PFU/mL and 100% accuracy. Furthermore, robust reactive oxygen species generated in situ by oxidized Amplex Red upon irradiation can completely kill the virus. This sensitive and efficient detection-inactivation integrated system will expand the viral diagnostic tools and reduce the risk of virus transmission in the environment.

A photothermal aptasensor based on rolling circle amplification-enriched DNAzyme for portable detection of ochratoxin A in grape juice.

Aptasensors for detection of ochratoxin A (OTA) have been extensively studied, but the majority of them require costly and large-scale equipment as signal readers. Herein, a photothermal aptasensor capable of portable detection of OTA through a thermometer was developed on basis of aptamer structural switching and rolling circle amplification (RCA)-enriched DNAzyme. Oligonucleotides and alkaline phosphatase (ALP) modified magnetic beads were prepared. The binding of aptamers to OTA led to the release of ALP labeled complementary DNA. After magnetic separation, ALP catalyzed the padlock dephosphorylation, inhibiting the subsequent RCA reaction. This process converted the OTA concentration into the amount of the photothermal reagent oxTMB produced from the catalytic reaction induced by RCA-enriched DNAzyme. Under the optimal conditions, the detection limit (LOD) of this aptasensor was 2.28 nM in a clean buffer, while the LOD reached 2.43 nM in 2 % grape juice. The good performance of the photothermal aptasensor makes it possible to measure OTA pollution in low resource environments.

Ratiometric fluorescence platform for the ultrasensitive detection of kanamycin based on split aptamer co-recognition triggers Mg2+-DNAzyme-driven DNA walker systems.

In this work, a novel 3D-DNA walker signal amplification strategy was designed to construct a fluorescent aptasensor for the detection of kanamycin (KAN). The aptasensor utilizes split aptamers for the synergistic recognition of KAN. The presence of KAN induces the split aptamers recombination to form the Mg2+-DNAzyme structure, which is activated by Mg2+ to drive the 3D-DNA walker process for cascading signal amplification. Employing gold nanoflowers (AuNFs) as walking substrate material increases the local DNA concentration to enhance the walker efficiency. The prepared fluorescent aptasensor achieved efficient and sensitive detection of KAN with satisfactory results in the concentration range of 1 × 10-8 - 1 × 10-3 µg/kg and the detection limit of 5.63 fg/kg. Meanwhile, the designed fluorescent aptasensor exhibited favorable specificity, anti-interference, storage stability and reproducibility, and verified the feasibility of its application in milk samples. The present work provides an effective tool for the regulation of KAN contamination in animal-derived foods with promising prospects.

Highly sensitive and selective demethylase FTO detection using a DNAzyme-mediated CRISPR/Cas12a signal cascade amplification electrochemiluminescence biosensor with C-CN/PCNV heterojunction as emitter.

Fat mass and obesity-associated protein (FTO) has gained attention as the first RNA N6-methyladenosine (m6A) modification eraser due to its overexpression being associated with various cancers. In this study, an electrochemiluminescence (ECL) biosensor for the detection of demethylase FTO was developed based on DNAzyme-mediated CRISPR/Cas12a signal cascade amplification system and carboxylated carbon nitride nanosheets/phosphorus-doped nitrogen-vacancy modified carbon nitride nanosheets (C-CN/PCNV) heterojunction as the emitter. The biosensor was constructed by modifying the C-CN/PCNV heterojunction and a ferrocene-tagged probe (ssDNA-Fc) on a glassy carbon electrode. The presence of FTO removes the m6A modification on the catalytic core of DNAzyme, restoring its cleavage activity and generating activator DNA. This activator DNA further activates the trans-cleavage ability of Cas12a, leading to the cleavage of the ssDNA-Fc and the recovery of the ECL signal. The C-CN/PCNV heterojunction prevents electrode passivation and improves the electron-hole recombination, resulting in significantly enhanced ECL signal. The biosensor demonstrates high sensitivity with a low detection limit of 0.63 pM in the range from 1.0 pM to 100 nM. Furthermore, the biosensor was successfully applied to detect FTO in cancer cell lysate and screen FTO inhibitors, showing great potential in early clinical diagnosis and drug discovery.

A dual amplified gold nanoparticle-based biosensor for ultrasensitive and selective detection of fibrin.

Ultrasensitive, selective, and non-invasive detection of fibrin in human serum is critical for disease diagnosis. So far, the development of high-performance and ultrasensitive biosensors maintains core challenges for biosensing. Herein, we designed a novel ribbon nanoprobe for ultrasensitive detection of fibrin. The probe contains gold nanoparticles (AuNPs) that can not only link with homing peptide Cys-Arg-Glu-Lys-Ala (CREKA) to recognize fibrin but also carry long DNA belts to form G-quadruplex-based DNAzyme, catalyzing the chemiluminescence of luminol-hydrogen peroxide (H2O2) reaction. Combined with the second amplification procedure of rolling circle amplification (RCA), the assay exhibits excellent sensitivity with a detection limit of 0.04 fmol L-1 fibrin based on the 3-sigma. Furthermore, the biosensor shows high specificity on fibrin in samples because the structure of antibody-fibrin-homing peptide was employed to double recognize fibrin. Altogether, the simple and inexpensive approach may present a great potential for reliable detection of biomarkers.

Autonomous enzymatic synthesis of functional nucleic acids for sensitive measurement of long noncoding RNA in human lung tissues.

Aberrant long noncoding RNA (lncRNA) expression is linked to varied pathological processes and malignant tumors, and lncRNA can serve as potential disease biomarkers. Herein, we demonstrate the autonomous enzymatic synthesis of functional nucleic acids for sensitive measurement of lncRNA in human lung tissues on the basis of multiple primer generation-mediated rolling circle amplification (mPG-RCA). This assay involves two padlock probes that act as both a detection probe for recognizing target lncRNA and a domain for producing complementary DNAzyme. Two padlock probes can hybridize with target lncRNA at different sites, followed by ligation to form a circular template with the aid of RNA ligase. The circular template can initiate mPG-RCA to generate abundant Mg2+-dependent DNAzymes that can specifically cleave signal probes to induce the recovery of Cy3 fluorescence. The inherent characteristics of ligase-based ligation reaction and DNAzymes endow this assay with excellent specificity, and the introduction of multiple padlock probes endows this assay with high sensitivity. This strategy can rapidly and sensitively measure lncRNA with a wide linear range of 1 fM - 1 nM and a detection limit of 678 aM within 1.5 h, and it shows distinct advantages of simplicity and immobilization-free without the need of precise temperature control and tedious procedures of nanomaterial preparation. Moreover, it enables accurate measurement of lncRNA level in normal cells and malignant tumor cells as well as differentiation of lncRNA expressions in tissues of non-small cell lung cancer (NSCLC) patients and normal individuals, with promising applications in biomedical studies and disease diagnosis.

Engineering of a DNAzyme-Based dimeric G-quadruplex rolling circle amplification for robust analysis of lead ion.

Detecting heavy metal pollution, particularly lead ion (Pb2⁺) contamination, is imperative for safeguarding public health. In this study, we introduced an innovative approach by integrating DNAzyme with rolling circle amplification (RCA) to propose an amplification sensing method termed DNAzyme-based dimeric-G-quadruplex (dimer-G4) RCA. This sensing approach allows for precise and high-fidelity Pb2⁺ detection. Strategically, in the presence of Pb2⁺, the DNAzyme undergoes substrate strand (S-DNA) cleavage, liberating its enzyme strand (E-DNA) to prime isothermal amplification. This initiates the RCA process, producing numerous dimer-G-Quadruplexes (dimer-G4) as the signal reporting transducers. Compared to conventional strategies using monomeric G-quadruplex (mono-G4) as the reporting transducers, these dimer-G4 structures exhibit significantly enhanced fluorescence when bound with Thioflavin T (ThT), offering superior target signaling ability for even detection of Pb2⁺ at low concentration. Conversely, in the absence of Pb2⁺, the DNAzyme structure remains intact so that no primers can be produced to cause the RCA initiation. This nucleic acid amplification-based Pb2⁺ detection method combing with the high specificity of DNAzymes for Pb2⁺ recognition ensures highly sensitive detection of Pb2+ with a detection limit of 0.058 nM, providing a robust tool for food safety analysis and environmental monitoring.

Programmable Entropy-Driven Circuit-Cascaded Self-Feedback DNAzyme Network for Ultra-Sensitive Fluorescence and Photoelectrochemical Dual-Mode Biosensing.

Inspired by natural DNA networks, programmable artificial DNA networks have become an attractive tool for developing high-performance biosensors. However, there is still a lot of room for expansion in terms of sensitivity, atom economy, and result self-validation for current microRNA sensors. In this protocol, miRNA-122 as a target model, an ultrasensitive fluorescence (FL) and photoelectrochemical (PEC) dual-mode biosensing platform is developed using a programmable entropy-driven circuit (EDC) cascaded self-feedback DNAzyme network. The well-designed EDC realizes full utilization of the DNA strands and improves the atomic economy of the signal amplification system. The unique and rational design of the double-CdSe quantum-dot-released EDC substrate and the cascaded self-feedback DNAzyme amplification network significantly avoids high background signals and enhances sensitivity and specificity. Also, the enzyme-free, programmable EDC cascaded DNAzyme network effectively avoids the risk of signal leakage and enhances the accuracy of the sensor. Moreover, the introduction of superparamagnetic Fe3O4@SiO2-cDNA accelerates the rapid extraction of E2-CdSe QDs and E3-CdSe QDs, which greatly improves the timeliness of sensor signal reading. In addition to the strengths of linear range (6 orders of magnitude) and stability, the biosensor design with dual signal reading makes the test results self-confirming.

Exonuclease-iii -propelled DNAzyme cascade for sensitive and reliable cervical cancer related miRNA analysis.

MicroRNAs (miRNAs) can serve as biomarkers for early-diagnosis, therapy, and postoperative care of cervical cancer. Sensitive and reliable quantification of miRNA remains a huge challenge due to its low expressing levels and background interference. Herein, we propose a novel exonuclease-III (Exo-III)-propelled DNAzyme cascade for sensitive and high-efficient miRNA analysis. This method involves the engineering of compact DNAzyme hairpin probes, including the H1 probe and H2 probe. The H1 probe is designed with exposed analyte recognition subunits that can specifically recognize target miRNA. This recognition triggers two processes: Exo-iii-assisted target regeneration and successive substrate cleavage catalyzed by DNAzyme. The unique character of Exo-III that catalyzes removal of mononucleotides from the blunt or recessed 3'-OH termini of dsDNA confers the approach with a minimal background signal. The multiple signal cycles provided an abundant signal amplification and consequently, the method exhibited a low limit of detection of 3.12 fM, and a better specificity over several homologous miRNAs. In summary, this powerful Exo-III driven DNAzyme cascaded system offers broader and more adaptable methods for comprehending the activities of miRNA in various biological occurrences.

An acid-responsive DNA hydrogel-mediated cascaded enzymatic nucleic acid amplification system for the sensitive imaging of alkaline phosphatase in living cells.

Alkaline phosphatase (ALP) is a class of hydrolase that catalyzes the dephosphorylation of phosphorylated species in biological tissues, playing an important role in many physiological and pathological processes. Sensitive imaging of ALP activity in living cells is contributory to the research on these processes. Herein, we propose an acid-responsive DNA hydrogel to deliver a cascaded enzymatic nucleic acid amplification system into cells for the sensitive imaging of intracellular ALP activity. The DNA hydrogel is formed by two kinds of Y-shaped DNA monomers and acid-responsive cytosine-rich linkers. The amplification system contained Bst DNA polymerase (Bst DP), Nt.BbvCI endonuclease, a Recognition Probe (RP, containing a DNAzyme sequence, a Nt.BbvCI recognition sequence, and a phosphate group at the 3'-end), and a Signal Probe (SP, containing a cleavage site for DNAzyme, Cy3 and BHQ2 at the two ends). The amplification system was trapped into the DNA hydrogel and taken up by cells, and the cytosine-rich linkers folded into a quadruplex i-motif in the acidic lysosomes, leading to the collapse of the hydrogel and releasing the amplification system. The phosphate groups on RPs were recognized and removed by the target ALP, triggering a polymerization-nicking cycle to produce large numbers of DNAzyme sequences, which then cleaved multiple SPs, restoring Cy3 fluorescence to indicate the ALP activity. This strategy achieved sensitive imaging of ALP in living HeLa, MCF-7, and NCM460 cells, and realized the sensitive detection of ALP in vitro with a detection limit of 2.0 × 10-5 U mL-1, providing a potential tool for the research of ALP-related physiological and pathological processes.