Search Results (133)

Magnesium ions (Mg²⁺) play an important role in animal health, with their concentration in the bloodstream serving as a key indicator for hypomagnesemia diagnosis. In this study, a flexible hydrophobic paper-based microfluidic field-effect biosensor was developed for point-of-care Mg²⁺ detection, which integrated flexible hydrophobic paper, semiconducting single-walled carbon nanotubes (SWNTs) and a Mg²⁺-specific RNA-cleaving DNAzyme(RCD)-based DNA nanostructure. Flexible hydrophobic paper was synthesized by using cellulose paper and octadecyltrichlorosilane, improving mechanical strength and decreasing biological interference. To achieve high sensitivity, the Mg²⁺-specific RCD was functionalized with SWNTs, and then repeatedly self-assembled two different Y-shaped DNAs to construct a DNA nanostructure based on a similar DNA origami technique. This proposed biosensor exhibited a linear detection range from 1 μM to 1000 μM, with a detection limit of 0.57 μM, demonstrating its great stability, selectivity, and anti-interference performance. This innovative design offers promising potential for Mg²⁺ monitoring in real applications. Full article

The B Cell Lymphoma-2 (BCL-2) family proteins are central regulators of apoptosis, and their dysregulation is frequently associated with cancer progression and resistance to therapy. While small molecules like venetoclax have shown promise, nucleic acid-based therapeutics targeting BCL-2 remain underexplored. Here, we report a novel in vitro evolution strategy to generate trans-acting RNA-cleaving DNAzymes targeting natural BCL-2 mRNA without requiring covalent substrate-linking. Using a 50-base region of BCL-2 mRNA as a selection target, we evolved several DNAzymes that demonstrate significant RNA cleavage activity. These DNAzymes downregulated BCL-2 expression, induced apoptosis, and reduced cell viability in HepG2 and MCF-7 cancer cells. In vivo, our novel DNAzymes significantly suppressed tumor growth in a syngeneic mouse breast cancer model, with efficacy comparable to 5-Fluorouracil. This study presents a proof of concept for a novel strategy to evolve functional DNAzymes against native mRNA sequences and highlights their potential as gene-silencing tools in cancer therapy. Future studies will explore the therapeutic potential of these findings in cancer patients. Additionally, investigating the underlying molecular mechanisms in more complex cancer models will further validate the observed effects. Full article

The rapid and ultrasensitive detection of Salmonella holds strategic significance for food safety surveillance and public health protection systems. This study innovatively developed a label-free biosensing platform based on the synergistic integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a and the fluorescent deoxyribozyme Aurora for the efficient detection of foodborne Salmonella. The detection mechanism operates through a molecular cascade reaction: target-activated Cas12a protein specifically degrades Aurora deoxyribozyme via its trans-cleavage activity, thereby abolishing the enzyme’s catalytic capability to convert 4-methylumbelliferyl phosphate (4-MUP) into the highly fluorescent product 4-methylumbelliferone (4-MU). This cascade ultimately enables quantitative target analysis through fluorescence signal attenuation. Following systematic optimization of critical reaction parameters, the biosensing system demonstrated exceptional analytical performance: a detection limit of 1.29 CFU/mL with excellent linearity (R² = 0.992) spanning six orders of magnitude (1.65 × 10¹–10⁶ CFU/mL), along with high specificity against multiple interfering bacterial strains. Spike-and-recovery tests in complex food matrices (milk, chicken, and lettuce) yielded recoveries of 90.91–99.40% (RSD = 3.55–4.72%), confirming robust practical applicability. Notably, the platform design allows flexible detection of other pathogens through simple replacement of CRISPR guide sequences. Full article

Electrochemiluminescence (ECL)/electrochemistry (EC) dual-mode sensors have garnered significant interest for their enhanced analytical reliability through the cross-verification of dual-signal outputs. However, conventional approaches necessitate two potential scans to acquire ECL and EC signals independently, resulting in temporal and environmental discrepancies between the two detection modes. In this paper, we present a novel synchronous ECL/EC dual-mode sensing platform for lead ion (Pb²⁺) detection via a one-step potential scan (0.2 to −0.4 V vs. Ag/AgCl) utilizing a G-quadruplex (G4)–hemin DNAzyme complex. This complex synergistically catalyzed the electrochemical reduction of dissolved oxygen, concurrently generating a distinct cathodic ECL emission from Ru(bpy)₃²⁺ and a synchronous reduction current peak at −0.25 V. Pb²⁺ quantification was achieved through its dose-dependent suppression of DNAzyme activity by destabilizing the G4–hemin interaction, thereby proportionally attenuating both ECL intensity and EC signal (reduction current). The integrated sensor demonstrated high sensitivity (detection limits of 1.51 nM for ECL detection and 2.03 nM for EC detection), robust anti-interference capability, and satisfactory reproducibility, with recoveries ranging from 95.5 to 103.1% in environmental water analysis. This work established a paradigm for one-step dual-mode sensor design, offering new prospects for environmental monitoring. Full article

The rapid and accurate detection of viral infections is of paramount importance, given their widespread impact across diverse demographics. Common viruses such as influenza, parainfluenza, rhinovirus, and adenovirus contribute significantly to respiratory illnesses. The pathogenic nature of certain viruses, characterized by rapid mutations and high transmissibility, underscores the urgent need for dynamic detection methodologies. Quantitative reverse transcription PCR (RT-qPCR) remains the gold-standard diagnostic tool. Its reliance on costly equipment, reagents, and skilled personnel has driven explorations of alternative approaches, such as catalytic DNA nanomachines. Diagnostic platforms using catalytic DNA nanomachines offer amplification-free nucleic acid detection without the need for protein enzymes and demonstrate feasibility and cost-effectiveness for both laboratory and point-of-care diagnostics. This study focuses on the development of multicomponent DNA nanomachines with catalytic proficiency towards a fluorescent substrate, enabling the generation of a fluorescent signal upon the presence of target nucleic acids. Specifically tailored variants are designed for detecting human parainfluenza virus type 3 (HPIV) and respiratory syncytial virus (RSV). The engineered DNA nanomachine features six RNA-binding arms for recognition and unwinding of RNA secondary structures, along with a catalytic core for nucleic acid cleavage, indicating potential utility in real clinical practice with minimal requirements. This research showcases the potential of DNA nanomachines as a reliable and sensitive diagnostic tool for RNA virus identification, offering promising prospects for clinical applications in the realm of infectious disease management. Full article

MircroRNA (miRNA) exhibits abnormal expression in many cancer diseases, and the detection and analysis of miRNA are significant for the early diagnosis of diseases and research on miRNA functions. In this work, we construct a UV-triggered DNAzyme (UTD) nanosensor for the early detection of miRNA in tumor cells. As the nanodevice was delivered into cells and irradiated by UV light, the controllable imaging of miRNA in living cells was achieved. This method effectively avoids false signal issues, providing a new strategy for high-spatiotemporal-resolution imaging of miRNA in living cells. Full article

Food safety is a pressing global concern due to the risks posed by contaminants such as pesticide residues, heavy metals, allergens, mycotoxins, and pathogenic microorganisms. While accurate, traditional detection methods like ELISA, HPLC, and mass spectrometry are often time-consuming and resource-intensive, highlighting the need for innovative alternatives. Biosensors based on biological recognition elements such as enzymes, antibodies, and aptamers, offer fast, sensitive, and cost-effective solutions. Using transduction mechanisms like electrochemical, optical, piezoelectric, and thermal systems, biosensors provide versatile tools for detecting contaminants. Advances in DNAzyme- and aptamer-based technologies enable the precise detection of heavy metals, while enzyme- and protein-based biosensors monitor metal-induced changes in biological activity. Innovations like microbial biosensors and DNA-modified electrodes enhance detection accuracy. Biosensors are also highly effective in identifying pesticide residues, allergens, mycotoxins, and pathogens through immunological, enzymatic, and nucleic acid-based techniques. The integration of nanomaterials and bioelectronics has significantly improved the sensitivity and performance of biosensors. By facilitating real-time, on-site monitoring, these devices address the limitations of conventional methods to ensure food quality and regulatory compliance. This review highlights the transformative role of biosensors and how biosensors are improved by emerging technologies in food contamination detection, emphasizing their potential to mitigate public health risks and enhance food safety throughout the supply chain. Full article

G-quadruplex (G4) DNAzymes with peroxidase activities hold potential for applications in biosensing. While these nanozymes are easy to assemble, they are not as efficient as natural peroxidase enzymes. Several approaches are being used to better understand the structural basis of their reaction mechanisms, with a view to designing constructs with improved catalytic activities. Spermine alters the structures and enhances the activities of some G4 DNAzymes. The reported effect of spermine in shifting the conformation of some G4 DNAzymes from antiparallel to parallel has not been tested on multimeric G4 DNAzymes. In this study, we examined the effects of spermine on the catalytic activities of multivalent constructs of Bcl2, c-MYC, PS2.M, and PS5.M. Our findings show that spermine significantly improved the peroxidase activity of PS2.M, an antiparallel G4 DNAzyme, while there was no significant effect on c-MYC, which already exists in a parallel conformation. The addition of spermine led to a substantial increase in the initial velocity of PS2.M and its multimeric form, enhancing it by approximately twofold. Therefore, spermine enhancement offers promise in expanding the range of DNAzymes available for use as biosensing tools. Full article

Vibrio harveyi is a serious bacterial pathogen which can infect a wide range of marine organisms, such as marine fish, invertebrates, and shrimp, in aquaculture, causing severe losses. In addition, V. harveyi can be transmitted through food and water, infecting humans and posing a serious threat to public safety. Therefore, rapid and accurate detection of this pathogen is key for the prevention and control of related diseases. In this study, nine rounds of in vitro screening were conducted with Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology using unmodified DNA libraries, targeting the crude extracellular matrix (CEM) of V. harveyi. Two DNAzymes, named DVh₁ and DVh₃, with high activity and specificity were obtained. Furthermore, a fluorescent biosensor with dual DNAzymes was constructed which exhibited improved detection efficiency. The sensor showed a good fluorescence response to multiple aquatic products (i.e., fish, shrimp, and shellfish) infected with V. harveyi, with a detection limit below 11 CFU/mL. The fluorescence signal was observed within 30 min of reaction after target addition. This simple, inexpensive, highly effective, and easy to operate DNAzymes biosensor can be used for field detection of V. harveyi. Full article

DNAzyme-based gene therapy has received great interest in the treatment of cancer and other diseases. Despite its considerable potential in clinical applications, the in vivo therapeutic efficacy of DNAzymes is still unsatisfactory, mainly due to challenges such as poor stability, inadequate catalytic activity, and insufficient target specificity. With the rapid development of nucleic acid chemistry and nanomedicine, substantial efforts have been dedicated in recent years to designing DNAzyme-based nanoplatforms with enhanced treatment efficacy. In addition, extensive combinations of DNAzymes with other treatment strategies have led to many synergistic therapies with improved therapeutic effects. This review focuses on the advance of DNAzyme-based nanoplatforms in cancer treatment. It first summarizes the solutions proposed in recent years to address each of the key challenges in DNAzyme-based tumor therapy, from the perspectives of DNA sequence design, the construction of organic/inorganic nanosystems, and regulation by environmental factors. Next, it reviews the different categories of combined treatment strategies using DNAzymes in cancer therapy. Finally, future trends as well as suggestions in the field are discussed in detail. Full article

Catalytic DNA has gained significant attention in recent decades as a highly efficient and tunable catalyst, thanks to its flexible structures, exceptional specificity, and ease of optimization. Despite being composed of just four monomers, DNA’s complex conformational intricacies enable a wide range of nuanced functions, including scaffolding, electrocatalysis, enantioselectivity, and mechano-electro spin coupling. DNA catalysts, ranging from traditional DNAzymes to innovative DNAzyme hybrids, highlight the remarkable potential of DNA in catalysis. Recent advancements in spectroscopic techniques have deepened our mechanistic understanding of catalytic DNA, paving the way for rational structural optimization. This review will summarize the latest studies on the performance and optimization of traditional DNAzymes and provide an in-depth analysis of DNAzyme hybrid catalysts and their unique and promising properties. Full article

The dynamic landscape of non-canonical DNA G-quadruplex (G4) folding into G-triplex intermediates has led to the study of G-triplex structures and their ability to serve as peroxidase-mimetic DNAzymes. Here we report the formation, stability, and catalytic activity of a 5′-truncated c-MYC promoter region G-triplex, c-MYC-G3. Through circular dichroism, we demonstrated that c-MYC-G3 adopts a stable, parallel-stranded G-triplex conformation. The chemiluminescent oxidation of luminol by the peroxidase mimicking DNAzyme activity of c-MYC-G3 was increased in the presence of Ca²⁺ ions. We utilized surface plasmon resonance to characterize both c-MYC-G3 G-triplex formation and its interaction with hemin. The detailed study of c-MYC-G3 and its ability to form a G-triplex structure and its DNAzyme activity identifies issues that can be addressed in future G-triplex DNAzyme designs. Full article

Viral hepatitis is a systemic infectious diseases caused by various hepatitis viruses, primarily leading to liver damage. It is widely prevalent worldwide, with hepatitis viruses categorized into five types: hepatitis A, B, C, D, and E, based on their etiology. Currently, the detection of hepatitis viruses relies on methods such as enzyme-linked immunosorbent assay (ELISA), immunoelectron microscopy to observe and identify viral particles, and in situ hybridization to detect viral DNA in tissues. However, these methods have limitations, including low sensitivity, high error rates in results, and potential false negative reactions due to occult serum infection conditions. To address these challenges, we have designed an AuNPs-DNA walker method that uses gold nanoparticles (AuNPs) and complementary DNA strands for detecting viral DNA fragments through a colorimetric assay and fluorescence detection. The DNA walker, attached to gold nanoparticles, comprises a long walking strand with a probe sequence bound and stem-loop structural strands featuring a modified fluorescent molecule at the 3′ end, which contains the DNAzyme structural domain. Upon the addition of virus fragments, the target sequence binds to the probe chains. Subsequently, the long walking strand is released and continuously hybridizes with the stem-loop structural strand. The DNAzyme undergoes hydrolytical cleavage by Mg²⁺, breaking the stem-loop structural strand into linear single strands. As a result of these structural changes, the negative charge density in the solution decreases, weakening spatial repulsion and rapidly reducing the stability of the DNA walker. This leads to aggregation upon the addition of a high-salt solution, accompanied by a color change. Virus typing can be performed through fluorescence detection. The innovative method can detect DNA/RNA fragments with high specificity for the target sequence, reaching concentrations as low as 1 nM. Overall, our approach offers a more convenient and reliable method for the detection of hepatitis viruses. Full article

Due to rapid industrialization, novel water-quality monitoring techniques for the detection of highly toxic and hazardous heavy metal ions are essential. Herein, a hybrid noble nanoparticle/DNAzyme electrochemical biosensor is proposed for the simultaneous and label-free detection of Pb²⁺ and Cr³⁺ in aqueous solutions. The sensor is based on the combination of a two-dimensional naked-platinum nanoparticle film and DNAzymes, whose double-helix configuration disassembles into smaller fragments in the presence of target-specific heavy metal ions. The electrochemical behavior of the fabricated sensor was investigated with non-faradaic electrochemical impedance spectroscopy (EIS), resulting in the successful detection of Pb²⁺ and Cr³⁺ well below their maximum permitted levels in tap water. So far, there has been no report on the successful detection of heavy metal ions utilizing the non-faradaic electrochemical impedance spectroscopy technique based on advanced nanomaterials paired with DNAzymes. This is also one of the few reports on the successful detection of chromium (III) via a sensor incorporating DNAzymes. Full article

Water is indispensable for life, yet many lack access to clean drinking water, resulting in fatalities from waterborne bacterial infections. Precise assessment of microbial abundance and viability in natural aquatic environments is vital. Adenosine triphosphate (ATP) serves as a parameter for viability assessments due to its presence in viable bacterial cells as an energy carrier. Traditional ATP detection methods involve chemical or enzymatic extraction, followed by measurement of light emission via the Luciferin–Luciferase complex. However, these methods are costly, present a low stability, require specialized equipment, and entail complex sample pretreatment. To overcome these limitations, we developed a biosensor based on aptamers, nucleic acid sequences with specific target-molecule-binding capabilities. Aptamers offer advantages such as an enhanced stability, a lower cost, and ease of design compared to antibodies. Recently, ATP has been used for aptamer selection testing. Our proposed biosensor utilizes a structure-switching ATP-binding DNA nanoswitch with two functional domains: a catalytic DNA-zyme domain and an ATP-binding aptamer domain. In the presence of ATP, its binding to the aptamer domain triggers the activation of the DNA-zyme domain, which is exploited for chemiluminescence (CL) detection. Integrating functional DNA biosensors with microfluidic paper-based analytical devices (µPADs) holds promise for point-of-care (POC) applications. However, achieving proper DNA binding on paper remains challenging, often requiring solution-based assay protocols, leaving µPADs for final signal readout. Here, we introduce an origami µPAD with preloaded dried reagents, allowing for on-paper assay execution upon sample addition and proper folding. Paper functionalization strategies and assay protocols were optimized to ensure simple and straightforward detection of ATP, employing a portable charge-coupled device (CCD) camera for CL detection. Calibration curves plotted against the logarithm of ATP concentration in the range of 1 to 500 µM facilitated determination of the assay’s limit of detection (LOD), which was found to be 3 µM. Full article