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Cationic porphyrins with large side arm substituents as resonance light scattering ratiometric probes for specific recognition of nucleic acid G-quadruplexes
摘要： Specific G-quadruplex-probing is crucial for both biological sciences and biosensing applications. Most reported probes are focused on fluorescent or colorimetric recognition of G-quadruplexes. Herein, for the first time, we reported a new specific G-quadruplex-probing technique—resonance light scattering (RLS)-based ratiometric recognition. To achieve the RLS probing of G-quadruplexes in the important physiological pH range of 7.4-6.0, four water soluble cationic porphyrin derivatives, including an unreported octa-cationic porphyrin, with large side arm substituents were synthesized and developed as RLS probes. These RLS probes were demonstrated to work well for ratiometric recognition of G-quadruplexes with high specificity against single- and double-stranded DNAs, including long double-stranded ones. The working mechanism was speculated to be based on the RLS signal changes caused by porphyrin protonation that was promoted by the end-stacking of porphyrins on G-quadruplexes. This work adds an important member in G-quadruplex probe family, thus providing a useful tool for studies on G-quadruplex-related events concerning G-quadruplex formation, destruction and changes in size, shape and aggregation. As a proof-of-concept example of applications, the RLS probes were demonstrated to work well for label-free and sequence-specific sensing of microRNA. This work also provides a simple and useful way for the preparation of cationic porphyrins with high charges.
关键词： G-quadruplex,ratiometric recognition,microRNA sensing,resonance light scattering,cationic porphyrin
更新于2025-11-19 16:56:35
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Amplification-free and direct fluorometric determination of telomerase activity in cell lysates using chimeric DNA-templated silver nanoclusters
摘要： A fluorogenic probe has been developed for determination of telomerase activity using chimeric DNA-templated silver nanoclusters (AgNCs). The formation of AgNCs was investigated before (route A) and after (route B) telomerase elongation reaction. Both routes caused selective quenching of the yellow emission of the AgNCs (best measured at excitation/emission wavelength of 470/557 nm) in telomerase-positive samples. The quenching mechanism was studied using synthetically elongated DNA to mimic the telomerase-catalyzed elongation. The findings show that quenching is due to the formation of parallel G-quadruplexes with a –TTA– loop in the telomerase elongated products. The assay was validated using different cancer cell extracts, with intra- and interassay coefficients of variations of <9.8%. The limits of detection for MCF7, RPMI 2650 and HT29 cell lines are 15, 22 and 39 cells/μL. This represents a distinct improvement over the existing telomeric repeat amplification protocol (TRAP) assay in terms of time, sensitivity and cost.
关键词： Biomarker,Telomers,HT29,Biosensor,G-quadruplex,MCF7,AgNCs,TRAP,RPMI 2650,Cancer probe
更新于2025-09-23 15:23:52
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G-quadruplex specific dye-based ratiometric FRET aptasensor for robust and ultrafast detection of toxin
摘要： G-quadruplex specific dyes are powerful tools for probing nucleic acid structures. Among nucleic acids, aptamers are of great interest, and widely exploited to construct versatile bioassays. Herein, based on G-quadruplex selective dye, thioflavin T (ThT), for probing the intrinsic structure of aptamers, we proposed a ratiometric fluorescence resonance energy transfer (FRET) aptasensor enabling robust and ultrafast detection of toxin. The binding of target ochratoxin A (OTA) would destruct the G-quadruplex structure of aptamer. It would lead to the detachment of ThT dye from aptamer which diminished the FRET effect between ThT and terminal-labeled dye, thus allowing quantification of OTA via FRET signals. The FRET aptasensor would confer an enhancement of 76.9% of signal to background ratio compared to the ThT-based non-FRET aptasensor. Remarkably, the FRET mechanism would eliminate the signal fluctuation resulted from varied probe concentration, thus benefiting the robustness of the assay. The aptasensor could achieve a detection of limit of 0.38 ng/mL for OTA detection. And the detection of OTA could be finished within 30 s. Besides, the assay was successful in analyzing OTA in coffee and oat samples with recoveries rate of 93.93%–107.59%. Therefore, G-quadruplex specific dye-based probing and FRET method would be a compelling design strategy for aptasensor, and may facilitate their practical application in food safety and environmental screening.
关键词： Fluorescence resonance energy transfer,G-quadruplex specific dyes,Homogeneous analysis,Toxin,Thioflavin T,Aptamer
更新于2025-09-23 15:23:52
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Tuning the selectivity of N-alkylated styrylquinolinium dyes for sensing of G-quadruplex DNA
摘要： Selective and sensitive detection of G-quadruplex DNA structures is an important issue and attracts extensive interest. To this end, numerous small molecular fluorescent probes have been designed. Here, we present a series of N-alkylated styrylquinolinium dyes named Ls-1, Ls-2 and Ls-3 with varying side groups at the chain end. We found that these dyes exhibited different binding behaviors to DNAs, and Ls-2 with a sulfonato group at the chain end displayed sensitivity and selectivity to G-quadruplex DNA structures in vitro. The characteristics of this dye and its interaction with G-quadruplex DNA were comprehensively investigated by means of UV–vis spectrophotometry, fluorescence, circular dichroism and molecular docking. Furthermore, confocal fluorescence images and MTT assays indicated dye Ls-2 could pass through membrane and enter the living HepG2 cells with low cytotoxicity.
关键词： N-Alkylated styrylquinolinium dye,Living HepG2 cell,G-quadruplex DNA,Cytotoxicity
更新于2025-09-23 15:22:29
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Turn-On Fluorescence Aptasensor on Magnetic Nanobeads for Aflatoxin M1 Detection Based on an Exonuclease III-Assisted Signal Amplification Strategy
摘要： In order to satisfy the need for sensitive detection of Aflatoxin M1 (AFM1), we constructed a simple and signal-on fluorescence aptasensor based on an autocatalytic Exonuclease III (Exo III)-assisted signal amplification strategy. In this sensor, the DNA hybridization on magnetic nanobeads could be triggered by the target AFM1, resulting in the release of a single-stranded DNA to induce an Exo III-assisted signal amplification, in which numerous G-quadruplex structures would be produced and then associated with the fluorescent dye to generate significantly amplified fluorescence signals resulting in the increased sensitivity. Under the optimized conditions, this aptasensor was able to detect AFM1 with a practical detection limit of 9.73 ng kg?1 in milk samples. Furthermore, the prepared sensor was successfully used for detection of AFM1 in the commercially available milk samples with the recovery percentages ranging from 80.13% to 108.67%. Also, the sensor performance was evaluated by the commercial immunoassay kit with satisfactory results.
关键词： aptasensors,signal amplification,G-quadruplex,magnetic nanobeads,aflatoxin M1
更新于2025-09-23 15:22:29
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Single-Sided Competitive Axial Coordination of G-Quadruplex/Hemin as Molecular Switch for Imaging Intracellular Nitric Oxide
摘要： Axial coordination is a crucial biological process to regulate biomolecules’ functions in natural enzymes. However, it is a great challenge to determine the single or dual axial interaction between the metal center of enzymes and the ligand. In this work, a controllable axial coordination system was developed based on G-quadruplex/hemin complex by designing a series of fluorescent derivatives. The mechanism on axial coordination of G-quadruplex/hemin with coumarin-imidazole ligands was proposed to be single-sided, and led to fluorescence quenching of ligands. Upon addition of nitric oxide, the fluorescence of ligands was recovered through competitive axial coordination, providing a “signal on” strategy for signal transduction. More significantly, the fluorescent imaging of intracellular nitric oxide was achieved after conjugating with gold nanoparticles. Also, the proposed protocol provided a smart strategy to monitor the relationship between nitric oxide and p53 protein activity in living cells.
关键词： axial coordination,imaging,G-quadruplex-hemin,biosensors,fluorescent probes
更新于2025-09-23 15:21:21
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A fluorescence method for homogeneous detection of influenza A (H1N1) DNA sequence based on Guanine(G)‐quadruplex‐ N‐methylmesoporphyrin IX (NMM) complex and Assistance‐DNA (A‐DNA) inhibition
摘要： In this work, we report a fluorescence method for homogeneous detection of influenza A (H1N1) DNA sequence based on G-quadruplex-NMM complex and Assistance DNA (A-DNA) inhibition. The quadruplex-based functional DNA (QBF-DNA), composed of complementary probe to the target H1N1 DNA sequence and G-rich fragment, was designed as the signal DNA. The A-DNA consisted of two parts, one part was complementary to target H1N1 DNA and the other part was complementary to the signal DNA. In the absence of target H1N1 DNA, the G-rich fragment of QBF-DNA can form G-quadruplex-NMM complex, which outputted a fluorescent signal. With the presence of target H1N1 DNA, QBF-DNA and A-DNA can simultaneously hybridize with target H1N1 DNA to form double-helix structure. In this case, the A-DNA partially hybridized with the QBF-DNA, which inhibited the formation of G-quadruplex-NMM complex, leading to the decrease of fluorescent signal. Under the optimum conditions, the fluorescence intensity was inversely proportional to the concentration of target H1N1 DNA over the range from 25pmol/L to 700pmol/L with a detection limit of 8pmol/L. In addition, the method is target specific and practicability, and would become a new diagnostic assay for influenza A (H1N1) DNA sequence and other infectious diseases.
关键词： G-quadruplex,H1N1 virus,Fluorescence method,DNA hybridization
更新于2025-09-19 17:15:36
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Facile Synthesis of Fluorescent Tungsten Oxide Quantum Dots for Telomerase Detection Based on the Inner Filter Effect
摘要： The traditional detection of telomerase activity is mainly based on the polymerase chain reaction (PCR), which has the disadvantages of being time-consuming and susceptible to interference, and we here propose a facile method to fabricate fluorescent tungsten oxide quantum dots (WOx QDs) and employ them for telomerase activity sensing. It is found that the fluorescence of WOx QDs can be significantly quenched by hemin based on the inner filter effect (IFE). However, in the presence of telomerase, the primer-DNA can be extended to generate repeating unit of TTAGGG to form G-quadruplex, and thus hemin can be encapsulated to reduce its absorbance, resulting in decreased IFE and efficient fluorescence recovery of WOx QDs. Based on the fluorescence changes of IFE between hemin and WOx QDs, the telomerase activity with the range of 50-30000 HeLa cells can be detected and the lowest detection amount can reach 17 cells. The method exhibits good versatility that can be also applied to the telomerase detection from A549 and L929 cells. In addition, because of the good biocompatibility of the sensor, it can be used for real-time monitoring of telomerase activity in living cells, showing great potential in tumor diagnosis and inhibitor drug screening.
关键词： inner filter effect,G-quadruplex,hemin,telomerase activity detection,fluorescent tungsten oxide quantum dots
更新于2025-09-19 17:13:59
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A turn-on graphene quantum dot and graphene oxide based fluorometric aptasensor for the determination of telomerase activity
摘要： A turn-on fluorometric assay is described for determination of the activity of enzyme telomerase. For this purpose, graphene quantum dots (GQDs) were first modified with the telomeric sequence (5′-amino-AATCCGTCGAGCAGAGTT-3′) via a condensation reaction. Injection of graphene oxide causes instant quenching of the blue fluorescence of the GQDs. Addition of cell extract containing telomerase, triggers the extension of telomer via addition of specific sequence (TTAGGG)n to its 3′ end. Fluorescence, best measured at excitation/emission wavelengths of 390/446 nm, is subsequently restored due to folding of the extended telomeric sequence into G-quadruplex structure. The method was applied to the determination of telomerase activity in crude cell extracts of as little as 10 HeLa cells. The linear dynamic range extends from 10 to 6500 cells.
关键词： G-quadruplex,Biosensors,Fluorescence,Optical sensing,Biomarker,Cancer detection,Nanoprobe
更新于2025-09-12 10:27:22
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Label-free optical biosensor for target detection based on simulation-assisted catalyzed hairpin assembly
摘要： Taking the advantage of the high selectivity of aptamers and enzyme-free catalyzed hairpin assembly (CHA) amplification strategy, we herein describe a label-free and enzyme-free sensitive fluorescent and colorimetric strategy for amplified thrombin detection in this paper. To support both biological inquiry and technological innovation, thermodynamic models are introduced to predict the minimum energy secondary structure of interacting nucleic acid strands and calculate the partition function and equilibrium concentration for complexes in our system. Then, the thermodynamics properties of interacting DNA strands and the reactions of toehold strand displacement-driven assembly have been simulated, validating the feasibility of the theory and optimizing the follow-up lab tests. Following that, our strategy for thrombin detection is proved to be feasible and effective in biological experiments.
关键词： Computation and simulation,G-quadruplex,catalyzed hairpin assembly,fluorescence biosensor,DNA strand displacement
更新于2025-09-10 09:29:36