基于CRISPR-Cas12a与G-四链体构建免标记电化学生物传感器检测microRNA

CRISPR-Cas12a是一种功能强大且可编程的分子诊断技术。本文基于CRISPR-Cas12a的附属切割活性与G-四链体/氯化血红素（Hemin）复合物,设计了一个免标记电化学生物传感器,实现对miRNA的强特异性检测。靶标miRNA-21与双链DNA探针上的Toehold区域结合并发生链置换反应,置换出双链DNA探针中较短的DNA。置换下来的DNA可以有效地激活CRISPR-Cas12a的附属切割活性。随后,具有附属切割活性的Cas12a切割电极表面上形成G-四链体/Hemin的DNA序列,导致电流信号减弱。在最优条件下,电流信号强度变化与10～100 pmol/L范围内的miRNA-21浓度呈良好的线性关系,检出限为4.2 pmol/L。该电化学生物传感器能够实现对单个碱基突变的miRNA-21或其它miRNA序列特异性识别,并可用于人血清样本（10%）中miRNA-21的检测。     

MicroRNA体外检测的研究进展

MicroRNA (miRNA)是一类内源性、进化高度保守的小分子非编码RNA,通过识别同源序列及干扰转录、翻译或表观遗传以调节基因的表达。研究发现,某些miRNA的异常表达与疾病相关,可作为生物标志物或药物靶点为疾病诊断、治疗及预后提供新思路,而准确测定miRNA的表达是其应用于临床的关键。本文结合近年来研究成果对传统检测方法及其改进和等温核酸扩增的新技术进行概述,分析这些方法的优势与不足。     

基于电化学技术的microRNA生物传感器

MicroRNA(miRNA)是一种内源性的非编码单链RNA,通过与mRNA的3'端非翻译区(UTR)的不完全互补或完全互补结合抑制靶mRNA的翻译或促使靶mRNA的降解来调控基因的表达,参与细胞的增殖、凋亡、分化和代谢等重要过程。MiRNA表达的变化可以起到癌基因和抑癌基因的作用,是一种潜在的肿瘤标志物,因此,miRNA的检测技术引起了人们的关注。由于电化学检测方法具有灵敏、快速、低成本和低能耗等特点,研究者广泛开展了应用电化学技术来发展miRNA检测的研究。本文将对基于电化学技术的miRNA检测方法进行综述。     

miRNA在β_2受体激动剂作用中的研究进展

β_2受体激动剂的滥用威胁着消费者的人身安全,也制约着食品工业和畜牧业的发展,然而药物种类繁多,更新迅速,如何对其有效监测一直是研究的重点小分子RNA(miRNA)是近年来在真核生物体内发现的一类长度约22个核苷酸的内源性非编码单链RNA,主要通过与靶基因mRNA靶标区域的互补配对,发挥降解靶mRNA或抑制mRNA翻译的作用。它能参与多种生物学过程包括细胞凋亡、分化和癌变等。β_2受体激动剂类药物有着共同的作用机理,近年来的研究表明,miRNA的异常表达与β_2受体激动剂的使用密切相关。本文主要对与β_2受体激动剂作用机理相关的miRNA的研究进展展开综述,以期为实现对该类药物的有效监测提供参考。     

电喷雾质谱法研究聚酰胺、酰亚胺衍生物与HIV-1基因DNA的相互作用

利用电喷雾质谱(ESI-MS)研究了6种识别分子与HIV-1基因调控区DNA的非共价键相互作用. 在研究中比较了不同识别分子与靶序列DNA结合的强弱, 发现双萘酰亚胺衍生物4对此序列DNA具有高亲合性的结合, 并分析了识别分子与DNA复合物的碎裂机理以及结合模式.     

基于三维有序金掺杂纳米二氧化钛的DNA生物传感器研究

用模板法在氧化铟锡(ITO)电极上制备具有三维有序多孔结构的金掺杂纳米二氧化钛修饰电极(3DOM GTD/ITO),扫描电镜(SEM)结果表明,制备的修饰电极三维结构规整有序、孔径均一。将标记有二茂铁(Fc)的DNA探针修饰到3DOM GTD/ITO电极上构建了一种新的标记型DNA生物传感器,通过Fc在DNA探针杂交前后的电化学信号变化可识别目标靶序列。采用循环伏安(CV)、示差脉冲(DPV)和交流阻抗(EIS)等方法对DNA探针在电极表面的固定和杂交进行表征。实验结果表明,该DNA生物传感器可以成功地识别乳腺癌基因靶序列,Fc的氧化还原电流与靶序列浓度在8.0×10-7～1.0×10-5 mol/L范围内呈线性关系,线性相关系数为0.9908,检测限为5.2×10-7 mol/L。     

氧化石墨烯增强荧光各向异性新策略在microRNA检测中的应用

microRNA(miRNA)是一类与癌症等重大疾病的发生密切相关的非编码小分子RNA.本文基于氧化石墨烯(GO)放大荧光各向异性(FA)原理,构建了GO/cDNA/Linker DNA/pDNA(DNA-GO)复合探针体系,结合靶物催化诱导信号循环放大策略,实现了miRNA-21的灵敏检测.在复合探针形成后,GO增加了荧光旋转体的体积和质量,Linker DNA上染料分子的荧光各向异性值增大.miRNA-21可通过"toehold"介导的链置换反应与Linker DNA杂交,使pDNA脱离Linker DNA.进一步加入Fuel DNA后,Fuel DNA与Linker DNA配对形成双链,从GO表面脱离,使Linker DNA上染料分子的荧光各向异性减小.被置换下来的miRNA-21进入下一循环,因此一个目标分子可以诱导形成多个Fuel DNA与Linker DNA的杂交双链,使荧光各向异性进一步减小.利用减小的荧光各向异性值实现了miRNA-21的灵敏检测.在本方法中,荧光各向异性减小值与miRNA-21浓度在10~330 nmol/L范围内呈线性关系,检测限为1.09 nmol/L.     

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.     

新型"三明治"结构DNA电化学传感器对急性早幼粒细胞白血病相关融合基因的检测

基于急性早幼粒细胞白血病(APL)中PML/RARα融合基因的碱基序列,设计了新型的锁核酸(LNA)修饰寡核苷酸作为捕获探针和信号探针,研究出一种基于"三明治"传感模式的电化学生物传感器对PML/RARα融合相关基因进行检测.靶序列分别与捕获探针和信号探针杂交后形成"三明治"结构.将修饰电极置于含有底物3,3′,5,5′-四甲基联苯胺(TMB)和过氧化氢的测定溶液中,用计时电流法检测靶序列.结果表明,该传感器可定量识别和检测溶液中人工合成的短链APL PML/RARα融合基因片段.经过条件优化,杂交前后电流值与靶标链浓度在1.0×10~(-12) ～2.5×10~(-11) mol/L范围内呈良好的线性关系,检出限为8.5×10~(-13) mol/L.该方法简单、特异性好,有望用于实际样品的检测.     

MicroRNA分析方法进展*

MicroRNAs（miRNAs）是一类对基因表达具有调控作用的非编码小分子RNA (18–23 碱基)。MiRNA在动植物中普遍存在,并且在动植物的生长、发育、分化和生殖等过程中发挥着重要作用。人类约30%的基因受miRNA调控,并且miRNA的表达水平与人类重大疾病密切相关。MiRNA的定量检测和表达分析对深入理解其作用机制、疾病的诊断与治疗以及相关基因药物的开发等具有重要意义。MiRNA检测一般是基于核酸杂交和扩增原理,方法主要有Northern印迹、微阵列芯片、原位杂交、实时反转录聚合酶链式反应（PCR）、滚环扩增和基于共轭聚合物的检测等。随着miRNA在不同生物中的大量发现和对其功能的深入研究,miRNA的检测方法不断改进和完善,新的扩增、探针标记技术和检测技术不断被开发。本文综述了miRNA检测方法的研究进展,评述了各类方法的优缺点,并对miRNA检测的发展趋势进行了展望。     

Analysis of the NCI-60 dataset for cancer-related microRNA and mRNA using expression profiles

BackgroundRecent studies have indicated that microRNA (miRNA) may play an oncogenic or tumor suppressor role in human cancer. To study the regulatory role of miRNAs in tumorigenesis, an integrated platform has been set up to provide a user friendly interface for query. The main advantage of the present platform is that all the miRNA target genes’ information and disease records are drawn from experimentally verified or high confidence records.ResultsMiRNA target gene results are annotated with reference to the disease gene as well as the pathway database. The correlation strength between miRNA and target gene expression profile is quantified by computing the correlation coefficient using the NCI-60 expression profiling data. Comprehensive analysis of the NCI-60 data found that the cumulative percentage of negative correlation coefficients for cleavage regulation is slightly higher than its positive counterpart; which indicated that the mRNA degradation mechanism is slightly dominant. In addition, the RNAHybrid and TargetScans scores are computed which potentially served as quantitative estimators for miRNA–mRNA binding events.Three scores are defined for each miRNA–mRNA pair, which are based on the disease gene and pathway information. These three scores allow user to sort out high confidence cancer-related miRNA–mRNA pairs.Statistical tests were applied to investigate the relations of three chromosomal features, i.e., CpG island, fragile site, and miRNA cluster, with cancer-related miRNAs. A web-based interface has been set up for query, which can be accessed at: http://ppi.bioinfo.asia.edu.tw/mirna_target/ConclusionsThe main advantage of the present platform on miRNA–mRNA targeting information is that all the target genes’ information and disease records are experimentally verified. Although this may limit the number of miRNA–mRNA relationships, the results provided here are more solid and have fewer false positive events. Certain novel cancer-related miRNA–mRNA pairs are identified and confirmed in the literature. Fisher's exact test suggests that CpG island and fragile site associated miRNAs tend to associate with cancer formation. In summary, the present platform provides an easy means of investigating cancer-related miRNAs.     

Fe_3O_4@ILs-β-CDCP磁固相萃取-电感耦合等离子体发射光谱法同时测定环境水样中铅和锑

本文制备了磁性固相萃取材料Fe_3O_4@ILs-β-CDCP,并对其进行了表征。详细考察了Fe_3O_4@ILs-β-CDCP萃取Pb(Ⅱ)和Sb(Ⅲ)的各种影响因素,如溶液pH值、样品体积、洗脱剂浓度和用量、洗脱剂类型、共存离子影响等。基于此,建立了磁固相萃取-电感耦合等离子体发射光谱法(ICP-OES)同时测定Pb(Ⅱ)和Sb(Ⅲ)的新方法。在优化的条件下,该方法对Pb(Ⅱ)和Sb(Ⅲ)的检出限分别为1.50和0.54ng/mL,相对标准偏差(RSDs)分别为5.9%和2.4%(n=6,c=100.0ng/mL)。将该方法应用于实际水样中Pb(Ⅱ)和Sb(Ⅲ)的测定,其回收率分别为77.8%~106%和82.4%~107%。方法具有检出限低、操作简便等优势。     

A computational model for non-conserved mature miRNAs from the rice genome

Several computational approaches employ the high complementarity of plant miRNAs to target mRNAs as a filter to recognize miRNA. Numerous non-conserved miRNAs are known with more recent evolutionary origin as a result of target gene duplication events. We present here a computational model with knowledge inputs from reported non-conserved mature miRNAs of Oryza sativa (rice). Sequence- and structure-based approaches were used to retrieve miRNA features based on rice Argonaute protein and develop a multiple linear regression (MLR) model (r² = 0.996, q²_cv = 0.989) which scored mature miRNAs as predicted by the MaturePred program. The model was validated by scoring test set (q² = 0.990) and computationally predicted mature miRNAs as external test set (q²_test = 0.895). This strategy successfully enhanced the confidence of retrieving most probable non-conserved miRNAs from the rice genome. We anticipate that this computational model would recognize unknown non-conserved miRNA candidates and nurture the current mechanistic understanding of miRNA sorting to unveil the role of non-conserved miRNAs in gene silencing.     

A Simple and Highly Sensitive Electrochemical Biosensor for microRNA Detection Using Target‐Assisted Isothermal Exponential Amplification Reaction

A simple and highly sensitive electrochemical biosensor for microRNA (miRNA) detection was successfully developed by integrating a target‐assisted isothermal exponential amplification reaction (EXPAR) with enzyme‐amplified electrochemical readout. The binding of target miRNA with the immobilized linear DNA template generated a part duplex and triggered primer extension reaction to form a double‐stranded DNA. Then one of the DNA strands was cleaved by nicking endonuclease and extended again. The short fragments with the same sequence as the target miRNA except for the replacement of uridines and ribonucleotides with thymines and deoxyribonucleotides could be displaced and released. Hybridization of these released DNA fragments with other amplification templates and their extension on the templates led to target exponential amplification. Integrating with enzyme‐amplified electrochemical readout, the electrochemical signal decreases with the increasing target microRNA concentration. The method could detect miRNA down to 98.9 fM with a linear range from 100 fM to 10 nM. The fabrication and binding processes were characterized with cyclic voltammetry and electrochemical impedance spectroscopy. The specificity of the method allowed single‐nucleotide difference between miRNA family members to be discriminated. The established biosensor displayed excellent analytical performance toward miRNA detection and might present a powerful and convenient tool for biomedical research and clinic diagnostic application.     

母乳中多种含卤持久性有机污染物的联合检测方法

建立了母乳中多种含卤持久性有机污染物(POPs)的联合检测方法,目标化合物主要包括六溴环十二烷(HBCDs)、多溴联苯醚(PBDEs)、多氯联苯(PCBs)和有机氯农药(OCPs)等.样品的前处理采用液液萃取、凝胶渗透色谱(GPC)净化和固相萃取(SPE)等技术,目标化合物经气相色谱-质谱联用仪(GC-MS)、液相色谱-三重四极杆串联质谱联用仪(LC-MS/MS)和气相色谱-三重四极杆串联质谱联用仪(GC-MS/MS)等进行检测.样品通过GPC除去脂肪,然后经SPE柱进一步净化并进行多组分分离,极大程度地减小了生物样品中复杂基质的干扰,适合样品量相对较小的人体样本中多种超痕量POPs的分析.应用灵敏度高、选择性更好的GC-MS/MS对样品中的PCBs和OCPs等进行分析,进一步降低基质的干扰.方法经过小牛血清加标实验验证,稳定可靠.POPs的加标回收率分别为88.7％～98.8％(PBDEs), 88.5％～92.5％(HBCDs), 67.9％～82.3％(PCBs)和81.7％～116.1％(OCPs),方法检出限分别为0.13～1.8 pg/mL(PBDEs), 0.31～1.2 pg/mL(HBCDs), 0.22～1.4 pg/mL(PCBs)和0.20～1.5 pg/mL(OCPs).采用本方法对潍坊地区20例母乳样品进行分析,结果显示,潍坊市母乳中HBCDs, PBDEs, PCBs、HCHs和DDTs的中值浓度分别为2.86, 7.76, 8.84、140和503 ng/g 脂重,此浓度水平与国内其它地区人群相当.     

QuEChERS结合柱净化的超高效液相色谱-串联质谱法测定有机肥中46种抗生素

建立了超高效液相色谱-串联质谱(UPLC-MS/MS)同时测定有机肥中磺胺类、喹诺酮类、大环内酯类46种抗生素的分析方法。样品用乙腈-EDTA缓冲溶液(p H 10.0)提取,盐析后离心分层,乙腈层按Qu ECh ERS法,采用吸附剂净化;缓冲溶液层经HLB柱净化。ACQUITY UPLC BEH C18柱用作色谱分离,以2mmol/L甲酸铵水溶液(含0.1%甲酸)-甲醇为流动相进行梯度洗脱;电喷雾电离源正离子(ESI+)多反应监测(MRM)模式检测,基质外标法定量。46种抗生素在1~200μg/L范围内线性关系良好,相关系数(r2)为0.996 6~0.999 9。在25,100,400μg/kg加标浓度下,3类抗生素的回收率分别为67.8%~95.6%,65.6%~89.4%和66.6%~107.8%,相对标准偏差为0.4%~11.9%;方法检出限(S/N=3)为0.6~4.6μg/kg,定量下限(S/N=10)为2.1~15.4μg/kg。     

Feature importance analysis in guide strand identification of microRNAs

MicroRNA (miRNA) is the negative regulator of gene expression, also known as guide strand of transient miRNA:miRNA* duplex. It is critical in maintaining the normal physiological processes such as development, differentiation, and apoptosis in many organisms. With increasing miRNA data, it is desirable to design methods to identify guide strand based on machine learning algorithms. In this study, the random forest models based on local sequence-structure features were proposed to identify miRNA in four species. The accuracies achieved were 86.51% for Homo sapiens, 81.66% for Ornithorhynchus anatinus, 82.33% for Mus musculus and 85.71% for Schmidtea mediterranea, respectively. Furthermore, the important analysis of feature elements was carried out by using the conditional feature importance strategy. The analysis results revealed that most of the significant elements were related to guanine-cytosine (GC) base pair. We believed that our method could be beneficial to annotate the function of miRNA and help the further understanding of the RNA interference mechanism.     

Detection of MicroRNAs using target-guided formation of conducting polymer nanowires in nanogaps

A nanogapped microelectrode-based biosensor array is fabricated for ultrasensitive electrical detection of microRNAs (miRNAs). After peptide nucleic acid (PNA) capture probes were immobilized in nanogaps of a pair of interdigitated microelectrodes and hybridization was performed with their complementary target miRNA, the deposition of conducting polymer nanowires, polyaniline (PAn) nanowires, is carried out by an enzymatically catalyzed method, where the electrostatic interaction between anionic phosphate groups in miRNA and cationic aniline molecules is exploited to guide the formation of the PAn nanowires onto the hybridized target miRNA. The conductance of the deposited PAn nanowires correlates directly to the amount of the hybridized miRNA. Under optimized conditions, the target miRNA can be quantified in a range from 10 fM to 20 pM with a detection limit of 5.0 fM. The biosensor array is applied to the direct detection of miRNA in total RNA extracted from cancer cell lines.