DNAzyme Biosensor Research Articles

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.

A Programmable DNAzyme for the Sensitive Detection of Nucleic Acids

AbstractNucleic acids in biofluids are emerging biomarkers for the molecular diagnostics of diseases, but their clinical use has been hindered by the lack of sensitive detection assays. Herein, we report the development of a sensitive nucleic acid detection assay named SPOT (sensitive loop‐initiated DNAzyme biosensor for nucleic acid detection) by rationally designing a catalytic DNAzyme of endonuclease capability into a unified one‐stranded allosteric biosensor. SPOT is activated once a nucleic acid target of a specific sequence binds to its allosteric module to enable continuous cleavage of molecular reporters. SPOT provides a highly robust platform for sensitive, convenient and cost‐effective detection of low‐abundance nucleic acids. For clinical validation, we demonstrated that SPOT could detect serum miRNAs for the diagnostics of breast cancer, gastric cancer and prostate cancer. Furthermore, SPOT exhibits potent detection performance over SARS‐CoV‐2 RNA from clinical swabs with high sensitivity and specificity. Finally, SPOT is compatible with point‐of‐care testing modalities such as lateral flow assays. Hence, we envision that SPOT may serve as a robust assay for the sensitive detection of a variety of nucleic acid targets enabling molecular diagnostics in clinics.

A Programmable DNAzyme for the Sensitive Detection of Nucleic Acids.

Nucleic acids in biofluids are emerging biomarkers for the molecular diagnostics of diseases, but their clinical use has been hindered by the lack of sensitive detection assays. Herein, we report the development of a sensitive nucleic acid detection assay named SPOT (sensitive loop-initiated DNAzyme biosensor for nucleic acid detection) by rationally designing a catalytic DNAzyme of endonuclease capability into a unified one-stranded allosteric biosensor. SPOT is activated once a nucleic acid target of a specific sequence binds to its allosteric module to enable continuous cleavage of molecular reporters. SPOT provides a highly robust platform for sensitive, convenient and cost-effective detection of low-abundance nucleic acids. For clinical validation, we demonstrated that SPOT could detect serum miRNAs for the diagnostics of breast cancer, gastric cancer and prostate cancer. Furthermore, SPOT exhibits potent detection performance over SARS-CoV-2 RNA from clinical swabs with high sensitivity and specificity. Finally, SPOT is compatible with point-of-care testing modalities such as lateral flow assays. Hence, we envision that SPOT may serve as a robust assay for the sensitive detection of a variety of nucleic acid targets enabling molecular diagnostics in clinics.

A Label‐Free Deoxyribozyme Sensor for m6A Demethylase Activity Detection†

Comprehensive SummaryA label‐free deoxyribozyme (DNAzyme) biosensor for m6A demethylase activity detection is developed. When demethylase FTO (Fat mass and obesity‐associated protein), an important m6A demethylase of ALKBH demethylase family is present, the m6A group is specifically demethylated, and the cleavage activity of DNAzyme is restored. This allows the cleavage product that contains G‐quadruplex sequence to bind to thioflavin T (ThT) and generate fluorescence signals. The biosensor shows high specificity and sensitivity, and fast reaction speed. Our study demonstrates a new design of allosteric DNAzyme for sensing. This method represents the first label‐free nucleic acid biosensor for FTO assay, providing a feasible route towards inhibitors screening.

Front Cover: Construction of an Oxidative Cleavage‐Activated DNAzyme Biosensor for Rapid Detection and Cellular Imaging of the Myeloperoxidase Activity (Anal. Sens. 3/2024)

Front Cover: Construction of an Oxidative Cleavage‐Activated DNAzyme Biosensor for Rapid Detection and Cellular Imaging of the Myeloperoxidase Activity (Anal. Sens. 3/2024)

Construction of an Oxidative Cleavage‐Activated DNAzyme Biosensor for Rapid Detection and Cellular Imaging of the Myeloperoxidase Activity

AbstractInvited for this month's cover is the collaborating group of Prof. Chun‐Yang Zhang at Southeast University, China and colleagues at Shandong Normal University. The cover picture shows the formation of oxidative cleavage‐activated DNAzyme that enables imaging of myeloperoxidase in living cells. More information can be found in the Research Article by Zi‐Yue Wang, Chun‐Yang Zhang, and co‐workers.

Construction of an Oxidative Cleavage‐Activated DNAzyme Biosensor for Rapid Detection and Cellular Imaging of the Myeloperoxidase Activity

AbstractMyeloperoxidase (MPO) is a mammalian pro‐oxidant protease and it is closely related to severe infections and diverse inflammatory diseases. Rapid and sensitive measurement of MPO activity is essential for anticancer drug discovery and inflammatory research. Herein, we demonstrate the construction of an oxidative cleavage‐activated deoxyribozymes (DNAzyme) biosensor for rapid detection and cellular imaging of the MPO activity. When target MPO is present, the phosphorothioate (PS)‐modified hairpin probe is site‐specifically cleaved by MPO, releasing the intact Mg2+‐dependent DNAzyme sequences. Subsequently, the activated DNAzyme initiates the cyclic cleavage of the signal probe with the assistance of cofactor Mg2+, liberating large numbers of Cy5 molecules. This assay possesses the characteristics of easy operation, low sample consumption, without the requirements of expensive radiolabeling, antibodies, and nanomaterials. Especially, this assay can be performed in one pot under isothermal conditions (37°C) within 60 min. Due to the high efficiency of DNAzyme‐based cyclic cleavage reaction and the intrinsic advantages of single‐molecule detection, this assay achieves high sensitivity with a limit of detection (LOD) of 2.74×10−3 ng μL−1. It can be applied to screen MPO inhibitors, measure cellular MPO activity at the single‐cell level, and image intracellular MPO in living cells, providing a powerful platform for early clinical diagnosis and drug discovery.

Development of a fluorescence-based DNAzyme biosensor to detect Pb2+ in tobacco leaf extracts for cleaner crop production

Lead ions (Pb2+) are widespread contaminants that threaten human health and enter the human body mainly through the consumption of crop plants grown on Pb2+-contaminated soil. The traditional methods used to monitor the Pb2+ content of plants are sensitive and accurate; however, there is still an urgent need to develop screening methods with high sensitivity and reliability to rapidly monitor Pb2+ from plant samples with a higher throughput by adapting simple extraction methods. Here, we developed a series of fluorescence-based Pb2+ biosensors by fusing DNAzymes with tetrahedral DNA nanostructures and demonstrated the ability of these biosensors to detect Pb2+ in plant extracts. We first fused one DNAzyme with the A-probe of the tetrahedral DNA nanostructure. The substrate strand of the DNAzyme, which contains a ribonucleotide at its center, is cleaved upon Pb2+ binding, resulting in the release of the FAM fluorophore and the production of a fluorescence signal. The biosensors showed great sensitivity and selectivity. The sensitivity of the biosensor was further improved by fusing four DNAzymes to the tetrahedral DNA nanostructure and using Alexa488 as the fluorophore, which has greater quantum yield than FAM. Remarkably, the improved biosensors were able to detect Pb2+ in plant extracts. Thus, the Pb2+ biosensors developed in this study exhibit great potential for the identification and tracking of polluted regions by rapidly monitoring Pb2+ levels in plants grown therein and facilitating cleaner crop production.

Ultra-sensitive DNAzyme-based optofluidic biosensor with liquid crystal-Au nanoparticle hybrid amplification for molecular detection

Due to the specific cofactor dependence and the potent catalytic capability, DNAzymes have shown great promise in the application of biosensors. However, it poses significant challenges for researchers to enhance the sensitivities of DNAzyme-based biosensing platforms. In this work, an ultra-sensitive, label-free, and quick-responsive DNAzyme biosensor is developed based on whispering gallery mode (WGM) optofluidic resonator with a hybrid amplification from liquid crystals (LCs) and Au nanoparticles (AuNPs). When a DNAzyme is cleaved by biotargets, the event of DNA hybridization is triggered and will result in the orientation transition of LC molecules (in the resonator core). According to the quadruple amplification from the disturbance of AuNPs, the excess polarizability of adhering biomolecules, the orientation variation of LC molecules, and the WGM resonance, the spectral wavelength shift can be employed as the sensing parameter to indicate the information of biotarget. L-histidine molecules are applied as the analyte. An ultra-low detection limit at a sub-femtomole level (~ 5 × 10−16 M) has been achieved using this highly-selective biosensor, which is eight orders of magnitude lower compared to the reported LC-based L-histidine sensor. In addition to L-histidine, this proposed optofluidic scheme with LC-AuNP hybrid amplification can also extend the applicability of DNAzyme-based biosensors for the detection of various molecules.

Near-infrared band Gold nanoparticles-Au film “hot spot” model based label-free ultratrace lead (II) ions detection via fiber SPR DNAzyme biosensor

Heavy metal lead ion (Pb2+) detection takes more and more attention due to its synergistic toxicity to human health and undegradability in environment. Currently, there are urgent demands for portable, anti-interference and economical Pb2+ sensors with high sensitivity and selectivity for environmental monitoring as well as modern healthcare in clinic. In this article, via compact and high performed titled fiber Bragg grating (TFBG) surface plasmon resonance (SPR) sensing platform, we present a novel DNAzyme biosensor for ultratrace Pb2+ detection on the basis of “hot spot” effect in near-infrared band. A gold nanoparticle (GNP)-on-Au film construction was built by DNAzyme and its substrate strand. When Pb2+ with high catalytic activity cleave the DNAzyme linked rA (ribonucleotide adenosine) containing substrate strand, making DNA double helix structure transform to a single strand, the GNPs that connected with DNAzyme fall down to the sensor surface. By FDTD simulation, we have demonstrated that the appearing “hot spot” effect due to the narrow gap (<2 nm) will eventually result in enhancement of the spectrum signal response which is directly related to the Pb2+ concentration. In experiments, the proposed fiber biosensor exhibits a competitive low limit of detection ∼ 8.56 pM, excellent selectivity against other environmentally related metal ions and the large dynamic response range from 10−11 M to 10−6 M. More significantly, clinical human serum samples were used to verify the practicability of this TFBG-SPR-DNAzyme-GNPs biosensor showing great potential value in various clinical applications as a high efficiency, portable and easy to miniaturization point-of-care testing technology.

Biosensors and Nanodevices Based on DNAzymes

This review mainly report the principle and development of biosensors and nanodevices based on DNAzymes, including fluorescent DNAzyme biosensor, colorimetric DNAzyme biosensor, Surface Enhanced Raman Scattering (SERS) DNAzymes biosensor, electrochemical DNAzymes biosensor, and catalytic biosensor using DNAzymes as signal amplifiers.

Temperature-Robust DNAzyme Biosensors Confirming Ultralow Background Detection.

Catalytic DNA/RNA, such as DNAzyme, has been widely adopted to construct biosensors, especially for metal ion analysis. However, traditional DNAzyme biosensors still suffer from fluctuating and relatively high background. Herein, we proposed a temperature-robust DNAzyme, conferring ultralow background in various temperatures, thus leading to highly sensitive and robust detection of metal ions. Instead of labeling substrate to directly output fluorescence signal, our proposed DNAzyme biosensor utilized a sequential detection process with a couple of proximity fluorescent probes, confirming very low background regardless of the conditions of cleavage reaction. This sequential DNAzyme biosensor conferred a signal to background ratio over 20 when the temperature of the catalytic reaction ranged from 20 to 41 °C. Benefitting from its ultralow background, it could confer a detection limit of 0.22 nM, which ranked as one of the highest sensitivity levels among DNAzyme-based fluorescent biosensors. This DNAzyme biosensor was over 6000 times more selective for Pb2+ against the most active interfering metal ions, Zn2+. Further, it has been successfully applied for analyzing lead pollution in tap water and eggs, with total recoveries ranging from 87% to 114%. This facile, simple, and effective design strategy would significantly improve the detection performance of DNAzyme biosensors, thus facilitating its practical applications for both food safety analysis and environment monitoring.

Colorimetric detection of microRNA based on DNAzyme and nuclease-assisted catalytic hairpin assembly signal amplification

Accurate and quantitative analysis of microRNA (miRNA) expression is critical for the diagnostics and theranostics of a disease. Herein, a proof-of-concept of a colorimetric horseradish peroxidase-mimicking DNAzyme (HRP-DNAzyme) biosensor for miRNA assay based on nuclease-assisted catalytic hairpin assembly (CHA) signal amplification was demonstrated. Duplex-specific nuclease (DSN) was employed to cleave the single-stranded DNA (ssDNA) chimeric probe (CP) on the magnetic bead (MB) surface via hybridization of the CP and target miRNA. The regenerated miRNA can cleave a large number of ssDNA CP to produce CHA initiator sequence fragments. The CP consists of two main regions: a target miRNA recognition DNA sequence at the 5′ end and a CHA initiator (CI) sequence at the 3′ end. The catalyzed assembly process of CHA produces a large amount of G-rich DNA. In the presence of hemin, the G-rich DNA forms G-quadruplex/hemin complex and mimics the horseradish peroxidase activity, which catalyzes a colorimetric reaction. For the proof-of-concept, microRNA-21 (miR-21) was selected as the model target to authenticate this strategy as a versatile assay platform. The proposed strategy allowed quantitation of the sequence specificity of miRNA-21 with a detection limit of 9.2 fM in a dynamic range from 10 fM–1 nM, with an excellent ability to discriminate the differences in miRNAs. Additionally, the miRNA assay in real samples was satisfactory, thereby confirming its applicability. Therefore, this method exhibited a great potential as a miRNA quantification method in biomedical research and clinical diagnosis.

A dual signal amplification-assisted DNAzyme biosensor for ultrasensitive detection of Argonaute 2 activity.

We develop a new DNAzyme biosensor for Argonaute 2 (Ago2) assay based on target-initiated rolling circle amplification in combination with an 8-17 DNAzyme-mediated dual signal amplification strategy. This biosensor can achieve ultrahigh sensitivity with a detection limit of 0.35 pM, and it can be further used for the screening of Ago2 inhibitors and the accurate measurement of endogenous Ago2 from HeLa cell extracts.

Portable and sensitive detection of copper(II) ion based on personal glucose meters and a ligation DNAzyme releasing strategy

Using a Cu2+-dependent DNA ligation DNAzyme, a portable and sensitive biosensor for Cu2+ detection based on personal glucose meters (PGMs) has been developed. In the presence of Cu2+, the DNAzyme catalyzed the ligation of nucleophilic attack of the phosphorus center of DNA1 by the hydroxyl group on DNA2 and forming a phosphodiseter bond. By introducing DNA-invertase conjugates and capture-DNA modified magnetic beads (MBs), the ligation product can hybridize with them and form invertase-functionalized MBs, which can be separated easily. The presence of invertase can convert sucrose to glucose and can be read out by the PGMs. With this method, a detection limit of 1nM could be achieved. Moreover, the method exhibited excellent metal ion selectivity and there was almost no effect of biological complex to the detection performance, which suggested our method can be successfully applied to in biological process researches, drug discovery, and clinic diagnostics.

A Sensitive Biosensor for Determination of Cu2+ by One‐step Electrodeposition

AbstractA simple one‐step electrodeposition method is described to fabricate three dimensional ordered macroporous chitosan−prussian blue−single walled carbon nanotubes (3DOM CS−PB−SWCNTs) film onto the gold electrode surface to fabricate a copper ions (Cu2+)‐specific DNAzyme biosensor. The new sensing strategy for sensitive and selective detection of Cu2+ was based on Au nanorods (AuNRs) as signal amplification labels. The electrochemical signal of glucose increased with the concentration of Cu2+ increasing. The morphologies and electrochemistry of the composites were investigated by using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and electrochemical techniques including cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) and so on. Linear correlations of copper ion concentration were obtained in the range from 10−18 M to 10−5 M, achieving with a limit of detection of 10−19 M (S/N=3). Parameters affecting the biosensor response such as temperature, the cleavage time and the time of hybridization were optimized. This biosensor showed a wide range, low detection limit, good reproducibility and high stability. Additionally, these striking properties endow the biosensor with a great promise for analytical applications.

A label-free DNAzyme fluorescence biosensor for amplified detection of Pb2+-based on cleavage-induced G-quadruplex formation

DNAzyme-based catalytic beacons have been widely studied for both in vivo and in vitro molecular detection. However, only a few label-free catalytic beacons with excellent analytical performance have been reported so far. In this work, by combining a catalytic DNAzyme for amplified sensing through enzymatic turnover with cleavage-induced G-quadruplex formation, a label-free DNAzyme biosensor was developed for amplified “turn-on” fluorescence detection of Pb2+ with a detection limit of 3nM. The method is very competitive compared to many other labeled or label-free methods with or without signal amplification. Due to the inherent specificity of the GR-5 DNAzyme, the method also exhibits excellent selectivity. This biosensor successfully detected Pb2+ in river water samples with high sensitivity and selectivity. Such a method might provide a universal DNAzyme-based sensing platform for sensitive detection of various targets both in environmental and biomedical fields.

A label-free hemin/G-quadruplex DNAzyme biosensor developed on electrochemically modified electrodes for detection of a HBV DNA segment

A label-free biosensor based on Au/G–CMWCNTs-GCE was proposed for the detection of a HBV DNA segment with a low LOD.

A target-triggered exponential amplification-based DNAzyme biosensor for ultrasensitive detection of folate receptors.

We develop a new method for ultrasensitive detection of folate receptors (FRs) using a target-triggered isothermally exponential amplification reaction (EXPAR)-based DNAzyme biosensor. This method exhibits excellent specificity and high sensitivity with a detection limit as low as 0.23 fM and a large dynamic range of 6 orders of magnitude from 1 fM to 1 nM. It might be further applied for the detection of various small molecule-binding proteins by simply changing the linked small molecule moiety of the hairpin probes.

Highly sensitive detection of telomerase using a telomere-triggered isothermal exponential amplification-based DNAzyme biosensor

A telomere-triggered isothermal exponential amplification-based DNAzyme biosensor is developed for highly sensitive detection of telomerase in cancer cells even at the single-cell level. This biosensor can be further applied for the screening of telomerase inhibitors for anticancer drug development.