基于激发态能量转移机理比率型荧光探针的研究进展

激发态能量转移(Excitation Energy Transfer,EET)作为一类重要的光物理现象,被广泛用于比率型荧光探针和分子灯标的设计以及DNA检测等多个领域.影响EET效率的两个重要因素是供受体间的空间距离和光谱交盖,通过调节供受体间的空间距离或光谱重叠程度来调控能量转移过程,实现对目标客体的双波长比率检测.综述了基于不同供受体荧光团的EET体系、供受体间的连接方式对能量转移效率的影响,以及通过调控供受体间光谱重叠程度或空间距离,获得识别不同客体的比率型荧光探针,并对EET机理的比率型荧光探针的设计以及未来在生物成像和医学检测等领域的应用进行了展望.     

罗丹明B-β-环糊精衍生物探针型主-客体分子相互识别作用的研究

应用光谱法研究了生物大分子探针型主体分子罗丹明B一β-环糊精衍生物与DNA的相互作用及温度对该探针特性的影响.探讨了相应的主体分子与DNA分子相互作用的方式、嵌插作用能力大小以及温度对主体分子含客体分子时的影响．进一步研究了探针型主一客体分子相互识别作用及作用能力的大小．探讨了主一客体分子相互识别作用的机制．     

基于氧杂蒽结构可控官能化的荧光探针研究新进展

荧光分子探针技术在表达分子间识别行为及复杂生命和环境体系的内状态信息方面具有非常优异的性能,氧杂蒽及其衍生物螺连隐色体结构变化伴随的分子荧光变换模型,广泛且深入的应用于构建新型功能光敏探针分子.近年来此类探针的合成设计及功能化调控研究异常活跃,新的突破不断涌现.综述了新近基于氧杂蒽及其衍生物的荧光探针在金属阳离子、pH、阴离子识别检测方面的研究进展,并简要阐释了该类探针分子的构筑,识别检测机理以及探针在生物成像和环境监测等方面的应用.     

非猝灭型Cu2+荧光分子探针的研究进展

Cu2+荧光分子探针的开发对高灵敏及高选择性检测Cu2+具有重要意义.本文按照探针的结构类型,综述了近些年来荧光增强及比率型Cu2+荧光分子探针的研究进展.引用文献40篇.     

罗丹明基“OFF-ON”型荧光探针研究的一些新进展

综述了近年来罗丹明衍生物在分子探针方面研究的一些新进展, 系统阐述了该类探针分子的构筑及探针分子片段由内酰胺螺环构架(无荧光/OFF型)到开环蒽醌体系(有荧光/ON型)转化过程中, 伴随着荧光的变化在离子和小分子检测方面的应用.     

分子实验室探针在生化分析中的应用研究进展

分子实验室探针（lab-on-a-molecule probe）是基于分子级别的检测,能够提供空间高密度的感应信号,其主要分为分子逻辑门、分子多模式检测以及兼具2者功能的探针,该探针在多分析物的定性以及竞争分析的定量灵敏检测中发挥了重要的作用。对上述3类探针在生化分析（金属阳离子、无机阴离子、氨基酸、蛋白质、致病菌等）中的应用进行了总结。     

基于大环分子的主客体超分子荧光探针

针对环境污染源的早期检测和疾病的预防与治疗已经研究开发出许多检测技术手段,其中,荧光探针作为一种方便、灵敏、可视化的检测技术得到了广泛关注与认可。大环分子荧光探针作为一类重要的荧光探针逐渐引起了研究者的关注。大环分子具有特定尺寸、可特异性配合某些基团的空腔。因此,在设计这类荧光探针时可以充分利用大环分子的空腔优势。此外,大环分子容易通过化学修饰制备多种功能化衍生物,这也为设计大环荧光探针提供了更多选择。本文回顾了大环分子荧光探针的设计策略,主要从探针的化学组成以及相互作用机理来阐述,为大环分子荧光探针的设计提供了系统的理论指导。     

选择性检测谷胱甘肽的荧光探针

本文设计合成了一种基于BODIPY衍生物选择性检测谷胱甘肽的比率式荧光探针1。荧光探针1中BODIPY的3位连有苯乙炔基团,5位连有咪唑盐离去基团,利用其与谷胱甘肽和半胱氨酸反应机理的不同实现了对谷胱甘肽的选择性检测。紫外可见吸收光谱和荧光光谱实验结果表明探针分子1与谷胱甘肽反应后的光谱发生明显红移,可以实现对谷胱甘肽的比率式检测。探针分子1对谷胱甘肽有极高的选择性,不受其它氨基酸尤其是半胱氨酸的干扰。荧光滴定实验表明探针分子1可实现对谷胱甘肽的定量检测,检测限为3.3×10-8 mol/L。探针分子成功地应用于活体细胞中检测谷胱甘肽。     

分子印迹荧光探针在农药检测中的应用进展北大核心CSCD

分子印迹聚合物由于可特异性地从样品溶液中富集目标物,已被广泛应用于粮食、果蔬等食品以及水、土壤等环境中农药的提取和检测.将分子印迹技术和量子点修饰技术相结合形成的分子印迹荧光探针,可以实现对目标物的高灵敏快速检测.据此,对分子印迹技术的原理、制备方法及应用进行了概述,并进一步阐述了量子点修饰的分子印迹荧光探针在农药检测中的应用和展望.     

罗丹明B—β—环糊精衍生物探针型主—客体分子相互识别？…

应用光谱法研究了生物大分子探针型主体分子罗丹明Ｂ－β－环糊精衍生物与ＤＮＡ的相互作用及温度对该探针特性的影响，探讨了相应的主体分子与ＤＮＡ分子相互作用的方式，嵌插作用能力大小以及温度对主体分子含客体分子时的影响，进一步研究了探针型主－客体分子相互识别作用及作用能力的大小，探讨了主－客体分子相互识别作用的机制。     

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.     

DNAzyme amplification of molecular beacon signal

This paper reports an improved catalytic molecular beacon. Addition of the target oligonucleotide activates a DNA enzyme (DNAzyme), which, in turn, activates multiple copies of molecular beacons (MB) and gives rise to a strong fluorescence signal. In a previous design, the activated DNAzyme could oligomerize, especially dimerize, and result in inactivation of the DNAzyme. The current design avoids this problem, upon activated by the target DNA, the DNAzyme will stay constantly active. With the improved method, a detection of 10 pM DNA has been demonstrated, which is 1000 times more sensitive than the method previously reported.     

Real time monitoring uracil excision using uracil-containing molecular beacons

As a highly conserved damage repair protein, UDG excises uracil bases through its glycosylase activity. We report here an alternative fluorescence method for UDG assay with high accuracy and sensitivity by applying uracil-modified molecular beacons as substrates. The detection limit of UDG is 0.005 U mL⁻¹. The K_M and k_cat are 0.89 ± 0.1 μM and 210 ± 10 min⁻¹, respectively. The method is applied to screening inhibitors and the results indicate that both of the 5-FU and cisplatin can inhibit UDG activity with the IC₅₀ values of 6.1 ± 0.52 mM and 3.2 ± 0.24 mM, respectively. Furthermore, the combination of uracil-modified molecular beacons and nuclease inhibitor makes the new method possible to specifically detect UDG activity in cell-free extracts and serum. Taken together, the simple, rapid and sensitive method has potential relevance for a variety of applications, such as molecular diagnosis and screening of UDG inhibitors.     

Inhibited aptazyme-based catalytic molecular beacon for amplified detection of adenosine

Combining the inhibited aptazyme and molecular beacon(MB),we developed a versatile sensing strategy for amplified detection of adenosine.In this strategy,the adenosine aptamer links to the 8-17 DNAzyme to form an aptazyme.A short sequence,denoted as inhibitor,is designed to form a duplex spanning the aptamer–DNAzyme junction,which blocks the catalytic function of the DNAzyme.Only in the presence of target adenosine,the aptamer binds to adenosine,thus the inhibitor dissociates from the aptamer portion of the aptazyme and can no longer form the stable duplex required to inhibit the catalytic activity of the aptazyme.The released DNAzyme domain will hybridize to the MB and catalyze the cleavage in the presence of Zn2+,making the fluorophore separate from the quencher and resulting in fluorescence signal.The results showed that the detection method has a dynamic range from 10 nmol/L to 1 nmol/L,with a detection limit of 10 nmol/L.     

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.     

基于分子信标末端现场标记三聚氰胺-Cu~(2+)电活性分子的高灵敏电化学传感器

本文构建了一种基于分子信标自由末端现场标记电活性信号分子的新型DNA传感器.首先将3′修饰巯基的分子信标通过Au–S键自组装到金电极表面,然后在修饰有羧基的5′自由末端通过共价偶合和配位作用依次组装上三聚氰胺(Mel)和铜离子(Cu2+),得到以Mel-Cu2+配合物为电活性信号源的分子信标.该方法简单实现了电活性分子信标的标记、分离和纯化.以[Fe(CN)6]3-/4-为电化学探针,采用循环伏安和电化学阻抗法对层层自组装过程进行了表征.杂交实验表明,Mel-Cu2+信号源所对应的峰电流强度随着杂交液浓度的增大逐渐降低,且氧化峰电流与互补序列浓度对数在1.0×10-15~1.0×10-9 mol/L范围内呈良好的线性关系.根据3σ计算得到检测限为2.4×10-16 mol/L.另外,由于分子信标特殊的茎环结构特征和Mel-Cu2+信号源稳定的无机配位组成,传感器显示了很高的特异性、再生性和稳定性.     

基于双重猝灭原理的分子信标定量检测凝血酶

利用G碱基和有机猝灭基团对荧光基团的双重猝灭作用构建了分子信标,建立了一种基于双重猝灭原理的检测凝血酶的简单方法.此分子信标中荧光基团设计为羧基荧光素(FAM),有机猝灭基团设计为Black Hole Quencher 1(BHQ-1),BHQ-1连接3个含有G碱基的核苷酸,分子信标的环设计为凝血酶的核酸适配体.体系中没有凝血酶时,分子信标呈茎环结构,荧光基团FAM与有机猝灭基团BHQ-1及G碱基相互靠近,FAM的荧光在BHQ-1及G碱基的双重猝灭下,其荧光信号很弱;当体系中有凝血酶存在时,分子信标与凝血酶特异性结合,形成G-四联体结构,茎-环结构被破坏,FAM远离猝灭基团BHQ-1及G碱基,其荧光得到恢复.在最适条件下,体系的荧光强度(△I)与凝血酶的浓度(C)在0.4~40 nmol/L范围内具有良好的线性关系,线性回归方程为△I=24.63C(nmol/L)+13.06(R2=0.9972),检出限为0.18 nmol/L(3σ,n=9).实际血样加标回收率为96.3%~98.7%.     

A novel sandwich assay with molecular beacon as report probe for nucleic acids detection on one-dimensional microfluidic beads array

A novel sandwich assay with molecular beacons as report probes has been developed and integrated into one-dimensional microfluidic beads array (1-D chip) to pursue a label-free and elution-free detection of DNA/mRNA targets. In contrast with the immobilized molecular beacons, this sandwich assay can offer lower fluorescence background and correspondingly higher sensitivity. Furthermore, this sandwich assay on 1-D chip operating in conjunction with molecular beacon technique allows multiple targets detection without the need of laborious and time-consuming elution, which makes the experiment process simple, easy to handle, and reproducible results. In the experiment, the synthesized DNA targets with different concentrations were detected with a detection limit of ∼0.05 nM. Moreover, the mRNA expression changes in A549 cells before and after anticancer drug 5-flouorouracil treatments were detected and the results were validated by the conventional RT-PCR method.     

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.     

Electrochemical detection of miRNA-21 based on molecular beacon formed by thymine-mercury(II)-thymine base pairing

We present a molecular beacon-based electrochemical biosensor with high sensitivity and specificity for the detection of microRNA-21. A special oligonucleotide probe was prepared containing a nucleotide sequence complementary to miR-21 and consecutively linking eight and six thymines to the 3′ and 5′ ends, respectively, to allow the formation of a T-Hg²⁺-T complex-based molecular beacon on the electrode surface by the selective binding of Hg²⁺ ions. The introduction of multiple thymines at the end of the probe avoids base overlapping between the miRNA sequence and the molecular beacon formation sequence, enabling a universal probe design that can detect all types of miRNAs. A ferrocene moiety was attached to the 5′-end of the specially designed probe as an electrochemical signal indicator. The molecular beacons are formed by six consecutive T-Hg²⁺-T pairs by Hg²⁺ addition, and the molecular beacons are destroyed by perfect hybridization between 22 bases as a result of miR-21 addition. Based on this detection mechanism, we were able to detect miR-21 with LODs of 0.64 pM and 1.08 pM in buffer solution and human serum, respectively. In addition, the specifically designed oligonucleotide probe showed perfect specificity in detecting only miR-21 without binding to other miRNAs. Finally, the sensor showed excellent miR-21 recovery ability from samples spiked into serum, indicating that the method described in this study worked perfectly, even in a turbid complex matrix such as human serum.