DNAzyme Amplification Research Articles

Nano-fuels-driven self-sacrificed ZIF-8@Apt integrated chip coupled with a DNAzyme system for the isolation and detection of glioblastoma-derived extracellular vesicles

Plasma extracellular vesicles (EVs) have emerged as promising biomarkers for the early diagnosis of glioblastoma. However, rapid and efficient isolation and detection of tumor-specific EV subsets from plasma remains challenging. To address this, we developed an innovative integrated chip based on self-sacrificial metal–organic frameworks and the epidermal growth factor receptor (EGFR) aptamers called ZIF-8@EGFR aptamer (ZIF-8@Apt), which further coupled with a “nano-fuels”-driven DNAzyme amplification strategy. The core functionality of the constructed chip lies in its ability to capture EVs with the ZIF-8@Apt module. Once captured, numerous DNA “nano-fuel” structures are in situ assembled on the EVs’ surfaces. Subsequently, the ZIF-8@Apt@EV@nano-fuels composites are lysed to release Zn2+, the EVs’ nucleic acids, and additional nano-fuels for subsequent detection. Finally, a DNAzyme detection system is introduced, where the released nano-fuels activate the DNAzyme, leading to multiple signal amplification aided by Zn2+ for highly sensitive detection of plasma EVs. This integrated approach effectively enriches and detects target EVs, achieving a significantly enhanced detection sensitivity of 500 particles/μL. In a clinical study involving glioblastoma patients, our chip demonstrated a remarkable ability to distinguish between cancer patients and healthy individuals, achieving an area under the curve (AUC) of 0.958. Moreover, the response signals of the chip can be conveniently read using fluorescence spectroscopy or microscope, offering a rapid, highly sensitive, and high-throughput analysis of plasma EVs for various application scenarios. Overall, this study introduces a comprehensive approach for the rapid isolation and accurate analysis of plasma EVs, holding significant promise for early diagnosis and disease monitoring.

Design of a triple-functional polyA-probe: Toward a facile electrochemical aptasensor for vanillin detection

This study successfully developed an electrochemical aptasensor based on a triple-functional biotin-modified poly-adenine (polyA) probe (Bio-TPP) for one-step ultrasensitive detection of vanillin (VAN). The Bio-TPP was self-assembled onto a gold electrode (AuE) via its polyA sequence, followed by conjugation with PtPd@Ni-Co layered double hydroxides labeled with streptavidin and thionine (SA/Thi/PtPd@Ni-Co LDH), which generated electrochemical signals. Importantly, in the presence of VAN, the specific substrate sequence of a Mg²⁺-dependent DNAzyme in the Bio-TPP hybridized with the output DNAzyme strand generated by the specific recognition between the aptamer (Apt) and VAN. And, with the help of Mg²⁺, the DNAzyme amplification reaction was triggered. This led to a significant reduction in the electrochemical signals, enabling highly sensitive detection of VAN. Benefiting from the precise and flexible design of the Bio-TPP, this aptasensor offered a new detection method, exhibiting excellent performance with a wide linear range of 1×10−6-10 μM and a detection limit of 0.30 pM. Additionally, this assay possessed good selectivity, reproducibility, and stability, which held great potential in bioanalysis.

Target-promoted autocatalytic hairpin assembly of bivalent DNAzymes for sensitive and label-free electrochemical metallothionein assay

Metallothionein (MT) has shown to be an important biomarker for environmental monitoring and various diseases, due to its significant binding ability to heavy metal ions. On the basis of such a characteristic and the Hg2+-stabilized DNA duplex (Hg2+-dsDNA) probe, as well as a new autocatalytic hairpin assembly (aCHA)/DNAzyme cascaded signal enhancement strategy, the construction of a highly sensitive and label-free electrochemical MT biosensor is described. Target MT molecules bind Hg2+ in Hg2+-dsDNA to disrupt the duplex structure and to release ssDNA sequences, which trigger subsequent aCHA for efficient production of mimic aCHA triggering strands and many bivalent DNAzymes. The signal hairpins on the electrode are then cyclically cleaved by DNAzyme amplification cascade to liberate plenty G-quadruplex sequences, which bind hemin and yield largely enhanced currents for sensitive assay of MT with a detection limit of 0.217 nM in a label-free approach. Such sensor also shows selective discrimination capability to MT against other interfering proteins and assay of MT in normal serums with dilution has also been verified, indicating its potential for highly sensitive detection of different heavy metal ion binding molecules for various application scenarios.

Electrochemical aptasensor based on CRISPR/Cas12a-mediated and DNAzyme-assisted cascade dual-enzyme transformation strategy for zearalenone detection

The combination of the CRISPR/Cas system and biosensing is of profound significance in small molecule diagnostics. Herein, we report a cascade dual-enzyme transformation strategy for ultrasensitive analysis of zearalenone (ZEN) by utilizing the CRISPR/Cas12a trans-cleavage activity and DNAzyme amplification. NH2-MnO2/Pd@Au NBs nanocomposites are greatly promising electrode modification material for improving electrode sensing performance. PtPd@Fe3O4 nanocomposites are synthesized as signal label materials to load the electroactive molecule methylene blue (MB). The recognition of ZEN target and aptamer is converted into DNA signal to regulate the trans-cleavage activity of the CRISPR/Cas12a on Mg2+-DNAzyme probes. After that, in the presence of DNAzyme probes and Mg2+, the MB signal changes caused by Mg2+dependent DNAzyme-assisted catalytic cleavage of signal labels on the electrode surface are evaluated. The quantitative detection of ZEN is ultimately achieved. Under optimal conditions, the as-prepared electrochemical aptasensor shows a wide linear detection range of 1 × 10-5 to 10 ng·mL−1, and the detection limit is 6.27 × 10-6 ng·mL−1. Moreover, the constructed aptasensor exhibits satisfactory selectivity, stability and repeatability, which also has a wide application prospect in the real sample analysis.

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.

Functional rolling circle amplification-based sensitive determination and low-speed centrifugation-based isolation of Staphylococcus aureus

The ability to quickly and accurately analyze Staphylococcus aureus (S. aureus) and isolate the bacteria in a simplified setting is crucial for the early identification and treatment of infectious illnesses. Here, we describe the development of a new aptamer-based detection and separation technique that combines Mg2+-dependent DNAzyme amplification cascades with catalytic hairpin assembly for enhanced sensitivity. This technique uses a rolling circle amplification procedure to build a detection scaffold with a repetitive functional hairpin structure that, upon identifying S. aureus, can launch a catalytic hairpin assembly-mediated DNAzyme-based cascade signal amplification. This allows S. aureus to be isolated using low-speed centrifugation and simultaneously quantified. The approach has a low limit of detection of 21 cfu/mL and a broad detection range of six orders of magnitude due to the inclusion of the catalytic hairpin assembly for signal amplification. In addition to high sensitivity, the method also demonstrates high selectivity for the identification and isolation of S. aureus, making it a useful instrument for reporting S. aureus infections.

DNAzyme-Amplified Cascade Catalytic Hairpin Assembly Nanosystem for Sensitive MicroRNA Imaging in Living Cells.

Sensitive imaging of microRNAs (miRNAs) in living cells is significant for accurate cancer clinical diagnosis and prognosis research studies, but it is challenged by inefficient intracellular delivery, instability of nucleic acid probes, and limited amplification efficiency. Herein, we engineered a DNAzyme-amplified cascade catalytic hairpin assembly (CHA)-based nanosystem (DCC) that overcomes these challenges and improves the imaging sensitivity. This enzyme-free amplification nanosystem is based on the sequential activation of DNAzyme amplification and CHA. MnO2 nanosheets were used as nanocarriers for the delivery of nucleic acid probes, which can resist the degradation by nucleases and supply Mn2+ for the DNAzyme reaction. After entering into living cells, the MnO2 nanosheets can be decomposed by intracellular glutathione (GSH) and release the loaded nucleic acid probes. In the presence of target miRNA, the locking strand (L) was hybridized with target miRNA, and the DNAzyme was released, which then cleaved the substrate hairpin (H1). This cleavage reaction resulted in the formation of a trigger sequence (TS) that can activate CHA and recover the fluorescence readout. Meanwhile, the DNAzyme was released from the cleaved H1 and bound to other H1 for new rounds of DNAzyme-based amplification. The TS was also released from CHA and involved in the new cycle of CHA. By this DCC nanosystem, low-abundance target miRNA can activate many DNAzyme and generate numerous TS for CHA, resulting in sensitive and selective analysis of miRNAs with a limit of detection of 5.4 pM, which is 18-fold lower than that of the traditional CHA system. This stable, sensitive, and selective nanosystem holds great potential for miRNA analysis, clinical diagnosis, and other related biomedical applications.

A novel electrochemical aptasensor based on rolling circle amplification-driven Ag+-DNAzyme amplification for ochratoxin A detection

A novel electrochemical aptasensor based on rolling circle amplification-driven Ag+-DNAzyme amplification for ochratoxin A detection

Fluorescent detection of brown spot of tobacco caused by Alternaria alternata based on lambda exonuclease-induced DNAzyme amplification.

A rapid, simple, and sensitive fluorescent detection method for brown spot of tobacco is established by lambda exonuclease-induced Mg2+-dependent DNAzyme amplification. It contains hybridization of the Alternaria alternata genome and HP1, digestion of the 5'-phosphorylated strand of the hybrid dsDNA by lambda exonuclease, acquisition of complete Mg2+-dependent DNAzyme, cleavage of the substrate modified with FAM and BHQ-1, and fluorescent detection. The proposed assay exhibits good sensitivity (10 pg L-1), selectivity and reproducibility. The method does not require pure DNA and expensive instruments, and can be performed within 2.5 hours. To the best of our knowledge, this is the first report of fluorescent detection of Alternaria alternata and its tobacco field samples. This method can be applied to the rapid and sensitive detection of Alternaria alternata in tobacco and its seedlings, and is particularly important for the green prevention and control of tobacco brown spot disease.

A versatile and smartphone-integrated detection platform based on Exo III-assisted recycling and DNAzyme amplification

Staphylococcus aureus (S. aureus) is a widespread foodborne pathogen, causing a serious threat to human health and public safety. Thus, it is significant for rapid S. aureus determination. Herein, a visual and smartphone-integrated aptasensor for intact S. aureus was fabricated based on the Exo III-assisted recycling and hemin/G-quadruplex DNAzymes for signal amplification. Different concentrations of S. aureus caused the difference in releasing different amounts of DNAzymes via Exo III-assisted recycling reaction. These DNAzymes catalyzed the oxidation of 2,2′-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS2-) to colored ABTS•−, which were respectively analyzed by smartphone and UV-Vis spectrophotometer for quantification. This assay exhibited good analytical performance with a detection limit of 32 CFU·mL−1 and a wide linear range. It also possesses the ability in complex sample matrices detection, low cost, and portability with the employment of a smartphone, making it a rapid, simple, and point-of-care test (POCT) method with great potential in food safety control.

Aptamer-based and sensitive label-free colorimetric sensing of phenylalanine via cascaded signal amplifications

The concentration variation of phenylalanine (Phe), an essential amino acid in humans, can cause metabolism disorders and even mental disability. Sensitive and convenient monitoring of Phe is therefore important for disease diagnosis. We describe here the establishment of a new aptamer-based, sensitive and label-free colorimetric Phe detection strategy by integrating catalytic hairpin assembly (CHA) and Mg2+-dependent DNAzyme amplification cascades. The target Phe coordinates with pentamethylcyclopentadienyl rhodium(III) chloride dimer [(Cp*RhCl2)2] to form a complex that has a high affinity to the corresponding aptamer sequence. Upon its binding to the aptamers in DNA duplex probes, ssDNA strands are released to trigger subsequent CHA reactions for the formation of many DNAzymes, which cleave the substrate signal probes to liberate lots of CHA initiation strands and free G-quadruplexes to realize the cascaded amplifications. Hemin further associates with the many G-quadruplexes to yield hemin/G-quadruplex mimicking peroxidases, which catalyze solution of substrate to exhibit highly enhanced UV–vis adsorption for detecting Phe at 0.19 μM level. At the meantime, the monitoring of Phe in diluted serums with high selectivity has also been demonstrated by the developed method, indicating its potential for simple diagnosis of Phe-related diseases.

Intracellular miRNA Imaging Based on a Self-Powered and Self-Feedback Entropy-Driven Catalyst-DNAzyme Circuit.

DNAzyme-based signal amplification circuits promote the advances in low-abundant miRNA imaging in living cells. However, due to the insufficient cofactor in living cells and unsustainable target utilization, self-powered and self-feedback DNAzyme amplification circuits have rarely been achieved. Here, a MnO2 nanosheet-mediated self-powered and self-feedback entropy-driven catalyst (EDC)-DNAzyme nanoprobe (MnPFEDz) was demonstrated for sensitive imaging of intracellular microRNA (miRNA). In this strategy, MnPFEDz was formed by adsorbing EDC modules and substrate probes on MnO2 nanosheets. The MnO2 nanosheets acted not only as glutathione (GSH)-responsive nanocarriers for efficient delivery of DNA probes but also as a DNAzyme cofactor supplier to power the DNAzyme biocatalysis and promote signal transduction in a feedback way. When entering the cells, GSH could decompose MnO2 nanosheets to generate numerous Mn2+ ion cofactors, leading to the release of DNA probes. Subsequently, the target miRNA initiated EDC cycles to generate amplified fluorescence signals and exposed the complete DNAzyme. Meanwhile, each of the exposed DNAzyme then cleaved the substrate probes with the help of Mn2+ ion cofactors and released a new trigger analogue for the next round of EDC cycles, initiating additional fluorescence signals in a feedback way. As a multiple signal amplification strategy, the MnPFEDz nanoprobe facilitated the effective detection of intracellular molecules with enhanced sensitivity and provided a versatile strategy for the construction of self-powered and self-feedback DNA circuits in living cells.

Recent advances of using personal glucose meter as a biosensor readout for non-glucose targets.

Personal glucose meter (PGM) has become the most successful biosensor in past decades due to its advantages of small size, convenient operation, and low cost. To take advantage of many years of research and development of PGMs, new signal transduction methods has been developed to expand the PGM from simple monitoring blood glucose to detection of numerous non-glucose targets. This review summarizes recent advance of PGM-based biosensors for non-glucose targets including signal transduction, signal amplification and target molecule recognition and analysis. Current challenges and future directions are also discussed. PGM can be used as biosensor readout to detect various non-glucose targets from metal ion, small molecule to protein and even living organisms such as bacteria and other pathogens by using different signal transduction elements such as invertase and amylase, and different signal amplification methods such as nanomaterials, nucleic acid reaction, liposome encapsulation, hydrogel trapping, DNAzyme amplification and biotin-streptavidin reaction.

A MnOx-nucleic acid nanoprobe with enzyme-free cascade signal amplification for ultrasensitive intracellular microRNA imaging

A novel MnOx-nucleic acid nanoprobe was constructed for catalytic imaging of microRNA in living cells based on the combination of catalytic hairpin assembly, hybridization chain reaction, and DNAzyme amplification. This nanoprobe exhibited ultrahigh sensitivity and specificity, and could distinguish tumor cells and normal cells by live cell microRNA imaging.

Advanced graphene oxide-based paper sensor for colorimetric detection of miRNA.

MicroRNAs (miRNAs), found in blood and body fluids, have emerged as potential non-invasive biomarkers for disease and injury. miRNAs are quantitatively evaluated using typical RNA analysis methods such as the quantitative reverse transcription polymerase chain reaction, microarrays, and Northern blot, all of which require complex procedures and expensive reagents. To utilize miRNAs as practical biomarkers, it will be helpful to develop simple and user-friendly sensors. In this study, a paper-based miRNA sensor was developed by combining two methods: (1) target-recycled DNAzyme (Dz) amplification and (2) graphene oxide-assisted Dz blotting on paper. The Dz spots on paper caused amiRNA-dependent color change in presence of colorimetric reagents and facilitated the quantification of absolute amount of the target miRNA, irrespective of the volume, with high reproducibility. This approach is technologically straightforward and enables quantification of as low as 7.75 fmol miRNA using a portable smartphone.

A Deoxyribozyme-Initiated Self-Catalytic DNA Machine for Amplified Live-Cell Imaging of MicroRNA.

Functional DNA nanostructures have been widely used in various bioassay fields. Yet, the programmable assembly of functional DNA nanostructures in living cells still represents a challenging goal for guaranteeing the sensitive and specific biosensing utility. In this work, we report a self-catalytic DNA assembly (SDA) machine by using a feedback deoxyribozyme (DNAzyme)-amplified branched DNA assembly. This SDA system consists of catalytic self-assembly (CSA) and DNAzyme amplification modules for recognizing and amplifying the target analyte. The analyte initiates the CSA reaction, leading to the formation of Y-shaped DNA that carries two RNA-cleaving DNAzymes. One DNAzyme can then successively cleave the corresponding substrate and generate numerous additional inputs to activate new CSA reactions, thus realizing a self-catalytic amplification reaction. Simultaneously, the other DNAzyme is assembled as a versatile signal transducer for cleaving the fluorophore/quencher-modified substrate, leading to the generation of an amplified fluorescence readout. By incorporating a flexible auxiliary sensing module, the SDA system can be converted into a universal sensing platform for detecting cancerous biomarkers, e.g., a well-known oncogene microRNA-21 (miR-21). Moreover, the SDA system realized the precise intracellular miR-21 imaging in living cells, which is attributed to the reciprocal amplification property between CSA reactions and DNAzyme biocatalysis. This compact SDA amplifier machine provides a universal and facile toolbox for the highly efficient identification of cancerous biomarkers and thus holds great potential for early cancer diagnosis.

Highly integrated and one-step triggered cascade DNA walker based on entropy-driven catalytic and DNAzyme amplification

As an amplification tool, DNA walker nanomachine exhibits promising potential in the fields of biosensing. To further improve amplification efficiency of DNA walker, a number of cascade amplification systems have been developed, but they still suffer from separated steps and long reaction time. Herein, by combining entropy-driven catalytic (EDC) amplification with DNAzyme reaction, a highly integrated cascade three-dimensional (3D) DNA walker is constructed, which allows the autonomous DNA circuit to be completed in one step. Further combined with element tag and inductively coupled plasma mass spectrometry (ICP-MS) detection, the cascade walker can be used for quantification of miRNA-21. Specifically, the reactant of EDC (WLT complex) and the substrate of DNAzyme (Substrate-Tb) are both fixed on magnetic beads (MBs) to assemble the track of walker. Once cascade DNA walker is triggered by target miRNA, EDC amplification runs first and keeps circulating to liberate many DNAzyme strands. DNAzyme can repeatedly cleave Substrate-Tb with autonomously walking along the substrate on MBs. As a result, a great number of fragments of Substrate-Tb, as output signal, are generated within 1 h, resulting in elemental signal response in ICP-MS and reflecting the amount of miRNA-21. A good linear relationship was obtained with miRNA-21 concentration in the range of 0.02−1 nmol L−1, and the detection limit of 5 pmol L−1 was achieved. The recoveries of miRNA-21 in human serum were 92.0–107 %. The developed method provided a novel idea for constructing highly integrated and efficient cascade DNA walker with ICP-MS strategy for bioanalysis.

Biodegradable Metal–Organic Frameworks Power DNAzyme for in Vivo Temporal-Spatial Control Fluorescence Imaging of Aberrant MicroRNA and Hypoxic Tumor

MicroRNAs (miRNAs) are involved in the essential progresses of many diseases and have emerged as therapeutic and diagnostic biomarkers. The combination of miRNA aberrant expression and tumor microenvironment (TME) features holds great potential for precise tumor imaging diagnosis but has been minimally explored. Herein, we rationally design a DNA@Cu-MOF nanosystem containing copper metal-organic frameworks (Cu-MOF) and a DNAzyme-assisted signal amplification procedure for deregulated miRNA-related hypoxic tumor diagnosis. The nanoprobes comprising a signal strand block Cu-specific DNAzyme precursor and a substrate strand are assembled on the surface of the hypoxia-responsive Cu-MOF. Under TME characterized by hypoxia, the DNA@Cu-MOF nanosystem disassociates and accomplishes the release of abundant Cu2+, DNAzyme precursor, and substrate strand. Target aberrant miRNA displaces the signal strand to recover one fluorescence signal for detection. Importantly, it activates the Cu-specific DNAzyme amplification, which produces miRNA aberrant expression-dependent fluorescence signal for hypoxic tumor diagnosis. Both in vitro and in vivo experiments validate its good performance for tumor cell diagnosis. The hypoxia-driven and miRNA-binding-induced self-powered and temporal-spatial fluorescence imaging nanosystem not only provides a great tool for aberrant miRNA-related hypoxic tumor diagnosis but also is readily applied for the control and modulation of biological functions.

Versatile Electrochemiluminescence and Photoelectrochemical Detection of Glutathione Using Mn2+ Substitute Target by DNA-Walker-Induced Allosteric Switch and Signal Amplification.

Glutathione (GSH) serves vital functions in biological systems and associates with various human diseases. In this work, a versatile electrochemiluminence (ECL) and a photoelectrochemical (PEC) "signal on" biosensing platform were developed for a sensitive assay of GSH by a Mn2+-powered DNAzyme amplification strategy combined with DNA-walker-triggered allosteric conversion. First, MnO2 nanosheets were reduced to Mn2+ by GSH; then, Mn2+ as a substitute target triggered DNAzyme-assisted cleavage-cycling amplification to generate numerous DNA output (s3). Meanwhile, the DNA molecular machine was introduced to bridge signal probes for versatile biosensing, which included hairpin DNA as a track and an arm as a walker. The presence of DNA output (s3) activated the swing arm to hybridize with hairpin DNA and then cut it by Nt.BbvCI, which initiated autonomous walking of the arm for forming a large number of streptavidin (SA) aptamers. Thus, a large number of CdS:Mn-SA tags as versatile signal probes was linked to the electrode by specific SA-aptamer binding, generating highly enhanced ECL and PEC signals for sensitive detection of the target. The present biosensing system take advantage of metal ion-based DNAzyme amplification, a DNA walker machine, multi-signals of QDs, and specificity of aptamers, which can provide a universal and efficient biosensing method for detecting various targets. The designed strategy demonstrated good performance for a GSH assay in human serum samples, showing more promising applications than other reported methods.

Target-induced steric hindrance protection of DNAzyme junctions for completely enzyme-free and amplified sensing of transcription factors

The interactions between proteins and DNA play critical roles in numerous essential cellular activities. Therefore, sensitive investigation of these interactions is crucial for the study of different biological activities. Hence, a convenient and enzyme-free sensing approach for homogeneous and ultra-sensitive assay of transcription factors was explored based on the steric hindrance-protected Mg2+-DNAzyme amplification strategy. The target proteins can specifically bind the unique sequences of the Y-shaped DNA probes with strong affinity to form the protein/DNA complexes and to protect them from being disrupted by the external invading DNA. The target-protected and intact Mg2+-DNAzymes further hybridize with the fluorescently quenched DNA hairpin substrate sequences and cleave them cyclically to yield substantially enhanced fluorescence response for amplified detection of transcription factors. Using nuclear factor kappa B p50 as the example target, a low detection limit down to 2.8 pM can be achieved. Besides, the approach also exhibits excellent selectivity due to the high specificity of the recognition regions and can be further applied in human serums. This sensing platform thus can expect to be a promising alternative for the sensitive analysis of multiple DNA-binding proteins by changing the corresponding recognition units.