Colorimetric detection of furin based on enhanced catalytic activity of G-quadruplex/hemin DNAzyme.

BACKGROUND: Rapid and sensitive colorimetric detection methods are crucial for diseases diagnosis, particularly those involving proteases like furin, which are implicated in various conditions, including cancer. Traditional detection methods for furin suffer from limitations in sensitivity and practicality for on-site detection, motivating the development of novel detection strategies. Therefore, developing a simple, enzyme-free, and rapid colorimetric analysis method with high sensitivity for furin detection is imperative. RESULTS: Herein, we have proposed a colorimetric method in this work for the first time to detect furin, leveraging the assembly of G-quadruplex/hemin DNAzyme with enhanced catalytic activity. Specifically, a peptide-DNA conjugate (PDC) comprising a furin-recognition peptide and flanking DNA sequences for signal amplification is designed to facilitate the DNAzyme assembly. Upon furin treatment, PDC cleavage triggers a cyclic catalytic hairpin assembly reaction to form the complementary double-stranded structures by hairpin 1 (HP1) and hairpin 2 (HP2), bringing the G-quadruplex sequence in HP1 closer to hemin on HP2. Moreover, the resulting G-quadruplex/hemin DNAzymes exhibit robust peroxidase-like activity, enabling the catalysis of the colorimetric reaction of ABTS2- for furin detection. Our method demonstrates high sensitivity, rapid response, and compatibility with complex sample matrices, achieving a detection limit as low as 1.1 pM. SIGNIFICANCE: The DNAzyme reported in this work exhibits robust catalytic activity, enabling high sensitivity and good efficiency for the detection. By eliminating the requirement for exogenous enzymes, our approach enables visual furin detection without expensive instrumentation and reagents, promising significant utility in biomedical and clinical diagnostic applications. Given the various design of peptide sequence and the programmability of DNA, it can be readily applied to analyzing other useful tumor biomarkers.

Simple and sensitive detection of Pseudomonas aeruginosa in neonatal infection based on a both-end blocked peroxidase-mimicking DNAzyme.

Developing a simple and highly sensitive approach for Pseudomonas aeruginosa (P. aeruginosa) detection is crucial, as it is closely associated with various disorders, such as newborn infections. Nevertheless, few of techniques have the capability to accurately identify P. aeruginosa with a high level of sensitivity and significantly improved stability. The employment of the both-end blocked peroxidase-mimicking DNAzyme significantly diminished the interferences from background signals, so conferring the approach with a high degree of selectivity and reproducibility. The proposed method is demonstrated with exceptional discernment capacity in differentiating interfering microorganisms. The simplicity, elevated sensitivity and high discerning capability make the method a highly promising alternative instrument for pathogenic bacteria detection.

This research presents a novel method for detecting P. aeruginosa using a combination of a simple molecular beacon (MB), duplex-specific nuclease (DSN), and both-end blocked peroxidase-mimicking DNAzyme. The MB probe utilized in this method can be shielded from DSN hydrolysis without requiring any additional modifications by regulating the number of stem bases to five. This assay is simple yet precise in its ability to quantitatively detect P. aeruginosa with a high level of sensitivity and specificity. In addition, the beacon enabled the identification of P. aeruginosa without the need for labeling, exhibiting a higher sensitivity over the conventional hairpin fluorescence beacon based methods.

Mirror-Image DNA Nanobox for Enhancing Environment Resistance of Nucleic Acid Probes.

Degradation and interference of the nucleic acid probes in complex biological environments like cytoplasm or body fluid can cause obvious false-positive signals and inefficient bioregulation in biosensing and biomedicine. To solve this problem, here, we proposed a universal strategy, termed L-DNA assembly mirror-image box-based environment resistance (L-AMBER), to protect nucleic acid probes from degradation and maintain their responsive activity in complex biological environments. Strand displacement reaction (SDR), aptamer, or DNAzyme-based D-DNA probes were encapsulated into an L-DNA box by using an L-D-L block DNA carrier strand to construct different kinds of L-AMBER probes. We proved that the L-DNA box could effectively protect the encapsulated D-DNA probes by shielding the interference of complex biological environments and only allowing small target molecules to enter for recognition. Compared with the D-AMBER probes, the L-AMBER probes can realize DNase I-assisted amplification detection of biological samples, low false-positive bioimaging, and highly efficient miRNA silence in living cells. Therefore, L-AMBER provided a universal and effective strategy for enhancing the resistance to environmental interference of nucleic acid probes in biosensing and biomedicine applications.

Dual-Function DNA Nanowires with Self-Feedback Amplification and Efficient Signal Transduction for Intracellular Imaging of MicroRNA-155.

Intracellular detection and imaging of microRNAs (miRNAs) with low expression usually face the problem of unsatisfactory sensitivity. Herein, a novel dual-function DNA nanowire (DDN) with self-feedback amplification and efficient signal transduction was developed for the sensitive detection and intracellular imaging of microRNA-155 (miRNA-155). Target miRNA-155 triggered catalytic hairpin assembly (CHA) to generate plenty of double-stranded DNA (dsDNA), and a trigger primer exposed in dsDNA initiated a hybridization chain reaction (HCR) between four well-designed hairpins to produce DDN, which was encoded with massive target sequences and DNAzyme. On the one hand, target sequences in DDN acted as self-feedback amplifiers to reactivate cascaded CHA and HCR, achieving exponential signal amplification. On the other hand, DNAzyme encoded in DDN acted as signal transducers, successively cleaving Cy5 and BHQ-2 labeled substrate S to obtain a significantly enhanced fluorescence signal. This efficient signal transduction coupling self-feedback amplification greatly improved the detection sensitivity with a limit of detection of 160 aM for miRNA-155, enabling ultrasensitive imaging of low-abundance miRNA-155 in living cells. The constructed DDN creates a promising fluorescence detection and intracellular imaging platform for low-expressed biomarkers, exhibiting tremendous potential in biomedical studies and clinical diagnosis of diseases.

High-Sensitivity Homogeneous Detection of miRNA-155 Governed by DNA Walker-Regulated Surface DNA Density of Magnetic Electrochemiluminescence Nanoparticles.

Homogeneous electrochemiluminescence (ECL) has gained attention for its simplicity and stability. However, false positives due to solution background interference pose a challenge. To address this, magnetic ECL nanoparticles (Fe3O4@Ru@SiO2 NPs) were synthesized, offering easy modification, magnetic separation, and stable luminescence. These were utilized in an ECL sensor for miRNA-155 (miR-155) detection, with locked DNAzyme and substrate chain (mDNA) modified on their surface. The poor conductivity of long-chain DNA significantly impacts the conductivity and electron transfer capability of Fe3O4@Ru@SiO2 NPs, resulting in weaker ECL signals. Upon target presence, unlocked DNAzyme catalyzes mDNA cleavage, leading to shortened DNA chains and reduced density. In contrast, the presence of short-chain DNA has minimal impact on the conductivity and electron transfer capability of Fe3O4@Ru@SiO2 NPs. Simultaneously, the material surface's electronegativity decreases, weakening the electrostatic repulsion with the negatively charged electrode, resulting in the system detecting stronger ECL signals. This sensor enables homogeneous ECL detection while mitigating solution background interference through magnetic separation. Within a range of 100 fM to 10 nM, the sensor exhibits a linear relationship between ECL intensity and target concentration, with a 26.91 fM detection limit. It demonstrates high accuracy in clinical sample detection, holding significant potential for clinical diagnostics. Future integration with innovative detection strategies may further enhance sensitivity and specificity in biosensing applications.

Exploring the catalytic mechanism of the 10-23 DNAzyme: insights from pH-rate profiles.

The 10-23 DNAzyme, a catalytic DNA molecule with RNA-cleaving activity, has garnered significant interest for its potential therapeutic applications as a gene-silencing agent. However, the lack of a detailed understanding about its mechanism has hampered progress. A recent structural analysis has revealed a highly organized conformation thanks to the stabilization of specific interactions within the catalytic core of the 10-23 DNAzyme, which facilitate the cleavage of RNA. In this configuration, it has been shown that G14 is in good proximity to the cleavage site which suggests its role as a general base, by activating the 2'-OH nucleophile, in the catalysis of the 10-23 DNAzyme. Also, the possibility of a hydrated metal acting as a general acid has been proposed. In this study, through activity assays, we offer evidence of the involvement of general acid-base catalysis in the mechanism of the 10-23 DNAzyme by analyzing its pH-rate profiles and the role of G14, and metal cofactors like Mg2+ and Pb2+. By substituting G14 with its analogue 2-aminopurine and examining the resultant pH-rate profiles, we propose the participation of G14 in a catalytically relevant proton transfer event, acting as a general base. Further analysis, using Pb2+ as a cofactor, suggests the capability of the hydrated metal ion to act as a general acid. These functional results provide critical insights into the catalytic strategies of RNA-cleaving DNAzymes, revealing common mechanisms among nucleic acid enzymes that cleave RNA.

Bioinspired Dual Hemin-Bonded G-Quadruplex and Histidine-Functionalized Metal-Organic Framework for Sensitive Biosensing.

Biomimetic enzymes have emerged as ideal alternatives to natural enzymes, and there is considerable interest in designing biomimetic enzymes with enhanced catalytic performance to address the low activity of the current biomimetic enzymes. In this study, we proposed a meaningful strategy for constructing an efficient peroxidase-mimicking catalyst, called HhG-MOF, by anchoring histidine (H) and dual hemin-G-quadruplex DNAzyme (double hemin covalently linked to 3' and 5' terminals of G-quadruplex DNA, short as hG) to a mesoporous metal-organic framework (MOF). This design aims to mimic the microenvironment of natural peroxidase. Remarkably, taking a terbium MOF as a typical model, the initial rate of the resulting catalyst was found to be 21.1 and 4.3 times higher than that of Hh-MOF and hG-MOF, respectively. The exceptional catalytic properties of HhG-MOF can be attributed to its strong affinity for substrates. Based on the inhibitory effect of thiocholine (TCh) produced by the reaction between acetylcholinesterase (AChE) and acetylthiocholine, a facile, cost-effective, and sensitive colorimetric method was designed based on HhG-MOF for the measurement of AChE, a marker of several neurological diseases, and its inhibitor. This allowed a linear response in the 0.002 to 1 U L-1 range, with a detection limit of 0.001 U L-1. Furthermore, the prepared sensor demonstrated great selectivity and performed well in real blood samples, suggesting that it holds promise for applications in the clinical field.

Colorimetric detection of single-nucleotide mutations based on rolling circle amplification and G-quadruplex-based DNAzyme.

In this work, we proposed a sensitive and selective colorimetric assay for single nucleotide mutation (SNM) detection combining rolling circle amplification (RCA) and G-quadruplex/hemin DNAzyme complex formation. In the detection principle, the first step involves ssDNA hybridization with a padlock probe (PLP) DNA, which can discriminate a single base mismatch. The successful ligation is followed by an RCA event to generate an abundance of G-quadruplexes (GQ-RCA) which are then transformed into a DNAzyme (G-quadruplex/hemin complex) by the addition of hemin. The color change from colorless 3,3',5,5'-tetramethylbenzidine (TMB) into colored oxTMB when hydrogen peroxide (H2O2) is added indicated the presence of a mutation. The assay had a limit of detection (LOD) of 2.14 pM. Mutations in samples from breast cancer patients were successfully detected with an accuracy of 100% when compared to Sanger sequencing results. The method is easily applicable even in resource poor setting regions given that it doesn't require any sophisticated or expensive instruments, and the signal readout is detectable simply by the naked eye. Our assay might be a useful tool for genetic analysis and clinical molecular diagnosis for breast cancer risk assessment and early detection.

Spatially Localized Entropy-Driven Evolution of Nucleic Acid-Based Constitutional Dynamic Networks for Intracellular Imaging and Spatiotemporal Programmable Gene Therapy.

The primer-guided entropy-driven high-throughput evolution of the DNA-based constitutional dynamic network, CDN, is introduced. The entropy gain associated with the process provides a catalytic principle for the amplified emergence of the CDN. The concept is applied to develop a programmable, spatially localized DNA circuit for effective in vitro and in vivo theranostic, gene-regulated treatment of cancer cells. The localized circuit consists of a DNA tetrahedron core modified at its corners with four tethers that include encoded base sequences exhibiting the capacity to emerge and assemble into a [2 × 2] CDN. Two of the tethers are caged by a pair of siRNA subunits, blocking the circuit into a mute, dynamically inactive configuration. In the presence of miRNA-21 as primer, the siRNA subunits are displaced, resulting in amplified release of the siRNAs silencing the HIF-1α mRNA and fast dynamic reconfiguration of the tethers into a CDN. The resulting CDN is, however, engineered to be dynamically reconfigured by miRNA-155 into an equilibrated mixture enriched with a DNAzyme component, catalyzing the cleavage of EGR-1 mRNA. The DNA tetrahedron nanostructure stimulates enhanced permeation into cancer cells. The miRNA-triggered entropy-driven reconfiguration of the spatially localized circuit leads to the programmable, cooperative bis-gene-silencing of HIF-1α and EGR-1 mRNAs, resulting in the effective and selective apoptosis of breast cancer cells and effective inhibition of tumors in tumor bearing mice.

Triple modal aptasensor arrays driven by CHA-mediated DNAzyme for signal-amplified atrazine pesticide accumulation monitoring in agricultural crops.

Developing sensors with high selectivity and sensitivity is of great significance for pesticide analysis in environmental assessment. Herein, a versatile three-way sensor array was designed for the detection of the pesticide atrazine, based on the integration of catalytic hairpin assembly (CHA) amplification and three-mode signal transducers. With atrazine, CHA was triggered to generate abundant G-quadruplex. The produced G-quadruplex hybrid could assemble with thioflavin T (TFT) or hemin to mimic enzyme and induce the fluorescence enhancement by TFT, or the colorimetric increase by the oxidized chromogenic substrate and the naked-eye color change by inhibiting the L-cysteine-mediated aggregation of gold nanoparticles. A distinctive three-mode array was successfully constructed with convenience, on-site accessibility and high sensitivity for enzyme-free practical analysis of atrazine. It is also effective and reliable for analyzing real samples including paddy water, paddy soil and polished rice. The detection limits for atrazine were as low as 7.4 pg/mL by colorimetric observation and 0.25 pg/mL by fluorescent detection. Furthermore, the array was exploited to monitor the residue, distribution and bioaccumulation of atrazine in maize and rice for food security and environmental assessment. Hence, this work presented a versatile example for sensitive and on-site all-in-one pesticide analysis arrays with multiple signal report modes.

Inhibition of Transmural Inflammation in Crohn's Disease by Orally Administered Tumor Necrosis Factor-Alpha Deoxyribozymes-Loaded Pyroptosis Nanoinhibitors.

Crohn's disease (CD) is a refractory chronic inflammatory bowel disease (IBD) with unknown etiology. Transmural inflammation, involving the intestine and mesentery, represents a characteristic pathological feature of CD and serves as a critical contributor to its intractability. Here, this study describes an oral pyroptosis nanoinhibitor loaded with tumor necrosis factor-α (TNF-α) deoxyribozymes (DNAzymes) (DNAzymes@degradable silicon nanoparticles@Mannose, Dz@MDSN), which can target macrophages at the site of inflammation and respond to reactive oxygen species (ROS) to release drugs. Dz@MDSN can not only break the inflammatory cycle in macrophages by degrading TNF-α mRNA but also reduce the production of ROS mainly from macrophages. Moreover, Dz@MDSN inhibits excessive pyroptosis in epithelial cells through ROS clearance, thereby repairing the intestinal barrier and reducing the translocation of intestinal bacteria to the mesentery. Consequently, these combined actions synergistically contribute to the suppression of inflammation within both the intestine and the mesentery. This study likely represents the first successful attempt in the field of utilizing nanomaterials to achieve transmural healing for CD, which also provides a promising treatment strategy for CD patients.

Redesigned Guide DNA Enhanced Clostridium butyricum Argonaute Activity for Amplification-Free and Multiplexed Detection of Pathogens.

Clostridium butyricum (CbAgo)-based bioassays are popular due to their programmability and directional cleavage capabilities. However, the relatively compact protein structure of CbAgo limits its cleavage activity (even at the optimal temperature), thus restricting its wider application. Here, we observed that guide DNA (gDNA) with specific structural features significantly enhanced CbAgo cleavage efficiency. Then, we invented a novel gDNA containing DNAzyme segments (gDNAzyme) that substantially enhanced the CbAgo cleavage efficency (by 100%). Using a molecular dynamics simulation system, we found that the augmented cleavage efficiency might be attributed to the large-scale global movement of the PIWI domain of CbAgo and an increased number of cleavage sites. Moreover, this gDNAzyme feature allowed us to create a biosensor that simultaneously and sensitively detected three pathogenic bacteria without DNA extraction and amplification. Our work not only dramatically expands applications of the CbAgo-based biosensor but also provides unique insight into the protein-DNA interactions.

Development of a DNAzyme-Driven Fluorescent Light-Up Aptasensor for Label-Free Detection of Multiple lncRNAs.

Long noncoding RNAs (lncRNAs) act as the dynamic regulatory molecules that control the expression of genes and affect numerous biological processes, and their dysregulation is associated with tumor progression. Herein, we develop a fluorescent light-up aptasensor to simultaneously measure multiple lncRNAs in living cells and breast tissue samples based on the DNAzyme-mediated cleavage reaction and transcription-driven synthesis of light-up aptamers. When target lncRNAs are present, they can be recognized by template probes to form the active DNAzyme structures, initiating the T4 PNK-catalyzed dephosphorylation-triggered extension reaction to generate double-strand DNAs with the T7 promoter sequences. The corresponding T7 promoters can initiate the transcription amplification catalyzed by the T7 RNA polymerase to generate abundant Broccoli aptamers and malachite green aptamers, which can bind DFHBI-1T and MG to generate strong fluorescence signals. Taking advantage of the good selectivity of DNAzyme-mediated cleavage of lncRNAs, high amplification efficiency of T7 transcription-driven amplification reaction, and bright fluorescence of the RNA aptamer-fluorophore complex, this method exhibits high sensitivity with a detection limit of 21.4 aM for lncRNA HOTAIR and 18.47 aM for lncRNA MALAT1, and it can accurately measure multiple lncRNAs in both tumor cell lines and breast tissue samples, providing a powerful paradigm for biomedical research and early clinic diagnostics.

A Dual-Recognition Fluorescence Enzyme-Linked Immunosorbent Assay for Specific Detection of Intact Lipid Nanoparticles via a Localized Scaffolding Autocatalytic DNA Circuit Amplifier.

Lipid nanoparticles (LNPs) are emerging as one of the most promising drug delivery systems. The long-circulating effect of intact LNPs (i-LNPs) is the key to efficacy and toxicity in vivo. However, the significant challenge is specific and sensitive detection of i-LNPs. Herein, a dual-recognition fluorescence enzyme-linked immunosorbent assay (DR-FELISA) was developed to directly isolate and detect i-LNPs by combining dual-recognition separation with a one-step signal amplification strategy. The microplates captured and enriched i-LNPs through antibody-antigen reaction. Dual-chol probes were spontaneously introduced into the lipid bilayer of captured i-LNPs, converting the detection of i-LNPs into the detection of double-cholesterol probes. Finally, the end of the dual-chol probes initiated the localized scaffolding autocatalytic DNA circuits (SADC) system for further signal amplification. The SADC system provides a sensitive and efficient amplifier through localized network structures and self-assembled triggers. Simultaneous recognition of i-LNPs surface PEG-lipid and lipid bilayer structures significantly eliminates interference from biological samples. i-LNPs were detected with high selectivity, ranging from 0.2 to 1.25 mg/mL with a limit of detection of 0.1 mg/mL. Moreover, this method allows the isolation and quantitative analysis of different formulations of i-LNPs in serum samples with a satisfactory recovery rate ranging from 94.8 to 116.3%. Thus, the DR-FELISA method provides an advanced platform for the exclusive and sensitive detection of i-LNPs, providing new insights for the study of the quality and intracorporal process of complex formulations.

Hook-Like DNAzyme-Activated Autocatalytic Biosensor for the Universal Detection of Pathogenic Bacteria.

DNAzyme-based assays have found extensive utility in pathogenic bacteria detection but often suffer from limited sensitivity and specificity. The integration of a signal amplification strategy could address this challenge, while the existing combination methods require extensive modification to accommodate various DNAzymes, limiting the wide-spectrum bacteria detection. We introduced a novel hook-like DNAzyme-activated autocatalytic nucleic acid circuit for universal pathogenic bacteria detection. The hook-like connector DNA was employed to seamlessly integrate the recognition element DNAzyme with the isothermal enzyme-free autocatalytic hybridization chain reaction and catalytic hairpin assembly for robust exponential signal amplification. This innovative autocatalytic circuit substantially amplifies the output signals from the DNAzyme recognition module, effectively overcoming DNAzyme's inherent sensitivity constraints in pathogen identification. The biosensor exhibits a strong linear response within a range of 1.5 × 103 to 3.7 × 107 CFU/mL, achieving a detection limit of 1.3 × 103 CFU/mL. Noted that the sensor's adaptability as a universal detection platform is established by simply modifying the hook-like connector module, enabling the detection of various pathogenic bacteria of considerable public health importance reported by the World Health Organization, including Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Salmonella typhimurium. Additionally, the specificity of DNAzyme in bacterial detection is markedly improved due to the signal amplification process of the autocatalytic circuit. This hook-like DNAzyme-activated autocatalytic platform presents a versatile, sensitive, and specific approach for pathogenic bacteria detection, promising to significantly expand the applications of DNAzyme in bacteria detection.

Fingerprinting DNAzyme Cross-Reactivity for Pattern-Based Detection of Heavy Metals.

Heavy metal contamination in food and water is a major public health concern because heavy metals are toxic in minute amounts. DNAzyme sensors are emerging as a promising tool for rapid onsite detection of heavy metals, which can aid in minimizing exposure. However, DNAzyme activity toward its target metal is not absolute and has cross-reactivity with similar metals, which is a major challenge in the wide-scale application of DNAzyme sensors for environmental monitoring. To address this, we constructed a four DNAzyme array (17E, GR-5, EtNA, and NaA43) and used a pattern-based readout to improve sensor accuracy. We measured cross-reactivity between three metal cofactors (Pb2+, Ca2+, and Na+) and common interferents (Mg2+, Zn2+, Mn2+, UO22+, Li+, K+, and Ag+) and then used t-SNE analysis to identify and quantify the metal ion. We further showed that this method can be used for distinguishing mixtures of metals and detecting Pb2+ in environmental soil samples at micromolar concentrations.

Controllable self-cleaning FET self-assembled RNA-cleaving DNAzyme based DNA nanotree for culture-free Staphylococcus aureus detection.

Staphylococcus aureus (SA) poses a serious risk to human and animal health, necessitating a low-cost and high-performance analytical platform for point-of-care diagnostics. Cellulose paper-based field-effect transistors (FETs) with RNA-cleaving DNAzymes (RCDs) can fulfill the low-cost requirements, however, its high hydrophilicity and lipophilicity hinder biochemical modification and result in low sensitivity, poor mechanical stability and poor fouling performance. Herein, we proposed a controllable self-cleaning FET to simplify biochemical modification and improve mechanical stability and antifouling performance. Then, we constructed an RCD-based DNA nanotree to significantly enhance the sensitivity for SA detection. For controllable self-cleaning FET, 1 H,1 H,2 H,2 H-perfluorodecyltrimethoxysilane based-polymeric nanoparticles were synthesized to decorate cellulose paper and whole carbon nanofilm wires. O2 plasma was applied to regulate to reduce fluorocarbon chain density, and then control the hydrophobic-oleophobic property in sensitive areas. Because negatively charged DNA affected the sensitivity of semiconducting FETs, three Y-shaped branches with low-cost were designed and applied to synthesize an RCD-based DNA-Nanotree based on similar DNA-origami technology, which further improved the sensitivity. The trunk of DNA-Nanotree was composed of RCD, and the canopy was self-assembled using multiple Y-shaped branches. The controllable self-cleaning FET biosensor was applied for SA detection without cultivation, which had a wide linear range from 1 to 105 CFU/mL and could detect a low value of 1 CFU/mL.

Recent progress on DNAzyme-based biosensors for pathogen detection.

Pathogens endanger food safety, agricultural productivity, and human health. Those pathogens are spread through direct/indirect contact, airborne transmission and food/waterborne transmission, and some cause severe health consequences. As the population grows and global connections intensify, the transmission of infectious diseases expands. Traditional detection methods for pathogens still have some shortcomings, such as time-consuming procedures and high operational costs. To fulfil the demands for simple and effective detection, numerous biosensors have been developed. DNAzyme, a unique DNA structure with catalytic activity, is gradually being applied in the field of pathogen detection owing to its ease of preparation and use. In this review, we concentrated on the two main types of DNAzyme, hemin/G-quadruplex DNAzyme (HGD) and RNA-cleaving DNAzyme (RCD), explaining their research progress in pathogen detection. Furthermore, we introduced two additional novel DNAzymes, CLICK 17 DNAzyme and Supernova DNAzyme, which showed promising potential in pathogen detection. Finally, we summarize the strengths and weaknesses of these four DNAzymes and offer feasible recommendations for the development of biosensors.

Site-specific N-alkylation of DNA oligonucleotide nucleobases by DNAzyme-catalyzed reductive amination.

DNA and RNA nucleobase modifications are biologically relevant and valuable in fundamental biochemical and biophysical investigations of nucleic acids. However, directly introducing site-specific nucleobase modifications into long unprotected oligonucleotides is a substantial challenge. In this study, we used in vitro selection to identify DNAzymes that site-specifically N-alkylate the exocyclic nucleobase amines of particular cytidine, guanosine, and adenosine (C, G and A) nucleotides in DNA substrates, by reductive amination using a 5'-benzaldehyde oligonucleotide as the reaction partner. The new DNAzymes each require one or more of Mg2+, Mn2+, and Zn2+ as metal ion cofactors and have kobs from 0.04 to 0.3 h-1, with rate enhancement as high as ∼104 above the splinted background reaction. Several of the new DNAzymes are catalytically active when an RNA substrate is provided in place of DNA. Similarly, several new DNAzymes function when a small-molecule benzaldehyde compound replaces the 5'-benzaldehyde oligonucleotide. These findings expand the scope of DNAzyme catalysis to include nucleobase N-alkylation by reductive amination. Further development of this new class of DNAzymes is anticipated to facilitate practical covalent modification and labeling of DNA and RNA substrates.

A portability self-powered sensor facilitates sensitive Cd2+ detection: Dual mechanism and three quantitative mode.

As a common heavy metal contaminant, Cd2+ has adverse effects on food safety and consumer health. It is very important for human health to realize highly sensitive Cd2+ detection methods. The self-powered sensing system based on enzyme biofuel cells (EBFCs) does not need an external power supply, which can simplify the experimental equipment and has great application value in portable detection. Thus, the biosensor is innovatively integrated into the screen-printed electrode to construct a new type of portable sensor suitable for on-site and real-time Cd2+ detection. Hybridization chain reaction (HCR) combined with the Cd2+-dependent deoxyribose (DNAzyme) signal amplification strategy is used to enhance the detection sensitivity while specifically recognizing the Cd2+. Moreover, the self-powered sensor combines with smartphones to realize quantitative Cd2+ detection without other instruments and has the characteristic of Effectively improving the hazard detection technology is essential to ensure food safety. Portability, simplicity, and speed are suitable for real-time Cd2+ detection in the field. The dual mechanism and three quantitative modes combining colorimetric and two electrical signals output modes are adopted to realize the visualization and accurate detection. A series of research results confirm that this strategy is of great significance to strengthen the development of intelligent Cd2+ technology, expand the application of self-powered sensing technology, and improve the safety detection system.