Proximity-Dependent Activation of Split DNAzyme Kinase.

Binary (also known as split) nucleic acid enzymes have emerged as novel tools in biosensors. We report a new split strategy to split the DNAzyme kinase into two independent and non-functional fragments, denoted Dk1sub and Dk1enz. In the presence of the specific target, their free ends are brought sufficiently close to interact with each other without the formation of Watson-Crick base pairings between Dk1sub and Dk1enz, thus allowing the DNA phosphorylation reaction. We term this approach proximity-dependent activation of split DNAzyme kinase (ProxSDK). The utility of ProxSDK is demonstrated by engineering a biosensing system that is capable of measuring specific DNA-protein interactions. We envision that the approach described herein will find useful applications in biosensing, imaging, and clinical diagnosis.

Target Recognition Triggered Split DNAzyme based Colorimetric Assay for Direct and Sensitive Methicillin-Resistance Analysis of Staphylococcus aureus.

The accurate and rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) holds significant clinical importance. This work presents a new method for detecting methicillin-resistant Staphylococcus aureus (S. aureus) in clinical samples. The method uses an aptamer-based colorimetric assay that combines a recognizing probe to identify the target and split DNAzyme to amplify the signal, resulting in a highly sensitive and direct analysis of methicillin-resistance. The identification of the PBP2a protein on the membrane of S. aureus in clinical samples leads to the allosterism of the recognizing probe, and thus provides a template for the proximity ligation of split DNAzyme. The proximity ligation of split DNAzyme forms an intact DNAzyme to identify the loop section in the L probe and generates a nicking site to release the loop sequence ("3" and "4" fragments). The "3" and "4" fragments forms an intact sequence to induce the catalytic hairpin assembly, exposing the G-rich section. The released the G-rich sequence of LR probe induces the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. The absorption intensity demonstrated a strong linear association with the logarithm of the S. aureus concentration across a wide range of 5 orders of magnitude dynamic range under the optimized experimental parameters. The limit of detection was calculated to be 23 CFU/ml and the method showed high selectivity for MRSA.

Microfluidic bead-based biosensor: Ultrasensitive ctDNA detection based on duplex-functional split-DNAzyme and dendritic enzyme-free signal amplification.

Circulating tumor DNA (ctDNA) is a crucial cancer biomarker for early or noninvasive monitoring, which is essential for developing ultrasensitive and selective assays in cancer diagnosis and treatment. Herein, a cascade signal amplification of duplex-functional split-DNAzyme and dendritic probes was proposed for ultrasensitive and specific detection of nasopharyngeal carcinoma-associated Epstein-Barr virus (EBV) DNA on microfluidic microbead array chips. With the assistance of Pb2+, the duplex-functional split-DNAzyme recognizes EBV DNA and then rapidly cleaves the substrate strand. Subsequently, the released target could be recycled, and its exposed capture probe, triggered the dendritic enzyme-free signal amplification. As the enhanced mass transfer capability, target recycling, and dendritic DNA structure signal amplification inherent to microfluidic bead arrays were integrated, it achieved an excellent detection limit of 0.36 fM and a wide linear range of 1 fM∼103 fM. Further, it was applied to content detect simulated samples of EBV DNA, recovery ranged from 97.2 % to 108.1 %, and relative standard deviation (RSD) from 3.3 % to 5.9 %, exhibiting satisfactory recovery results. The developed microfluidic biosensor was a high-sensitivity and anti-interference system for ctDNA analysis, with minimal reagent volumes (microlitres) required. Thus, it is a promising platform for ctDNA at the lowest level at their earliest incidence.

Asymmetrically split DNAzyme-based colorimetric and electrochemical dual-modal biosensor for detection of breast cancer exosomal surface proteins.

Exosomal surface proteins are potentially useful for breast cancer diagnosis and awareness of risk. However, some detection techniques involving complex operations and expensive instrumentation are limited to advance to clinical applications. To solve this problem, we develop a dual-modal sensor combining naked-eye detection and electrochemical assay of exosomal surface proteins from breast cancer. Most of existing sensors rely on aptamers recognizing exosomes and generating amplified signals at the same time, which require well-designed aptamer probes to avoid difficulties in identifying exosomes. In our work, aptamers not bound by the exosomes can serve as complete templates to induce formation of G quadruplexes. The peroxidase activity of the G-quadruplex/hemin DNAzyme catalyze substrates can generate both color and electrochemical signals. The developed dual-modal sensor offers a remarkable capability to differentiate nonmetastatic, metastatic breast cancer patients, and healthy individuals through the analysis of exosomal surface proteins. The sensor's distinctive features, including its universality, simplicity, and cost-effectiveness, position it as a promising diagnostic tool in breast cancer research and clinical practice.

Smartphone-based chemiluminescence detection of aflatoxin B1 via labelled and label-free dual sensing systems.

To develop a sensing platform for onsite determination of AFB1 in foodstuffs, we developed smartphone-based chemiluminescence detection of AFB1 via labelled and label-free dual modes. The labelled mode was characteristic of double streptavidin-biotin mediated signal amplification, obtaining limit of detection (LOD) of 0.04 ng/mL in the linear range of 1-100 ng/mL. To reduce the complexity in the labelled system, a label-free mode based on both split aptamer and split DNAzyme was fabricated. A satisfactory LOD of 0.33 ng/mL was generated in the linear range of 1-100 ng/mL. Both labelled and label-free sensing systems achieved outstanding recovery rate in AFB1-spiked maize and peanut kernel samples. Finally, two systems were successfully integrated into smartphone-based portable device based on custom-made components and android application, achieving comparable AFB1 detection ability to a commercial microplate reader. Our systems hold huge potential for AFB1 onsite detection in food supply chain.

DNAzyme cascade circuits in highly integrated DNA nanomachines for sensitive microRNAs imaging in living cells.

DNA molecular probes have emerged as powerful tools for fluorescence imaging of microRNAs (miRNAs) in living cells and thus elucidating functions and dynamics of miRNAs. In particular, the highly integrated DNA probes that can be able to address the robustness, sensitivity and consistency issues in a single assay system were highly desired but remained largely unsolved challenge. Herein, we reported for the first time that the development of the novel DNA nanomachines that split-DNAzyme motif was highly integrated in a single DNA triangular prism (DTP) reactor and can undergo target-activated DNAzyme catalytic cascade circuits, allowing amplified sensing and imaging of tumor-related microRNA-21 (miR-21) in living cells. The DNA nanomachines have shown dynamic responses for target miR-21 with excellent sensitivity and selectivity and demonstrated the potential for living cell imaging of miR-21. With the advantages of facile modular design and assembly, high biostability, low cytotoxicity and excellent cellular internalization, the highly integrated DNA nanomachines enabled accurate and effective monitoring of miR-21 expression levels in living cells. Therefore, our developed strategy may afford a reliable and robust nanoplatform for tumor diagnosis and for related biological research.

A visual and sensitive Hg2+ detection strategy based on split DNAzyme amplification and peroxidase-like activity of hemin-graphene composites.

A visual and sensitive Hg2+ detection strategy was developed based on split DNAzyme amplification and hemin-graphene oxide composites (H-GNs). Two split DNAzyme sequences can form two entire enzyme-strands DNA (E-DNA) by T-Hg2+-T interaction. The E-DNA can bind with the loop of molecular beacon (MB) to form Mg2+-dependent DNAzyme structure. The formed DNAzyme can circularly cleave the loop of MB, resulting large amount of DNA fragments. The resultant DNA fragments can prevent H-GNs from aggregation by adsorbing on its surface. Consequently, the supernate with large amount of H-GNs shows dark blue color after chromogenic reaction. This strategy shows a linear range from 50 pM to 1200 pM. The limit detection can be low to 33 pM. This strategy provides a visual and enzyme-free amplification mode for quick and sensitive screen of Hg2+.

A catalytic cleavage strategy for fluorometric determination of Hg(II) based on the use of a Mg(II)-dependent split DNAzyme and hairpins conjugated to gold nanoparticles.

A catalytic cleavage strategy was developed for the fluorometric determination of Hg(II). The method is based on the use of a Mg(II)-dependent split DNAzyme. Fluorophore labeled hairpins were conjugated to gold nanoparticles (AuNPs) upon which fluorescence is quenched. Thymine-Hg(II)-thymine (T-Hg(II)-T) interaction causes the two DNA sequences to form an entire enzyme-strand DNA (E-DNA). The E-DNA bind to the hairpins on the AuNPs to form a Mg(II)-dependent DNAzyme structure. The circular cleavage of hairpins results in a signal amplification and in the recovery of fluorescence. The assay has a limit of detection (LOD) as low as 80 pM of Hg(II). This LOD is comparable to those obtained with other amplification strategies. The method was successfully applied to the determination of Hg(II) in Chinese herbs (Atractylodes macrocephala Koidz). Graphical abstract Schematic of a catalytic cleavage strategy based on Mg(II)-dependent split DNAzyme for fluorometric determination of Hg(II).

A structure-switchable aptasensor for aflatoxin B1 detection based on assembly of an aptamer/split DNAzyme.

An ultrasensitive, colorimetric and homogeneous strategy for aflatoxin B1 (AFB1) detection, which uses a DNA aptamer and two split DNAzyme halves, has been developed. Split halves of a hemin-binding DNAzymes is combined with an AFB1 aptamer to generate a homogeneous colorimetric sensor that undergoes an AFB1 induced DNA structural change. In the absence of AFB1, the split probes have peroxidase mimicking DNAzyme activity associated with catalysis of a color change reaction. Specific recognition of AFB1 by the aptamer component leads to structural deformation of the aptamer-DNAzyme complex, which causes splitting of the DNAzyme halves and a reduction in peroxidase mimicking activity. Therefore, a decrease of colorimetric signal arising from the catalytic process takes place upon in the presence of AFB1 in a concentration dependent manner in the 0.1-1.0 × 10(4) ng/mL range and with a colorimetric detection limit of 0.1 ng/mL. The new assay system exhibits high selectivity for AFB1 over other mycotoxins and can be employed detect the presence of AFB1 in ground corn samples. Overall, the strategy should serve as the basis for the development of rapid, simple and low-cost methods for detection of mycotoxins.