Voltammetric determination of DNA based on regulation of DNA strand displacement using an allosteric DNA toehold

An electrochemical biosensor for determination of DNA is described that is based on the reaction of regulated DNA (reg-DNA) first with substrated DNA (subs-DNA) to form a reaction intermediate. The intermediate binds target DNA (T) by hybridization and initiates a branch migration leading to the production of complex of substrated DNA and target DNA (TC). Once TC is produced, it reacts with assisted DNA (ass-DNA) through a toehold exchange mechanism, yielding the product complex of substrated DNA and assisted DNA (CS). The target is then released back into the solution and and catalyzes the next cycle of toehold-exchange with the reaction intermediate of substrated DNA and regulated DNA (CPR). Unlike in a conventional DNA toehold that is hardwired with the branch migration domain, the allosteric DNA toehold is designed into a reg-DNA which is independent of the branch migration domain. Under the optimal experimental conditions and at a working potential as low as 0.18 V, response to DNA is linear in the 1 fM to 1000 pM concentration range, and the detection limit is 0.83 fM. The assay is highly specific and can discriminate target DNA even from a single-base mismatch. It was applied to the analysis of DNA spiked plasma samples.

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Graphical abstract Schematic illustration of the electrochemical strategy for target DNA detection based on regulation of DNA strand displacement using an allosteric DNA toehold strategy. It can be used to analyze DNA-spiked plasma samples and has a low detection limit of 0.83 fM.

     

Preparative separation of isomeric caffeoylquinic acids from Flos Lonicerae by pH-zone-refining counter-current chromatography

This work concentrates on pH-zone-refining counter-current chromatography of two isomeric dicaffeoylquinic acids, 3,5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid along with 3-caffeoylquinic acid, from crude extracts of Flos Lonicerae. The elution sequence of the isomeric dicaffeoylquinic acids, the mixing zone and mechanism of separation are discussed. The separation of 2.136g of the crude sample from Flos Lonicerae yielded two isomeric compounds: 0.289g 3,5-dicaffeoylquinic acid and 0.106g 3,4-dicaffeoylquinic acid plus 0.690g 3-caffeoylquinic acid at a high purity of over 92.9%, 94.2% and 97.5%, respectively.     

Miniaturized matrix solid-phase dispersion and solid-phase clear-up combined with capillary electrophoresis for efficient determination of trace bioactive components in complicated sample matrix: Take Wubi Shanyao Pill as an example

Accurate quantitative analysis of trace analytes in a complicated matrix is a challenge in modern analytical chemistry. An appropriate analytical method is considered to be one of the most common gaps during the whole process. In this study, a green and efficient strategy based on miniaturized matrix solid-phase dispersion and solid-phase extraction combined with capillary electrophoresis was first proposed for extracting, purifying and determining target analytes from complicated matrix, using Wubi Shanyao Pill as an example. In detail, 60 mg of samples were dispersed on MCM-48 to obtain high yields of analytes, then the extract was purified with a solid-phase extraction cartridge. Finally, four analytes in the purified sample solution were determined by capillary electrophoresis. The parameters affecting the extraction efficiency of matrix solid-phase dispersion, purification efficiency of solid-phase extraction and separation effect of capillary electrophoresis were investigated. Under the optimized conditions, all analytes demonstrated satisfactory linearity (R²>0.9983). What's more, the superior green potential of the developed method for the determination of complex samples was confirmed by the Analytical GREEnness Metric Approach. The established method was successfully applied in the accurate determination of target analytes in Wubi Shanyao Pill and thus provided reliable, sensitive, and efficient strategy support for its quality control.     

High-performance liquid chromatography micro-fraction bioactive evaluation combined with countercurrent chromatographic separation of antioxidants from Citrus peel and their tyrosinase inhibition activities

In the present study, high-performance liquid chromatography micro-fraction bioactive evaluation and high speed countercurrent chromatography were performed on screening, identification and isolation of antioxidants from Citrus peel. Three compounds were screened as antioxidants and tyrosinase inhibitors using 2,2′-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase activity test, then they were identified as eriocitrin, narirutin and hesperidin. Moreover, the solvent system ethyl acetate-n-butanol-water (6:4:10, v/v/v) was used for separation of ethyl acetate extract of Citrus peel by high speed countercurrent chromatography. In total, 0.45 mg of eriocitrin with 87.10% purity, 2.04 mg of narirutin with 95.19% purity and 1.35 mg of hesperidin with 95.19% purity were obtained from 20 mg of ethyl acetate extract of Citrus peel in a single run and then each component was subjected to 2,2′-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase inhibition assay. Eriocitrin showed great antioxidant activity (the half-maximum concentration: 3.65 µM) and tyrosinase inhibition activity (the half-maximum concentration: 115.67 µM), while narirutin and hesperidin exhibited moderate activity. Tyrosinase inhibition activity for eriocitrin in vitro was reported for the first time. Furthermore, molecular docking between eriocitrin and mushroom tyrosinase was also studied.     

Matrix solid-phase dispersion combined with micro-fractionation bioactivity evaluation screening polymethoxylated flavones from Citrus peels

Polymethoxyflavones were a unique class of natural and safe flavonoids containing two or more methoxy groups, which were also the most abundant edible part in Citrus peel. The optimum condition in the process of selective extraction of polymethoxylated flavones from Citrus peel by matrix solid-phase dispersion (MSPD) was as follows: SBA-15 as adsorbent, ethyl acetate as eluent, the mass ratio of adsorbent to sample 1:1, and the mixture of sample and adsorbent was ground for 3 min. Twelve antioxidants were successfully screened by micro-fractionation bioactivity evaluation assay, in which four of them were flavonoid glycosides, seven of them were polymethoxylated flavones, and one was phenylpropanoid. 1-sinapoly-β-D-glucopyranoside (1) was reported for the first time in Citrus peel. And antioxidant capacity of 1-sinapoly-β-D-glucopyranoside, 5, 7, 8, 3′, 4′, 5′-hexamethoxyflavone (6), hexamethoxyflavone (11), and 5, 6, 7, 4′-tetramethoxyflavone (7) were reported for the first time. Nobiletin (compound 8), 3, 5, 6, 7, 8, 3′, 4′-heptamethoxyflavone (9) and tangeretin (10) were isolated and purified by countercurrent chromatography combined with preparative liquid chromatography. Antioxidant activity evaluation indicated that the three isolated polymethoxylated flavones owned similar antioxidant activity. This study indicated that MSPD combined with micro-fractionation bioactive evaluation was efficient in screening bioactive compounds for rapid extraction and effective pinpointing bioactive substances in natural products.     

Large-scale separation of hydroxyanthraquinones from Rheum palmatum L. by pH-zone-refining counter-current chromatography

pH-zone-refining counter-current chromatography was successfully applied to purify four hydroxyanthraquinones, rhein, emodin, aloe-emodin and chrysophanol, from three crude extracts of Rheum palmatum L.. After the two-phase solvent system methyl tert-butyl ether-tetrahydrofuran-water at an optimized volume ratio of 2:2:3 (v/v) was equilibrated, trifluoroacetic acid (10 mM) was added to the organic phase as a retainer and ammonia (10 mM), sodium carbonate (15 mM) and sodium hydroxide (15 mM) were added to the aqueous phase as the eluter, respectively, for three individual runs. Three separation runs of 1.25, 1.53 and 1.41 g of the three crude samples yielded four hydroxyanthraquinones: 0.70 g rhein, 0.81 g emodin, 0.41 g aloe-emodin and 0.94 g chrysophanol at a high purity of over 99.0, 98.5, 98.2 and 97.8% (determined by HPLC), respectively. The structures were identified by electrospray ionization MS-MS and (1)H NMR.     

含吡唑环的1,2,4-三唑希夫碱类衍生物的合成及生物活性

以自制的4-氨基-4,5-二氢-3-[(3,5-二甲基吡唑-1-基)甲基]-1,2,4-三唑-5-硫酮衍生物为中间体, 与取代苯甲醛反应合成了一系列新型含吡唑环的1,2,4-三唑希夫碱衍生物. 通过红外光谱、 核磁共振波谱、 高分辨质谱、 元素分析及X射线单晶衍射对目标化合物进行了结构表征, 并初步测试了其生物活性. 结果表明, 大部分化合物表现出较好的抑菌活性, 其中化合物G9, G10和G15的抑菌效果优于对照药三唑酮.     

含嘧啶环的1,3,4-噁二唑Mannich碱的合成及生物活性

以芳香酸为原料, 通过酯化、 肼解及环化反应制得中间体5-芳基取代-1,3,4-噁二唑-2-硫酮(C1~C3), 然后中间体与甲醛和取代氨基嘧啶(D1~D5)发生Mannich反应得到一系列新型含有嘧啶环的1,3,4-噁二唑类衍生物(E1~E15). 所得目标化合物的结构经元素分析、 IR及 ¹H NMR确认. 初步的生物活性测定结果表明, 大部分目标化合物对植物病原菌有很好的抑制作用, 其中化合物E3和E8的抑菌效果优于对照药三唑酮.     

一种针对弱场搜寻态冷极性分子的可控静电表面囚禁方案

提出一种使用带电金属环和六个球电极和一个外加偏置电场实现对冷极性分子静电囚禁的新方案.计算装载和囚禁时空间电场分布.囚禁中心距离芯片表面的高度可以通过外电场和环形电极所加电压来操控.蒙特卡罗模拟表明对于中心速度为15 m·s^-1的ND₃分子束,装载效率可以达到70%,得到冷分子的温度大约为45 mK.当继续增加偏置电场强度时,单阱分裂为对称的两个阱.如果同时改变球形电极上所加电压,得到不对称的两个阱,可以借此来调节两个阱中所囚禁的冷分子数目的比例.为了易于理解,用蒙特卡罗方法模拟了装载、囚禁、分裂冷分子波包的动力学过程.