基于分子信标定量检测肿瘤细胞p21 mRNA表达变化

根据p21基因序列设计合成特异性检测p21 mRNA的分子信标, 发展了一种体外快速、定量检测p21 mRNA的新方法, 将其用于肿瘤细胞p21 mRNA表达水平的检测. 结果发现: 抑制p53表达引起CNE2细胞中p21 mRNA表达水平降低, 转染ING1诱导MCF-7细胞中p21 mRNA表达上调, 5-氟尿嘧啶处理MCF-7细胞后p21 mRNA表达水平呈浓度和时间相关性变化. 这种特异性强、操作快速、简便的新方法有望广泛用于各类样品中p21 mRNA表达水平检测.     

基于滚环合成的聚分子信标用于 凝血酶的靶向检测

采用滚环扩增(RCA)合成得到的DNA长链打开带适配体的分子信标, 由于RCA长链上带有多个与分子信标(MB)互补的重复序列, 其打开分子信标的能力比单一互补短链提高了上百倍. 所形成的聚多价分子信标组装体, 在分子信标浓度相同的情况下, 打开后的荧光强度也大幅上升; 并且由于组装体上多价适配体的存在, 聚分子信标对凝血酶的靶向能力显著增强. 实验结果表明, 聚分子信标结合凝血酶后, 其荧光信号与凝血酶浓度呈线性关系, 检测灵敏度达到0.2 nmol/L, 该体系的构建有利于实现对凝血酶的高灵敏、 特异性检测.     

多元线性模型计算基于荧光猝灭原理的膜传感器对分析物的响 …

根据基于荧光猝灭原理的化学传感膜的结构特征和发光机理,推导并提出量化作用于膜传感器的多种荧光猝灭原因的多元线性模型。结合三杯法,该模型可简便地求出反映不同荧光猝灭机理的特征参数。通过建立的数学模型,测定并计算了芘丁酸膜传感器对应于呋喃妥因等２３种药物多种荧光猝灭因素的响应数据和响应参数。结果表明,传感膜对分析物的响应特征往往能得到主要猝灭因素响应模型的近似反映,这为基于荧光猝灭原理的化学传感器定量     

基于双分子识别荧光猝灭法高选择性测定多巴胺

制备了以二硫基琥珀酰亚胺丙酸酯,4-巯基苯硼酸功能化CdTe量子点探针,对其进行了X-射线光电子能谱和透射电镜表征。在431 nm激发波长下,对探针及加入多巴胺后进行发射光谱扫描,最大发射峰红移,在最大发射波长处,多巴胺对合成的探针具有猝灭效应,且多巴胺对体系的猝灭程度与多巴胺的量呈良好的线性关系,据此建立了一种直接、灵敏、选择性好的测定多巴胺的方法。在最佳条件下,多巴胺的其线性范围为0.02~20.0 mmol/L,线性回归方程为ΔF=20.1+31.7C(mmol/L),相关系数为R2=0.987,检出限为4.9 nmol/L。本方法已成功用于多巴胺注射液、血清及尿样中多巴胺的测定,结果满意。     

基于氧荧光猝灭速率法的生化需氧量检测

利用氧荧光猝灭速率的方法,结合自行构建的BOD光纤传感装置进行海水中生化需氧量(BOD)含量检测。考察了四种筛选的海洋耗氧菌种在四甲基硅氧烷(TMOS)、二甲基二甲氧基硅烷(Di Me-DMOS)和聚乙烯醇(PVA)包埋固定情况下,对不同浓度的葡萄糖-谷氨酸(GGA)标准溶液的荧光响应情况。BOD敏感膜的荧光响应在0·2~30mg/L浓度范围内呈良好的线性关系,对2mg/L标准溶液测定的相对标准偏差为2·5%(n=6),响应时间(t95%)为4·0min,BOD敏感膜使用寿命大于12个月。实际海水样品检测表明,利用BOD敏感膜检测得到的结果与国标BOD5方法之间存在较好的一致性。     

厚朴酚在乙醇中的荧光自猝灭及猝灭机理

系统研究了厚朴酚的荧光光谱和吸收光谱随浓度的变化, 揭示了厚朴酚具有较强的自猝灭特性. 其猝灭包括静态和动态机制: 随着浓度增加, 厚朴酚发生聚集; 同时, 单体和聚集体对荧光的猝灭表现为动态猝灭, 由单体、二聚体、三聚体造成的荧光猝灭速率常数kqm、k qd、k qt值远大于扩散控制的荧光猝灭速率常数. 说明除扩散控制的短程能量转移引起的猝灭外, 厚朴酚还存在长程能量转移导致的猝灭机制.     

基于荧光猝灭的铜传感器研究

研究了过渡金属离子对荧光分子 1 萘胺二乙酸钠 (NADA)的荧光猝灭现象及其猝灭机理 ,并基于此建立铜离子传感体系。该铜离子传感器的检测限为8.2 7× 1 0 - 6mol/L ,定量检测范围为 2 .6 9× 1 0 - 5～ 1 .2 0× 1 0 - 4 mol/L。     

分子信标用于p53 mRNA的体外定量检测

根据p53基因的序列设计并合成了能特异性检测p53 mRNA的分子信标(MB), 发展了一种快速定量测定细胞内总RNA提取物中p53 mRNA的方法. 采用鼻咽癌(CNE2)细胞系和经RNA干扰技术降低p53基因表达的CNE2-p53RNAi细胞系, 抽提总RNA并用MB检测, 验证了MB的检测对象是p53 mRNA. 将该方法应用于多种肿瘤细胞内p53基因表达水平的分析, 表达变化趋势与经典的mRNA分析方法RT-PCR检测结果相符. 在此基础上, 用MB对5-氟尿嘧啶(5-Fu)处理的肺腺癌细胞(A549)进行了p53 mRNA的体外定量检测, 结果表明采用MB能够快速地获知该药物对细胞内p53 mRNA表达影响的信息.     

荧光猝灭法结合分子对接研究胰蛋白酶与蒙花苷相互作用

以荧光猝灭法与分子对接为主要研究手段,研究了在模拟生理条件下蒙花苷对胰蛋白酶荧光发射的猝灭作用,并探究了二者之间的相互作用类型与复合物的动力学稳定性。实验结果表明,pH 8.4时在不同温度下蒙花苷均可有效地猝灭胰蛋白酶的荧光发射,并且猝灭类型均为静态猝灭,即蒙花苷与胰蛋白酶间形成了稳定的复合物。使用双对数方程计算可知复合物为1∶1型,根据Van’t Hoff方程对不同温度下蒙花苷猝灭胰蛋白酶的光谱数据分析可知复合物的生成是熵增放热的自发过程。同步荧光光谱结果表明蒙花苷对胰蛋白酶的发光猝灭主要是通过影响其Trp残基的荧光发射实现的。分子对接研究表明蒙花苷可结合在胰蛋白酶表面由His57、Thr98、Gln175、Trp215、Gln192、Ser195等残基所形成的疏水性口袋中,并通过氢键、芳环堆积、疏水、静电吸引等弱作用与胰蛋白酶形成非共价复合物,蒙花苷与Trp215残基间存在明显的疏水与氢键作用,这些作用可减弱这个残基微环境的极性并最终导致其荧光发射的猝灭。30 ns分子动力学模拟表明胰蛋白酶与蒙花苷所形成的复合物具有良好的稳定性,验证了分子对接结果的稳定性和合理性。     

采用纳米金和荧光标记的适配体检测凝血酶

将荧光染料分子标记的含29个碱基的可识别凝血酶的DNA适配体非特异吸附到纳米金表面,荧光发生猝灭,加入凝血酶后,凝血酶与适配体特异性结合,使适配体空间结构发生改变,荧光染料分子远离纳米金表面,荧光恢复,因此可以实现对凝血酶的检测。实验结果表明,这种检测方法简便、快速、特异性强,检出限为0.54 nmol/L(对应样品体积为200μL)。     

Quantitative Detection of PremicroRNA-21 Based on Chimeric Molecular Beacon

As an important tumor marker, the expression level of premicroRNA-21 (pre-miR-21) can provide important information for early diagnosis, drug treatment and prognosis of tumor. Thus, developing pre-miR-21 detection is of great importance for diagnosis of disease. Herein, a direct, simple and rapid method for quantitative detection of pre-miR-21 was developed. This detecting system contained the tool of molecular beacon which could hybridize specifically with pre-miR-21 and form duplex to induce fluorescence signal change. When loop bases hybridized with the pre-miR-21 in the solution to form a double-stranded duplex, the fluorophore and the quencher was separated and caused fluorescence recovery. We developed the new method for pre-miR-21 assay with detection limit of 0.5 nM. Under the optimal conditions, the method was used to detect the level of pre-miR-21 in different tumor cells and tissues. The results showed that pre-miR-21 level was tightly related with tumor cells origins and malignancy degree. In 16 cases of gastric cancer tissues and adjacent tissues, the level of pre-miR-21 in 12 cases of cancer tissues was higher than that of adjacent tissues; 3 cases had lower expression level than that of adjacent tissues, and 1 case had no significant difference. Furthermore, Quantitative real-time PCR (qRT-PCR) method was used in to confirm the reliability of the detection results. The consistent results of the two different methods clearly showed that the molecular beacon assay with advantages of simplicity and rapidity for pre-miR-21 detection was hopeful for the wide usability in the function research and clinical diagnosis of pre-microRNA.     

Detection of PCB77 by Antibody-Coated Competitive Fluorescent Quantitative Immuno-PCR Using Molecular Beacon

Abstract

A rapid and quantitative technique is urgently needed in detecting toxicological and carcinogenic polychlorinated biphenyls (PCBs) in the environment. In this study, a high sensitive real time fluorescent quantitative immuno-PCR (FQ-IPCR) approach using molecular beacon (MB) was developed to detect PCB77, which was classified as a human carcinogen. MB-based FQ-IPCR was then performed on serial dilutions of known PCB77 concentrations equivalent to 10-fold dilutions of 10–10⁵ fg/mL. A correlation coefficient of 0.997 was identified, and a linear relationship between 10 fg/mL and 100 pg/mL, with y = 0.543x + 21.779, was obtained. Three soil samples were determined by MB-based FQ-IPCR to proof the validity of the method, and the results of which were further confirmed by conventional enzyme-linked immuno sorbent assay (ELISA). Based on sensitivity and reproduction, the MB-based real time FQ-IPCR technique will become a promising monitoring tool for environmental endocrine disruptors.     

分子信标荧光探针用于抑癌基因ING1表达产物的定量测定

根据抑癌基因ING1基因的序列设计并合成了检测ING1转录产物的分子信标核酸探针,发展了一种快速测量从正常细胞系和鼻咽癌肿瘤细胞系提取的ING1转录产物的方法,所得结果与用逆转录结合PCR法(RT-PCR)得到的结果相吻合.并且,将能表达ING1基因的质粒转入肿瘤细胞进行培养后,再将分子信标转入肿瘤细胞,发现转导了质粒的肿瘤细胞比未转导质粒的肿瘤细胞内的荧光明显增强,从而进一步证实了所设计的分子信标核酸探针与ING1转录产物的结合.     

Label-Free Fluorescence Molecular Beacon Probes Based on G-Triplex DNA and Thioflavin T for Protein Detection

Protein detection plays an important role in biological and biomedical sciences. The immunoassay based on fluorescence labeling has good specificity but a high labeling cost. Herein, on the basis of G-triplex molecular beacon (G3MB) and thioflavin T (ThT), we developed a simple and label-free biosensor for protein detection. The biotin and streptavidin were used as model enzymes. In the presence of target streptavidin (SA), the streptavidin hybridized with G3MB-b (biotin-linked-G-triplex molecular beacon) perfectly and formed larger steric hindrance, which hindered the hydrolysis of probes by exonuclease III (Exo III). In the absence of target streptavidin, the exonuclease III successively cleaved the stem of G3MB-b and released the G-rich sequences which self-assembled into a G-triplex and subsequently activated the fluorescence signal of thioflavin T. Compared with the traditional G-quadruplex molecular beacon (G4MB), the G3MB only needed a lower dosage of exonuclease III and a shorter reaction time to reach the optimal detection performance, because the concise sequence of G-triplex was good for the molecular beacon design. Moreover, fluorescence experiment results exhibited that the G3MB-b had good sensitivity and specificity for streptavidin detection. The developed label-free biosensor provides a valuable and general platform for protein detection.     

Aptamer Molecular Beacon Sensor for Rapid and Sensitive Detection of Ochratoxin A

Ochratoxin A (OTA) is a carcinogenic fungal secondary metabolite which causes wide contamination in a variety of food stuffs and environments and has a high risk to human health. Developing a rapid and sensitive method for OTA detection is highly demanded in food safety, environment monitoring, and quality control. Here, we report a simple molecular aptamer beacon (MAB) sensor for rapid OTA detection. The anti-OTA aptamer has a fluorescein (FAM) labeled at the 5′ end and a black hole quencher (BHQ1) labeled at the 3′ end. The specific binding of OTA induced a conformational transition of the aptamer from a random coil to a duplex–quadruplex structure, which brought FAM and BHQ1 into spatial proximity causing fluorescence quenching. Under the optimized conditions, this aptamer sensor enabled OTA detection in a wide dynamic concentration range from 3.9 nM to 500 nM, and the detection limit was about 3.9 nM OTA. This method was selective for OTA detection and allowed to detect OTA spiked in diluted liquor and corn flour extraction samples, showing the capability for OTA analysis in practical applications.     

分子信标用于不对称PCR检测乙型肝炎病毒

为了缓解竞争杂交对分子信标检测的影响，将分子信标与不对称聚合酶链式反应(PCR)联用进行血清中乙型肝炎病毒(HBV)的检测。并采用更为直接的荧光方法，将不对称PCR扩增中的引物浓度比确定为5：1，该结果与电泳方法得到的结果有所不同。文中结合对称及不对称PCR过程中荧光强度的变化情况，对其原因进行了详细的探讨。分子信标与不对称PCR联用，可以专一地检测出血清中HBV的存在，与分子信标／对称PCR相比．其检测效果明显增强。     

Determination of Sodium Hexametaphosphate in Beverages Based on the Fluorescence Quenching of Acridine Orange

Sodium hexametaphosphate was shown to form a complex with acridine orange by electrostatic interactions that induce fluorescence quenching. Analysis of fluorescence intensity showed that the process was dominated by static quenching, which was confirmed by absorption spectra and lifetime of the excited state. The decreased fluorescence intensity was directly proportional to the concentration of sodium hexametaphosphate between 8.0 × 10^?7 and 1.1 × 10^?5 mol L^?1 with a limit of detection of 5.3 × 10^?7 mol L^?1. Beverages were analyzed for sodium hexametaphosphate with recoveries between 91.7% and 108.3%.     

A Label-free and Functional Fluorescent Oligonucleotide Probe Based on a G-Quadruplex Molecular Beacon for the Detection of Kanamycin

A label-free and turn-off fluorescent method for the quantitative detection of kanamycin based on a functional molecular beacon was developed. The molecular beacon consists of two hairpin structures with a split G-rich oligonucleotide in the middle. The kanamycin's aptamer formed the loops portion for recognizing kanamycin, and the G-quadruplex bound by Thioflavin T(ThT) was employed as the reporter. In the absence of target, the molecular beacon folded into double stem-loops and the splited G-rich oligonucleotid came close to form a G-quadruplex. When ThT bound to the G-quadruplex, the fluorescence intensity of the solution increased. Upon the addition of kanamycin, the function between kanamycin and aptamer unfolded the hairpin and disassembled the G-quadraplex structure, resulting in a significant decrease in the fluorescence intensity. A good linear relationship ranging from 0.7 nmol/L to 10 nmol/L was achieved and the limit of detection was 0.37 nmol/L. Besides, it could efficiently recognize kanamycin in real samples.     

TaqMan-分子灯标：一种新型的荧光基因检测探针

在TaqMan及分子灯标的基础上开发了一类新型的均相荧光检测探针—— TaqMan-分子灯标(TaqMan-MB)，该探针集合了分子灯标的发夹结构及TaqMan探针降 解作用的工作原理，使检测效果更好．与实时PCR仪联用，可用于靶基因的定量检 测．