Chemical-Oxidation Cleavage Triggered Isothermal Exponential Amplification Reaction for Attomole Gene-Specific Methylation Analysis

Genomic 5-methylcytosine (5-mC) modification is known to extensively regulate gene expression. The sensitive and convenient analysis of gene-specific methylation is wishful but challenging due to the lack of means that can sensitively and sequence-selectively discriminate 5-mC from cytosine without the need for polymerase chain reaction. Here we report a chemical-oxidation cleavage triggered exponential amplification reaction (EXPAR) method named COEXPAR for gene-specific methylation analysis. EXPAR was proved to not only have rapid amplification kinetics under isothermal condition but also show excellent sequence-selectivity and linear-dependence on EXPAR trigger. Further initiation of EXPAR by chemical-cleavage of DNA at 5-mC, the COEXPAR showed high specificity for methylated and nonmethylated DNA, and ∼10⁷ copies of triggers were replicated in 20 min, which were used to quantify the methylation level at the methylation loci. As a result, the gene-specific methylation level of a p53 gene fragment, as a target model, was analyzed in two linear ranges of 10 fM–1 pM and 1 pM–10 nM, and limits of detection of 411 aM (<i>S</i>/<i>N</i> = 3) by fluorescence, and 576 aM (<i>S</i>/<i>N</i> = 3) by electrochemistry. The method fulfilled the assay in an isothermal way in ∼5 h without the need for tedious sample preparation and accurate thermocycling equipment, which is likely to be a facile and ultrasensitive way for gene-specific methylation analysis

Construction of a Zinc Porphyrin–Fullerene-Derivative Based Nonenzymatic Electrochemical Sensor for Sensitive Sensing of Hydrogen Peroxide and Nitrite

Enzymatic sensors possess high selectivity but suffer from some limitations such as instability, complicated modified procedure, and critical environmental factors, which stimulate the development of more sensitive and stable nonenzymatic electrochemical sensors. Herein, a novel nonenzymatic electrochemical sensor is proposed based on a new zinc porphyrin–fullerene (C₆₀) derivative (ZnP–C₆₀), which was designed and synthesized according to the conformational calculations and the electronic structures of two typical ZnP–C₆₀ derivatives of <i>para</i>-ZnP–C₆₀ (ZnP_p–C₆₀) and <i>ortho</i>-ZnP–C₆₀ (ZnP_o–C₆₀). The two derivatives were first investigated by density functional theory (DFT) and ZnP_p–C₆₀ with a bent conformation was verified to possess a smaller energy gap and better electron-transport ability. Then ZnP_p–C₆₀ was entrapped in tetraoctylammonium bromide (TOAB) film and modified on glassy carbon electrode (TOAB/ZnP_p–C₆₀/GCE). The TOAB/ZnP_p–C₆₀/GCE showed four well-defined quasi-reversible redox couples with extremely fast direct electron transfer and excellent nonenzymatic sensing ability. The electrocatalytic reduction of H₂O₂ showed a wide linear range from 0.035 to 3.40 mM, with a high sensitivity of 215.6 μA mM^–1 and a limit of detection (LOD) as low as 0.81 μM. The electrocatalytic oxidation of nitrite showed a linear range from 2.0 μM to 0.164 mM, with a sensitivity of 249.9 μA mM^–1 and a LOD down to 1.44 μM. Moreover, the TOAB/ZnP_p–C₆₀/GCE showed excellent stability and reproducibility, and good testing recoveries for analysis of the nitrite levels of river water and rainwater. The ZnP_p–C₆₀ can be used as a novel material for the fabrication of nonenzymatic electrochemical sensors

Attomolar Determination of Coumaphos by Electrochemical Displacement Immunoassay Coupled with Oligonucleotide Sensing

Coumaphos, an organophosphorus pesticide (OP) used worldwide, has raised serious public concerns due to its positive association with major types of cancer. Herein, a novel method for attomolar coumaphos detection was developed on the basis of an electrochemical displacement immunoassay coupled with oligonucleotide sensing. An optimized displacement immunoassay was constructed to improve the binding efficiency of an antigen–antibody pair, and a guanine-rich single-strand DNA label, in combination with oligonucleotide sensing, was used to amplify the detection signal with “direct” relationship to the analyte. As a result, coumaphos was sensitively determined from the enhanced catalytic cycle of guanine-Ru­(bpy)₃²⁺ by chronoamperometry. The limit of detection (LOD) was down to 0.18 ng L^–1 (S/N = 3), which is equal to 49.6 amol in a sample solution of 100 μL. In comparison with conventional methods, the proposed method has the lowest LOD and better accessibility to high-throughput sensing systems. Besides, it can complete the whole analysis process in under 50 min and exhibits good performance of excellent selectivity to the OPs. With regard to the advantages of rapidity, convenience, low cost, and ease of operation, the proposed method has provided a promising platform capable of fast and in-field OP detection, which may make the system promising for potential applications in the detection of other small molecules

Real-Time Sensing of TET2-Mediated DNA Demethylation In Vitro by Metal–Organic Framework-Based Oxygen Sensor for Mechanism Analysis and Stem-Cell Behavior Prediction

Active DNA demethylation, mediated by O₂-dependent ten–eleven translocation (TET) enzymes, has essential roles in regulating gene expression. TET kinetics assay is vital for revealing mechanisms of demethylation process. Here, by a metal–organic framework (MOF)-based optical O₂ sensor, we present the first demonstration on real-time TET2 kinetics assay in vitro. A series of luminescent Cu­(I) dialkyl-1,2,4-triazolate MOFs were synthesized, which were noble-metal-free and able to intuitively response to dissolved O₂ in a wide range from cellular hypoxia (≤15 μM) to ambient condition (∼257 μM). By further immobilization of the MOFs onto transparent silicon rubber (MOF@SR) to construct O₂ film sensors, and real-time monitoring of O₂ consumption on MOF@SR over the reaction time, the complete TET2-mediated 5-methylcytosine (5mC) oxidation process were achieved. The method overcomes the limitations of the current off-line methods by considerably shortening the analytical time from 0.5–18 h to 10 min, and remarkably reducing the relative standard deviation from 10%–68% to 0.68%–4.2%. As a result, the Michaelis–Menten constant (<i>K</i>_m) values of TET2 for 5mC and O₂ in ascorbic acid-free (AA^–) condition were precisely evaluated to be 24 ± 1 and 43.8 ± 0.3 μM, respectively. By comparative study on AA-containing (AA⁺) conditions, and further establishing kinetics models, the stem-cell behavior of TETs was successfully predicted, and the effects of key factors (AA, O₂, Fe²⁺) on TETs were revealed, which were fully verified in mouse embryonic stem (mES) cells. The method is promising in wide application in kinetics analysis and cell behavior prediction of other important O₂-related enzymes