双链特异性核酸酶介导的高灵敏度microRNA分析

基于氧化石墨烯(GO)对荧光标记单链DNA探针的荧光猝灭效应以及双链特异性核酸酶(DSN)选择性切割DNA/RNA杂合结构中DNA单链的特性,本文建立了一种新型恒温信号放大方法用于microRNA(miRNA)的高灵敏度检测.靶标miRNA首先与荧光DNA探针杂交,DSN能够特异性地将杂合双链中的DNA探针水解为碎片但不会降解miRNA,GO对酶切产生的寡核苷酸碎片吸附能力显著降低,使得荧光基团远离GO表面而不被猝灭.释放出的miRNA可再次发生与荧光DNA探针杂交、DSN酶切等反应,如此反复,可实现恒温条件下一个miRNA分子与多个探针杂交、酶切、释放荧光基团的循环过程,最终体系的荧光信号得到显著放大,通过记录体系的荧光信号即可实现对靶标miRNA的灵敏检测.     

基于磁珠和双链特异性核酸酶辅助目标循环技术构建荧光传感器用于MicroRNA的检测

基于磁珠(MBs)的分离富集和双链特异性核酸酶(DSN)选择性切割DNA单链的特性,建立了信号增强型荧光生物传感器用于microRNA-21(miR-21)的检测。荧光素(FAM)修饰的捕获探针(Cps),通过亲和素-生物素的特异识别作用固定在磁珠表面。当miR-21存在时,Cps与其杂交形成DNA/RNA双螺旋结构,DSN能特异性水解杂合双链中的DNA,同时释放出荧光标记片段和完整的miR-21。被释放出来的miR-21与另一Cp再次杂交并被DSN酶切,如此循环,从而实现恒温条件下一个miR-21与多个Cp杂交、酶切,释放出大量的荧光标记片段的循环过程,最终使体系的荧光强度明显变大。相反,当miRNA-21不存在时,Cps无法形成双螺旋结构,DSN对单链DNA无酶切作用,不能水解Cps,经磁分离,上清液没有荧光标记片段,所以检测不到荧光信号。最佳条件下,miR-21浓度在100~5×104fmol/L范围内,荧光强度与其浓度呈良好的线性关系,检测限达80 fmol/L。该传感器可以识别单碱基错配序列,有望为肿瘤早期诊断提供新思路。     

单碱基多样性的非测序检测

单碱基多样性（SNP）是最常见的基因突变形式之一,经研究证明与很多疾病相关。虽然测序是检测SNP的重要方法,但其需要检测仪器,且检测时间较长,限制了其临床应用。本文综述了SNP的常见非测序分析方法。首先讨论了检测的热力学问题,并归纳了主要的检测策略：基于杂交的检测,基于链取代反应的检测和酶介导的检测。在三维均相检测方法中,主要介绍了不同信号开关策略,如荧光开关、酶识别开关和场效应开关。三维原位检测不仅能检测SNP,还能提供其细胞定位信息,在细胞异质性较高时更具优势。二维界面检测的识别反应速率和杂交效率受到一定影响,但界面检测能进一步减小干扰,亦便于实现高通量检测。以DNA正四面体探针界面为代表的改良界面具有优良的灵敏度和特异性。同时本文亦讨论了现有方法的局限性,并对SNP非测序检测研究进行展望。     

双链特异性核酸切割酶等温循环扩增结合 高效液相色谱高灵敏检测双目标microRNA

通过双链特异性核酸切割酶(DSN)循环扩增策略结合高效液相色谱(HPLC)法,实现了对两种疾病标志物microRNA(miRNA)的选择性分离和高灵敏检测.采用酶循环等温扩增策略增强了目标miRNA的信号,提高了HPLC法检测核酸的灵敏度.利用固定在磁珠上的不同长度和碱基序列的DNA探针实现了不同miRNA信号的色谱分...     

酶放大DNA电化学传感器的研究进展

DNA电化学传感器灵敏度高、选择性好、分析时间短和检测成本低,极大地推动了生物传感器的发展. 结合蛋白质酶、功能核酸酶的催化效率高与特异性好,可提高检测灵敏度和选择性. 本文评述了酶放大DNA电化学传感器的研究进展,并分析现存问题,展望发展趋势.     

基于纳米金与纳米银簇间表面等离子增强能量转移效应特异性检测microRNA

microRNAs(miRNAs)的灵敏检测对临床诊断具有十分重要的意义.本研究采用偶联DNA聚合酶和核酸内切酶介导的恒温扩增反应实现靶标循环再生的策略,利用纳米金(AuNPs)与纳米银簇(AgNCs)间表面等离子增强能量转移效应,开发了一种miRNA定量检测方法.在AuNPs表面组装两种探针(Probe a和Probe b)制备响应元件Probe b-Probe a-AuNP,其中Probe a通过3′端巯基共价偶联到AuNPs表面,此外具有靶标miRNA互补序列、核酸内切酶酶切序列和Probe b互补序列,Probe b为荧光AgNCs合成模板.靶标miRNA存在时,启动酶级联恒温扩增反应,导致Probe b脱离AuNPs表面,抑制了Probe b为模板合成的AgNCs与AuNPs间表面等离子增强能量转移效应,使得反应体系荧光信号增强.本方法的检出限为2.5×10-11 mol/L,与miRNAs商业化检测试剂盒相比,避免了逆转录反应,而且操作简单,检测成本低,可应用于生物样本中miRNAs分析.     

基于illumina测序技术的IGROV-1/CP细胞中miRNA种类鉴定及相对丰度分析

从正常培养的IGROV-1/CP细胞中提取小RNA,构建小RNA的cDNA文库,然后用illumina通用测序平台对上述cDNA文库进行测序.从测序获得的序列数据中去除冗余数据后,与已知人类miRNA序列数据库进行比对,获得IGROV-1/CP细胞miRNA表达谱.以所获的序列长度与已知人类miRNA数据库中序列长度差异不大于2和无碱基错配为限制条件,共找到53种已知miRNA.按在cDNA文库测序中的出现频率计算,出现频率不大于10的低拷贝miRNA最多,占检测到所有miRNA种类的56.6%.说明illumina通用测序技术能够在检测细胞内的各种小RNA序列的同时反应相对丰度信息,该研究利用上述新技术获得了顺铂耐药性IGROV-1/CP细胞系miRNA表达种类和相对丰度的实验数据.     

基于核酸外切酶Ⅲ信号放大技术的荧光适配体传感器用于卡那霉素的检测

建立了一种基于核酸外切酶Ⅲ(Exo Ⅲ)和碳纳米颗粒(CNPs)的信号放大体系用于卡那霉素(KAN)检测的新方法。合成了水溶性的CNPs,并设计合成了不同序列的DNA,具体包括:卡那霉素适配体(Apt),羧基荧光素标记的信号DNA探针(FAM-DNA)和互补链cDNA。当体系中不存在KAN时,Apt与cDNA可以杂交形成双链DNA,体系中FAM-DNA处于单链状态,Exo Ⅲ不能水解单链DNA;此时,体系中加入CNPs,单链FAM-DNA被CNPs吸附,荧光发生淬灭;在KAN存在下,Apt与其靶标KAN特异性结合,此时FAM-DNA与cDNA杂交形成双链DNA,由于CNPs对双链DNA吸附较弱,DNA探针的荧光不发生淬灭。ExoⅢ可以特异性的从3’-端对FAM-DNA降解,释放FAM荧光团和cDNA,该体系通过"降解-杂交"循环,最终释放出大量的FAM荧光团。由于CNPs对FAM具有较低的亲和力,释放出的FAM不能吸附在CNPs表面,FAM荧光不会发生淬灭,实现荧光信号放大扩增作用。方法线性范围为50～100 nmol/L,检测限为2.5 nmol/L。该方法可用于实际样品牛奶中卡那霉素的检测。     

单碱基多样性的非测序检测（英文）

单碱基多样性(SNP)是最常见的基因突变形式之一,经研究证明与很多疾病相关。虽然测序是检测SNP的重要方法,但其需要检测仪器,且检测时间较长,限制了其临床应用。本文综述了SNP的常见非测序分析方法。首先讨论了检测的热力学问题,并归纳了三类主要的检测策略:基于杂交的检测、基于链取代反应的检测和酶介导的检测。在三维均相检测方法中,主要介绍了不同信号开关策略,如荧光开关、酶识别开关和场效应开关。三维原位检测不仅能检测SNP,还能提供其细胞定位信息,在细胞异质性较高时更具优势。二维界面检测的识别反应速率和杂交效率受到一定影响,但界面检测能进一步减小干扰,亦便于实现高通量检测;以DNA正四面体探针界面为代表的改良界面具有优良的灵敏度和特异性。同时,本文亦讨论了现有方法的局限性,并对SNP非测序检测研究进行展望。     

焦测序技术及其在遗传分析中的应用

焦测序技术是一种新的实时DNA测序技术。它在DNA聚合酶、三磷酸腺苷硫酸化酶、荧光素酶和三磷酸腺苷双磷酸酶4种酶的协同作用下,使引物延伸聚合脱氧核糖核酸(dNTP)释放焦磷酸盐(PP i)、PP i转换三磷酸腺苷(ATP)、ATP产生荧光信号与dNTP和ATP的降解等化学反应偶联起来,检测结果准确可靠。本文综述了焦测序技术的基本原理、操作步骤和它在单核苷酸多态性(SNP)研究、微生物的分型鉴定和基因甲基化检测等遗传分析中的应用,并对焦测序技术的发展作了展望。     

Indirect Electrosorptive Detection via Kinetic Facilitation in Ion Chromatography

Abstract

A new technique based on electrosorption is presented for the determination of selected anions in ion chromatography. Unlike conventional methods, which are based on the measurement of nonfaradaic currents, the present method utilizes a kinetic facilitation imparted to the electroreduction of a cationic “adsorbate probe” by specifically adsorbed anions, and hence involves the measurement of faradaic currents. Amminated, transition-metal salts of Co(III) have been found most useful as adsorbate probes for this application. The current enhancement-analyte concentration response curves obtained were determined to be linear over a limited range at mercury, but show curvature at virtually all concentrations at silver. Detection limits for this technique are slightly higher than those realized using more conventional double-layer capacitance methods. A brief discussion of the future prospects for this new approach is given.     

A 2D cadmium metal–organic framework: Synthesis,structure and luminescence sensing for chromate,permanganate, cupric,silver and ferric

A multi-responsive Cd metal–organic framework {[Cd (ttpe)(H₂O)(ip)]•4H₂O•DMAC}_n ( 1•4H ₂ O•DMAC ) was synthesized using hydrothermal method (ttpe = 1,1,2,2-tetra(4-(1H-1,2,4-triazol-1-yl)phenyl)ethylene, ip = isophthalate, DMAC = N,N-dimethylacetamide), and characterized. 1 exhibits a 2D (4,4) network. The luminescent sensing experimrnts showed that 1•4H ₂ O•DMAC as a new MOF luminescent sensor can detect Cr₂O₇²⁻, CrO₄²⁻, MnO₄⁻, Cu²⁺, Ag⁺ and Fe³⁺ in aqueous solution with simultaneously high efficiency and high sensitivity. The quenching constants K_sv for Cr₂O₇²⁻, CrO₄²⁻, MnO₄⁻, Cu²⁺, Ag⁺ and Fe³⁺ are 4.231 × 10⁴ M⁻¹, 2.471 × 10⁴ M⁻¹, 6.459 × 10³ M⁻¹, 7.617 × 10³ M⁻¹, 1.563 × 10⁴ M⁻¹ and 3.574 × 10⁴ M⁻¹, respectively. The detection limits are 0.094 μM for Cr₂O₇²⁻, 0.108 μM for CrO₄^{2 −} , 0.346 μM for MnO₄⁻, 0.302 μM for Cu²⁺, 0.221 μM for Ag ⁺ , and 0.100 μM for Fe³⁺. 1•4H ₂ O•DMAC exhibits high photocatalytic efficiency for degradation of methylene blue under visible light irradiation.     

Surface-enhanced Raman scattering for protein detection

Proteins are essential components of organisms and they participate in every process within cells. The key characteristic of proteins that allows their diverse functions is their ability to bind other molecules specifically and tightly. With the development of proteomics, exploring high-efficiency detection methods for large-scale proteins is increasingly important. In recent years, rapid development of surface-enhanced Raman scattering (SERS)-based biosensors leads to the SERS realm of applications from chemical analysis to nanostructure characterization and biomedical applications. For proteins, early studies focused on investigating SERS spectra of individual proteins, and the successful design of nanoparticle probes has promoted great progress of SERS-based immunoassays. In this review we outline the development of SERS-based methods for proteins with particular focus on our proposed protein-mediated SERS-active substrates and their applications in label-free and Raman dye-labeled protein detection. Figure Protein-mediated SERS-active substrates for protein detection     

Integration of Separation Capillary with a Au Film Electrode and an Etched Decoupler in Amperometric Capillary Electrophoresis

Introduced in this article is fabrication of an integrated capillary for the applications of electrochemical detection in capillary electrophoresis. The separation section, voltage decoupler, and working electrode were composed into a single section of capillary. The porous decoupler was constructed by HF etching till the thickness of the capillary wall less than 20 μm. The working electrode was prepared by sputtering Au-films on the outlet of the capillary. The integration of the separation capillary with a complete detection assembly improves the convenience for the routine application of electrochemical measurements in capillary electrophoresis. For a 100-μm-i.d. capillary, the theoretical plate number of catechol and the migration time reproducibility can reach 120,000 and 1.9% RSD, respectively. The linear range exceeds 3 order of magnitude and the detection limit is lower than 0.65 μM.     

Amperometric multidetection with composite enzyme electrodes

The use of composite biosensors for multianalyte detection strategies is discussed. Graphite–Teflon rigid composite biosensors offer the possibility of coimmobilization of several enzymes by simple physical inclusion in the bulk of the electrode matrix with no covalent linkages. A novel trienzyme graphite–Teflon–glucose oxidase (GOD)–alcohol oxidase (AOD)–peroxidase (HRP)–ferrocene bisosensor yielded amperometric steady-state currents similar to those obtained with graphite–Teflon–GOD–HRP–ferrocene and graphite–Teflon–AOD–HRP–ferrocene electrodes for the same concentration of glucose and ethanol, respectively. The performance of the trienzyme biosensor for multianalyte detection was evaluated with the simultaneous determination of glucose and ethanol after separation by HPLC, in samples of sweet wine. The simultaneous analysis of several analytes in the same sample should imply that, with an adequate dilution, the concentration levels of the analytes can be included within the ranges of linearity of the corresponding calibration plots. The use of two composite biosensors in a parallel configuration, so that different analytes can be simultaneously detected with no need of chromatographic separation, is also discussed. The usefulness of this approach was evaluated by the simultaneous analysis of glucose and ethanol in sweet wine, and of glucose and lactic acid in red wine.     

光照度探测器设计与精度校正

助航灯光系统为夜间或能见度较低环境下飞机的安全起降提供安全保障。快速准确地测量和判断其光强等级、光照射角等是否符合国际民用航空组织(ICAO)的标准,是保障机场安全运行的重要工作。本文设计了基于硅光电池的照度探测器,通过余弦校正器和不同滤光片的组合完成视见函数校正和照度探测的精度标定,实现了灯光的高速动态检测;完成了光照度探测器的线性标定和动态精度测量实验,照度测量误差率和动态测量误差均满足ICAO规定的要求;实现了助航灯光平行截面内光强的高速动态检测。     

Chromenone-rhodamine conjugate for naked eye detection of Al3+ and Hg2+ ions in semi aqueous medium

Chromenone-rhodamine conjugate 1 has been synthesized and its metal ion binding properties have been studied in CH₃CN/water (3:1, v/v; 10 mM HEPES buffer; pH = 6.85). Compound 1 senses multiple metal ions such as Al³⁺ and Hg²⁺ by exhibiting turn on fluorescence and color change (colorless to pink). Al³⁺ and Hg²⁺ ions have been distinguished with the aid of tetrabutylammonium iodide (TBAI). While in the presence of I^? the pink color of the 1.Hg²⁺ complex was completely discharged; under identical conditions the pink color of 1.Al³⁺ complex was retained.     

A review of reaction detection in HPLC

Summary In the past decade, reaction detection has rapidly gained importance as a means to improve the sensitivity and selectivity of detection in HPLC. Today, most attention is being paid to post-column reaction detection with final analysis via fluorescence monitoring. Novel developments involve the use of solid-phase reactors for reagent addition or catalysis (ion-exchange resins, immobilized enzymes), and the increasing use of systems based on peroxyoxalate chemiluminescence detection. The rapidly growing number of reported applications indicates that commercialization of reaction-detection equipment is urgently needed.