滚环扩增阳离子共轭聚合物均相检测microRNA

结合恒温滚环扩增(rolling circle amlification,RCA)、单链特异性核酸外切酶Ⅰ(exonucleaseⅠ,ExoⅠ)和阳离子共轭聚合物(Cationic conjugated polymer,CCP)荧光共振能量转移(fluorescence reso-nance energy transfer,FRET)技术,建立了一种特异、灵敏的均相检测microRNA(miRNA)的新方法.该方法应用荧光标记探针与RCA扩增的长链DNA产物杂交,当加入CCP时,其与杂交的标记探针通过静电力结合,发生高效的FRET.未杂交的标记探针利用ExoⅠ水解成单核苷酸,其与CCP相互作用力弱,不能发生有效的FRET.基于此,无需分离和洗涤步骤,实现了RCA扩增miRNA的均相检测.方法特异性好,灵敏度高,线性为0.5～20pmol/L,检出限为0.2pmol/L.方法为miRNA均相检测和原位成像分析以及临床诊断提供了新策略.     

基于金纳米粒子的催化发夹DNA组装检测microRNA

结合金纳米粒子和催化发夹DNA组装,发展了一种检测microRNA(miRNA)的新方法.首先,在金纳米粒子表面修饰荧光素标记的发夹DNA探针P1,P1荧光被金纳米粒子猝灭.当加入目标miRNA分子时,其与P1杂交并形成P1-miRNA中间体,同时打开P1发夹结构.继而P1-miRNA与P2发夹探针结合,形成稳定的P1-P2双链结构,导致P1上的荧光素远离金纳米粒子而释放荧光,同时顶替下miRNA.随后,miRNA又可以引发金纳米粒子表面多个P1与P2探针的组装,如此循环,实现了对miRNA的放大检测.方法中miRNA在50pmol/L~2nmol/L之间呈现良好的线性关系,最低检出限为39pmol/L.方法简便,成本低,应用于HeLa细胞和HepG 2细胞中miRNA的检测,结果满意.     

可单波长激发的荧光纳米粒子的合成与表征

根据荧光共振能量转移(FRET)原理,先选定了能级匹配的给体(4-乙氧基-9-(2-羟乙基)-1,8-萘二甲酰亚胺(EHNI))和受体(硝基苯并二恶唑基染料(NBD)),采用一步细乳液聚合方法将这2种生色团引入单个的聚合物纳米粒子.制得的荧光纳米粒子兼有EHNI和NBD 2种染料的光谱特性,证明了2种生色团已经结合进入纳米粒子中.并且通过改变这2种染料的掺杂比例,在单一波长激发下,通过发生荧光共振能量转移,聚合物纳米粒子表现出多色,及可调控、可区分的荧光发射信号.     

含ESIPT染料的光开关多色 荧光聚合物纳米粒子

采用一步细乳液聚合法,将激发态分子内质子转移(ESIPT)荧光染料给体4-甲基苯胺基苯并噻唑(ABT)和4-甲基苯酚基苯并噻唑(HBT)与光致变色受体2-(3′,3′-二甲基-6-硝基螺\[苯并吡喃-2,2′-吲哚啉\]-1′-基)辛基-甲基丙烯酸酯(SP8MA)引入单个聚合物纳米粒子中,合成一系列基于荧光共振能量转移(FRET)的新型光开关多色荧光聚合物纳米粒子.在365 nm紫外光和525 nm可见光的交替照射下,通过选择性发生给受体间的FRET,聚合物纳米粒子表现出多色及可开关调控和可区分的荧光发射信号.该纳米粒子在信息加密、动态防伪等方面有着潜在的利用价值.     

一种新型太阳能电池菁染料光敏剂的合成及其性能研究

设计合成了Cy3菁染料太阳能电池光敏剂,其结构特点是分子中吲哚环氮原子上的取代基为对羧苄基。本文通过自行合成的染料中间体N-对羧苄基-2,3,3-三甲基-3H-吲哚啉-5-磺酸钾在超声波作用下与原甲酸三乙酯缩合制备得到了该染料。结果表明,超声波法具有反应时间短、温度低和收率高等优点;探讨了3种洗脱剂对产品的柱分离效果,其中异丙醇∶水=5∶1效果最佳;分析测试了Cy3菁染料的光谱性能,在水溶液中Cy3的最大紫外-可见吸收波长为551 nm,最大荧光发射波长为565 nm;与其相比,在-环糊精水溶液中,最大吸收和荧光发射波长不变,荧光发射强度有所增加。     

基于PFP荧光共振能量转移原理检测氧化损伤DNA

提出一种基于水溶性共轭聚合物PFP荧光共振能量转移原理检测氧化损伤DNA的方法。通过加入DNA修复酶来识别并切除被氧化的DNA碱基,获得含磷酸基团核苷酸空隙的DNA,再通过羟基引入荧光标记物,加入PFP后可得到PFPDNA复合物,通过荧光共振能量转移(fluorescence resonance energy transfer,FRET)进行氧化损伤DNA的检测。对方法的影响因素进行了考查和优化,包括PFP的浓度、DNA修复酶的种类、DNA聚合酶I的用量以及芬顿反应的时间等。研究表明,基于PFP荧光共振能量转移原理检测氧化损伤DNA方法具有灵敏度高和特异性强的优势,可确保检测的准确性,对老年疾病的预防医学具有很好的应用前景。     

一种新型聚合物颗粒的制备及其在氨基乙酸测定中的应用

设计一种基于荧光增强原理检测伯胺的新方法.利用邻苯二甲醛与烯丙基硫醇、乙二醇二甲基丙烯酸酯(EDMA)在偶氮二异丁腈(AIBN)引发下热聚合制得了一种新型聚合物颗粒.该聚合物颗粒本身的荧光很弱,但可与伯胺作用,形成具有强荧光的异吲哚类化合物.利用聚合物颗粒的这一性质测定氨基乙酸.实验结果表明:在pH值为8.0的磷酸盐缓冲溶液/乙醇(体积比4:1)混合介质中,控制聚合物颗粒质量浓度为1.5 g/L,聚合物颗粒与氨基乙酸反应3 h以上,反应可进行完全,混合悬浮液的荧光强度可达到稳定值.聚合物颗粒悬浮液的相对荧光强度对氨基乙酸的浓度为5.0×10-6～7.O×10-5 mol/L时有线性响应,线性相关系数为0.997 3,检测限为1.4×10-6mol/L,回收率为98.1%～103.3%.此聚合物颗粒也可以用于其他伯胺化合物的测定.     

一种新型荧光传感器用于果糖的识别与检测

以间氨基苯硼酸和间苯二胺为功能单体,过硫酸铵为引发剂,在水溶液中通过自由基聚合反应得到(间氨基苯硼酸–间苯二胺)聚合物.通过荧光光度计检测该聚合物激发波长为297,nm,发射波长为375,nm.实验证明糖类可以淬灭聚合物的荧光,其荧光猝灭程度具有果糖甘露糖葡萄糖蔗糖的规律.在pH 10.0条件下,聚合物的相对荧光强度随果糖浓度的增加而降低,线性范围为10-7~10-2,mol/L,最低检测限为10-8,mol/L,可用于饮料中果糖含量的测定,具有一定的应用价值.     

Integration of rolling circle amplification and cationic conjugated polymer for the homogeneous detection of single nucleotide polymorphisms

A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and tested. Mutant DNA serves as the template for specifically circularizing a padlock probe (PLP) with a sequence that is complementary to the mutant DNA. Afterwards, the mutant DNA directly acts as the primer to initiate the RCA reaction in the presence of phi29 DNA polymerase that generates a long, tandem single-strand DNA product. During the RCA reaction, fluorescein-labeled dUTPs are incorporated into the RCA products. When the CCP is introduced, efficient FRET from CCP to fluorescein occurs as a result of the strong electrostatic interactions between the CCP and the DNA produced by RCA. The wild-type DNA contains a single base mismatch with PLP with the result that the PLP is not circularized, RCA is not triggered and inefficient FRET results. By measuring the change of the emission intensities of CCP and fluorescein, it was possible to detect the SNP in a homogeneous manner. The method is sensitive and specific enough to detect 0.1 pmol/L mutant DNA and to determine a mutant allele frequency as low as 2.0%.     

高致病性猪繁殖与呼吸综合征RPA-LFD诊断方法的建立及初步应用

高致病性猪繁殖与呼吸综合征(HP-PRRS)给我国养猪业带来了巨大的损失.为快速、便捷诊断该病,建立重组酶聚合酶扩增结合侧流层析技术(RPA-LFD)的HP-PRRS诊断方法.针对HP-PRRSV Nsp2基因特异序列,设计生物素(Biotin)标记的特异性引物和FAM荧光素标记的探针,对RPA-LFD的反应体系和条件进行优化,结果显示,HP-PRRSV RPA-LFD最佳反应体系为：上下游引物(0.42 pmol/μL)各2.1μL,探针(0.3 pmol/μL)0.6μL,Rehydration buffer 29.5μL,cDNA模板1μL,ddH₂O 12.2μL,MgOAc 2.5μL,共50μL;最佳反应条件为：37℃,20 min.该方法同时检测高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)、猪繁殖与呼吸综合征病毒VR2332株(PRRSV VR2332)和NADC30-like株等10种常见感染猪的病原,结果显示,该方法除对HP-PRRSV检测呈阳性外,对PRRSV VR2332株、NADC30-like和其它9种感染猪的病原检测均为阴性,特异...     

Multiplex detection of KRAS and BRAF mutations using cationic conjugated polymers

The mutation detections of KRAS and BRAF genes are of significant importance to predict the responses to anti-cancer therapy and develop new drugs. In this paper, we developed a multi-step fluorescence resonance energy transfer (FRET) assay for multiplex detection of KRAS and BRAF mutations using cationic conjugated polymers (CCP). The newly established detection system could detect as low as 2% mutant DNAs in DNA admixtures. By triggering the emission intensity change of CCP and the dyes labeled in the DNA, four possible statuses (three mutations and one wildtype) can be differentiated in one extension reaction. The detection efficiency of this new method in clinical molecular diagnosis was validated by determining KRAS and BRAF mutations of 51 formalin-fixed paraffin-embedded (FFPE) ovary tissue samples. Furthermore, the result of the CCP-based multi-step FRET assay can be directly visualized under UV light so that no expensive instruments and technical expertise are needed. Thus, the assay provides a sensitive, reliable, cost-effective and simple method for the detection of disease-related gene mutations.     

Magnetically assisted fluorescence ratiometric assays for adenosine deaminase using water-soluble conjugated polymers

A magnetically assisted fluorescence ratiometric technique has been developed for adenosine deaminase assays with high sensitivity using water-soluble cationic conjugated polymers (CCPs). The assay contains three elements: a biotin-labeled aptamer of adenosine (biotin-aptamer), a signaling probe single-stranded DNA-tagged fluorescein at terminus (ssDNA-Fl) and a CCP. The specific binding of adenosine to biotin-aptamer makes biotin-aptamer and ssDNA-Fl unhybridized, and the ssDNA-Fl is washed out after streptavidin-coated magnetic beads are added and separated from the assay solution under magnetic field. In this case, after the addition of CCP to the magnetic beads solution, the fluorescence resonance energy transfer (FRET) from CCP to fluorescein is inefficient. Upon adding adenosine deaminase, the adenosine is converted into inosine, and the biotin-aptamer is hybridized with ssDNA-Fl to form doubled stranded DNA (biotin-dsDNA-Fl). The ssDNA-Fl is attached to the magnetic beads at the separation step, and the addition of CCP to the magnetic beads solution leads to efficient FRET from CCP to fluorescein. Thus the adenosine deaminase activity can be monitored by fluorescence spectra in view of the intensity decrease of CCP emission or the increase of fluorescein emission in aqueous solutions. The assay integrates surface-functionalized magnetic particles with significant amplification of detection signal of water-soluble cationic conjugated polymers. Supported by the “100 Talents” Program of the Chinese Academy of Sciences, and the National Natural Science Foundation of China (Grant No. 20574073)     

过氧化草酸酯化学发光检测DNA荧光探针

根据过氧化草酸酯与双氧水作用产生的高能中间体激发荧光物质发光的原理,研究了利用过氧化草酸酯TCPO-H2O2化学发光体系测定Cy5标记的DNA探针的新方法,探讨了溶剂、酸度、催化剂及发光溶液浓度对化学发光信号的影响,确立了最佳分析条件.其线性范围为8.4×10-9~2.8×10-6mol/L,检出限(3σ)为2.8×10-9mol/L.据此建立了操作简便、灵敏度高的测定Cy5标记的DNA探针的新方法.     

基于芘希夫碱的汞离子荧光探针的合成与性能研究

以4-氯-7-硝基苯并呋咱（NBD-Cl）和芘甲醛为原料,设计合成了一种荧光化合物芘苯并呋咱类希夫碱,并通过核磁、质谱对其结构进行了表征. 结果表明：在二甲基亚砜（DMSO）水溶液中,该化合物能够对Hg2+表现出荧光增强响应,而相同条件下其他重金属离子对该化合物几乎没有类似的荧光识别现象. 因此,该荧光化合物对Hg2+具有专一荧光识别作用,是一种有效的Hg2+荧光探针.     

Fluorescent immunosensor based on fluorescence resonance energy transfer between CdSe/ZnS quantum dots and Au nanorods for PRRSV detection

An ultrasensitive and selective sandwich-type fluorescent immunosensor based on fluorescence resonance energy transfer (FRET) between CdSe/ZnS quantum dots (QDs) and Au nanorods (AuNRs) was developed for the rapid detection of porcine reproductive and respiratory syndrome virus (PRRSV). Modified CdSe/ZnS QDs and AuNRs were bioconjugated with primary antibodies M and GP5, respectively. When the target antigen was present, a well-known sandwich-type form CdSe/ZnS QDs-M antibody-antigen-GP5 antibody-AuNRs was produced, and thus reducing the distance between the QDs and NRs. As a result, FRET occurred and the fluorescence intensity of CdSe/ZnS QDs decreased, and this decrease was used to determine the antigen concentration. Ultrasensitive detection of PRRSV at low concentrations in swine serum was achieved with a limit of detection of 0.55 TCID₅₀/mL and a linear detection range of 10¹–3.5 ?× ?10⁴ TCID₅₀/mL. Therefore, the fluorescent immunosensor is selective and highly sensitive; specifically, it can detect PRRSV at low concentrations in swine serum over a large concentration range. This proposed detection method can facilitate the development of high-performance fluorescent immunosensors for the detection of PRRSV and other antigens.     

氧化石墨烯对DNA荧光分子探针FRET效应的研究

研究了氧化石墨烯的浓度、氧化石墨烯与DNA分子荧光探针的反应温度、DNA链长度对FRET效应的影响.研究表明,氧化石墨烯的浓度、氧化石墨烯与DNA分子荧光探针的反应温度、DNA链长度对FRET效应都有着重要的影响.氧化石墨烯浓度越大,其对DNA分子探针的荧光猝灭效率越高;氧化石墨烯与DNA分子荧光探针的反应温度越高,淬灭效率越高;DNA链越长,氧化石墨烯淬灭的效率越低.因此,氧化石墨烯的浓度、氧化石墨烯与DNA分子荧光探针的反应温度、DNA链长度对FRET效应都应该是设计生物传感器时考虑的因素.     

苔藓植物RAPD应体系的建立及遗传多样性分析

以多枝青藓为材料优化RAPD反应条件,在此基础上对11种苔藓植物进行遗传多样性分析.结果表明:苔藓植物RAPD反应(25μl体系)的最佳条件为,Taq酶,1.0U;Mg2+浓度,2.0 mmol/L;dNTP浓度,0.2 mmol/L;模板DNA,60 ng;引物浓度,10 pmol.用40条引物进行扩增筛选,有6条引物扩增条带清晰,重复性好,共扩增出77条带.通过SPSS11.5分析软件对扩增结果进行聚类分析,结果与形态学分类基本一致,说明RAPD技术可用于苔藓植物的遗传多样性研究.     

辣根过氧化物酶催化合成聚-4-羟基苯乙烯基吡啶

利用辣根过氧化物酶催化合成了新型共轭聚合物----聚-4-羟基苯乙烯基吡啶, 辣根过氧化物酶的催化氧化反应使底物中的苯环直接聚合. 荧光光谱研究结果表明, 聚合物具有良好的光致发光特性, 辣根过氧化物酶能有效地催化4-羟基苯乙烯基吡啶的聚 合反应.     

Highly sensitive biosensors based on water-soluble conjugated polymers

Conjugated, conductive polymers are a kind of important organic macromolecules, which has found applications in a variety of areas. The application of conjugated polymers in developing fluorescent biosensors represents the merge of polymer sciences and biological sciences. Conjugated polymers are very good light harvesters as well as fluorescent polymers, and they are also “molecular wires”.Through elaborate designs, these important features, i.e.efficient light harvesting and electron/energy transfer, can be used as signal amplification in fluorescent biosensors. This might significantly improve the sensitivity of conjugated polymer-based biosensors. In this article, we reviewed the application of conjugated polymers, via either electron transfer or energy transfer, to detections of gene targets, antibodies or enzymes. We also reviewed recent efforts in conjugated polymer-based solid-state sensor designs as well as chip-based multiple target detection. Possible directions in this conjugated polymer-based biosensor area are also discussed.