Single Methylation Sensitive Restriction Endonuclease-Based Cascade Exponential Amplification Assay for Visual Detection of DNA Methylation at Single-Molecule Level.

Function as a potential cancer biomarker, DNA methylation shows great significance in cancer diagnosis, prognosis, and treatment monitoring. While the lack of an ultrasensitive, specific, and accurate method at the single-molecule level hinders the analysis of the exceedingly low levels of DNA methylation. Herein, based on the outstanding recognition and digestion ability of methylation-sensitive restriction endonuclease (MSRE), we established a single MSRE-based cascade exponential amplification method, which requires only two ingeniously designed primers and only one recognition site of MSRE for the detection of DNA methylation. Differentiated by MSRE digestion, the cleaved unmethylated DNA is too short to induce any amplification reactions, while methylated DNA remains intact to trigger cascade exponential amplification and the subsequent CRISPR/Cas12a system. By integrating the two exponential amplification reactions, as low as 1 aM methylated DNA can be accurately detected, which corresponds to 6 molecules in a 10 µL system, indicating that our method is more sensitive than single amplification-based methods with the ability to detect DNA methylation at the single-molecule level. In addition, 0.1% methylated DNA can be effectively distinguished from large amounts of unmethylated DNA. Our method is further introduced to exploit the expression difference of DNA methylation among normal cells and cancer cells. Moreover, the visual detection of DNA methylation is also realized by the full hybridization between amplification products and the crRNA of CRISPR/Cas12a. Therefore, the proposed method has great potential to be a promising and robust bisulfite-free method for the detection of DNA methylation at the single-molecule level, which is of great importance for early diagnosis of cancer.

Preparation of a stoichiometric molecularly imprinted polymer for auramine O and application in solid-phase extraction.

We describe a stoichiometric approach to the synthesis of molecularly imprinted polymers specific for auramine O. Using the stoichiometric interaction in molecular imprinting, no excess of binding sites is necessary and binding sites are only located inside the imprinted cavities. The free base of the template was obtained to facilitate the interaction with the monomers. Itaconic acid was selected as the functional monomer, and stoichiometric ratio of the interaction with the free base was investigated. The molecularly imprinted polymer preparation conditions such as cross-linker, molar ratio, porogen were optimized as divinylbenzene, 1:2:20 and chloroform/N,N-dimethylformamide, respectively. Under the optimum conditions, a good imprinting effect and very high selectivity were achieved. A solid-phase extraction method was developed using the molecularly imprinted polymers as a sorbent and extraction procedure was optimized. The solid-phase extraction method showed a high extraction recovery for auramine O in its hydrochloride form and free form compared to its analogues. The results strongly indicated that stoichiometric imprinting is an efficient method for development of high selectivity molecularly imprinted polymers for auramine O.

Membrane-Protected Molecularly Imprinted Polymer for the Microextraction of Indole-3-butyric Acid in Mung Bean Sprouts.

Based on the hollow fiber protected molecularly imprinted polymer, a micro-solid-phase extraction (µ-SPE) method was developed and applied for the analysis of indole-3-butyric acid in mung bean sprouts by high-performance liquid chromatography. The extraction conditions of the µ-SPE method were optimized using L9(34) orthogonal, and optimum conditions were found as follows: pH of sample solution was 2.0, chloroform was the organic solvent for embedding the µ-SPE bars, and acetonitrile was the desorption solvent. In addition, the extraction time was 80 min, desorption time was 5 min, stirring speed was 800 rpm, and concentration of NaCl was 10%. Under the optimum conditions, a standard curve was established for IBA, with a correlation coefficient of 0.9999. After extraction with phosphate buffer solution (pH = 9.0), successful pretreatment of mung bean sprouts was achieved by the µ-SPE method. The limit of detection was 0.075 mg/kg, and the recoveries were found to be in the range of 88.9-106.4%. This method is simple, environmentally friendly, and can be used for the determination of indole auxin contents in green bean sprouts quickly and accurately.

In Situ Liquid-Phase-Adsorption Measurement System Based on Fiber-Optic Sensing with the Aid of Membranes.

At present, liquid phase adsorption (LPA) is still being quantitatively characterized in the way of manual sampling and off-line determination because of the complexity of the system comparing to gas adsorption. This paper describes a novel method for in situ, real-time measurement of LPA in general based on fiber-optic sensing (FOS) with the aid of membranes for the first time. A self-made measurement vessel was assembled from an adsorption bag, thermostatic devices with a stirrer, and a fiber-optic dipping probe. Also, macroporous adsorption resins (MARs) and rutin were chosen as model adsorbent and adsorbate to establish the FOS system. Here, in situ light absorption measurement was achieved by eliminating interference of adsorbent particles via encapsulating them with a membrane into the adsorption bag. In situ LPA measurement of rutin solution on MARs was obtained by detecting light absorption at 353 nm using dipping probe, in the broad concentration range from 0.3 to 60 mg/L with excellent linearity (R 2 = 0.9996). In situ measurements of adsorption and desorption kinetics on five kinds of MARs with different polarities were systematically carried out, showing that the adsorption process obeyed the pseudo-second-model. As well as, the system was proved to be highly accurate and reproducible. More importantly, this method enabled to study the initial stage of the adsorption process, starting from the time of the first second, which is the most important part in the adsorption kinetics, and this is impossible for traditional sampling methods. The successful application of FOS to in situ measurement of LPA not only contributes to fast, automatic, and real-time monitoring of LPA process but also enriches the research connotation of adsorption.