Phosphorylation of DNA with 5'-hydroxyl termini plays a critical role in a majority of normal cellular events, including DNA recombination, DNA replication, and repair of DNA during strand interruption. Determination of nucleotide kinase activity and inhibition is under intense development due to its importance in regulating nucleic acid metabolism. Here, by using T4 polynucleotide kinase (PNK) as a model, which plays an essential role in cellular nucleic acid metabolism, particularly in the cellular responses to DNA damage, we describe a strategy for simply and accurately determining nucleotide kinase activity and inhibition by means of a coupled λ exonuclease cleavage reaction and graphene oxide (GO) based platform. The dye attached dsDNA preserves most of the fluorescence when mixed with GO. While dsDNA is phosphorylated by PNK and then immediately cleaved by λ exonuclease, fluorescence is greatly quenched. Because of the super quenching ability and the high specific surface area of GO, the as-proposed platform presents an excellent performance with wide linear range and low detection limit in the cell extracts environment. Additionally, inhibition effects of adenosine diphosphate, ammonium sulfate, and sodium hydrogen phosphate have also been investigated. The method not only provides a universal platform for monitoring activity and inhibition of nucleotide kinase but also shows great potential in biological process researches, drug discovery, and clinic diagnostics.
Construction of an electrical signal-sensitive nanoreactor in response to small molecule remains a challenge in the developing fields of biomimetic device. Solid nanochannels are considered as promising candidates for constructing smart systems, which is highly sensitive to biochemical stimulus. Here, we report an hourglass shaped nanochannel reactor based on cascade enzymatic catalysis and cation-selective nanochannel system. The employed glucose-specific dual-enzyme combination, glucose oxidase (GOx) and horseradish peroxidase (HRP), ensures the glucose catalytic efficiency and selectivity. Presence of glucose immediately induced the bienzymatic sequential reaction. The yielding gluconic acid decreased the microenvironmental pH in the channel gradually. Different concentration of glucose produced different amount of acid and thus altered the negative charge density inside the nanochannel to different extent. Modification convenience and mechanical robustness also ensure the stability of the test platform. Owing to its unique cation-selective property and high sensitivity toward microenvironmental alteration, this nanodevice shows robust glucose-responsive properties through monitoring ionic current signatures.
Sensitive and selective detection of DNA is in urgent need due to its important role in human bodies. Many disorders, such as Alzheimer's disease and various cancers, are closely related with DNA damage. In this work, a novel electrochemical DNA biosensor was constructed on a DNA-assembling graphene platform which provided a robust, simple and biocompatible platform with large surface area for DNA immobilization. The as-designed DNA sensor was fabricated by directly assembling captured ssDNA on a graphene-modified electrode through the π-π stacking interaction between graphene and ssDNA bases. Then, the target DNA sequence and oligonucleotide probes-labeled AuNPs were able to hybridize in a sandwich assay format, following the AuNPs-catalyzed silver deposition. The deposited silver was further detected by differential pulse voltammetry. Owing to the high DNA loading ability of graphene and the distinct signal amplification by AuNPs-catalyzed silver staining, the resulting biosensor exhibited a good analytical performance with a wide detection linear range from 200 pM to 500 nM, and a low detection limit of 72 pM. Additionally, the biosensor was proved to be able to discriminate the complementary sequence from the single-base mismatch sequence. The simple biosensor is promising in developing electronic, on-chip assays in clinical diagnosis, environmental control, and drug discovery.
5'-Polynucleotide kinase is a crucial class of enzyme that catalyzes the phosphorylation of nucleic acids with 5'-hydroxyl termini. This process regulates many important cellular events, especially DNA repair during strand damage and interruption. The activity and inhibition of nucleotide kinase have proven to be an evident effect on cellular nucleic acid regulation and metabolism. Here, we describe a novel nanochannel biosensor for monitoring the activity and inhibition of T4 polynucleotide kinase (PNK), a famous member of the 5'-kinase family playing a major role in the cellular responses to DNA damage. On the basis of the functionalized nanochannel system and coupled λ exonuclease cleavage reaction, the nanochannel-sensing platform exhibits high sensitivity and convenience toward kinase analysis. Biotin-labeled dsDNA effectively blocks the streptavidin-modified nanochannel through forming a closely packed arrangement of DNA structure inside the channel. When dsDNA is phosphorylated by PNK and then immediately cleaved by λ exonuclease, the pore-blocking effect almost disappears. This PNK-induced microstructural distinctness can be directly and accurately monitored by the nanochannel system, which benefits from its high sensitivity to the change of the effective pore size. Furthermore, modification convenience and mechanical robustness also ensure the stability of the test platform. This as-proposed strategy exhibits excellent analytical performance in both PNK activity analysis and inhibition evaluation. The simple and sensitive nanochannel biosensor shows great potential in developing on-chip, high-throughput assays for fundamental biochemical process research, molecular-target therapies, and clinic diagnostics.
Two of the most persistent challenges for the sensing applications of luminescent carbon nitride-based materials are poor quantum yields and aggregation-induced luminescence quenching in aqueous environments. Herein, a highly emissive oxygen-doped carbon nitride composite (OCNP@M7) was synthesized, with a metal−azolate framework (MAF-7) serving as a luminous booster. Both experimental studies and theoretical calculations suggest that the MAF-enhanced electron-donating effect dramatically promoted the electron density on the πstructure of oxygen-doped carbon nitride. In addition, the structural rigidity of MAF-7 effectively inhibits both aggregation and nonradiative energy dissipation. Consequently, OCNP@M7 exhibits strong and stable blue emission under UV light irradiation and an absolute quantum yield up to 95.2%, which is, as far as we know, the highest value among fluorescent carbon nitride materials in solution ever reported. OCNP@M7 could further function as a high-efficiency fluorescent probe for the sensitive detection of sulfadimethoxine residues in complex environments. It is anticipated that this strategy can be extended to fabricate various carbon nitride-based antibiotic monitoring systems with tailor-made functions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.