基于纳米金和脱氧核酶荧光探针检测铅离子

本文以"8-17"脱氧核酶为识别靶标的配体,设计了一种基于纳米金(AuNPs)和脱氧核酶的荧光信号放大法检测Pb2+的探针。"8-17"脱氧核酶由一条底物链(17DS)和一条酶链(17E)组成。在底物链的一端修饰荧光基团6-羧基荧光素(FAM),而酶链通过巯基修饰到AuNPs表面。当底物链与酶链相混合时,酶链与底物链杂交,AuNPs与FAM靠近,导致FAM的荧光被AuNPs猝灭。当向该体系加入Pb2+时,"8-17"脱氧核酶被Pb2+激活,酶链将底物链剪切为两段,破坏了杂交的刚性结构,从而使得FAM的荧光恢复。基于此原理,构建了一种定量检测Pb2+的高灵敏传感器,该传感器对Pb2+的检测限达0.6nmol/L。     

基于脱氧核酶的新型铅离子荧光探针

将荧光猝灭基团修饰的17E脱氧核酶(17E DNAzyme)与荧光基团修饰的底物链通过6个脱氧核苷酸相连, 得到了一种新型的对Pb2+敏感的荧光探针. 由于DNAzyme与底物链发生分子内杂交, 荧光基团与猝灭基团相互靠近, 导致荧光猝灭. 当Pb2+存在时, DNAzyme被激活, 底物链被切断后释放出荧光基团标记的DNA片段, 从而产生明显的荧光信号. 据此可在常温下快速检测Pb2+, 检测下限为10 nmol/L. 在Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Mg2+和Ni2+等多种二价金属离子中, 除Zn2+, Mn2+和Cd2+略有干扰外, 其它几种金属离子均无响应, 表明该荧光探针对Pb2+具有良好的选择性.     

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.     

基于双酶切级联信号放大的核酸检测方法

利用茎环结构定位探针构建了一个基于双酶切反应的级联信号放大体系,并将其用于核酸的检测.在该体系中,茎环结构定位探针首先是内切酶Tth EndonucleaseⅣ的作用底物,被剪切后又作为定位探针介导切口酶Nt.Bst NBI对分子信标实施剪切,将这2步剪切反应结合起来可有效克服切口酶对于目标核酸中特定识别序列的依赖,同时进一步提高了检测灵敏度.实验结果表明,荧光信号与目标DNA浓度的对数值呈线性相关,响应范围为1 pmol/L～1 nmol/L,并且具有良好的识别单碱基变异的能力.此外,本方法序列设计简单,通用性强,仅改变定位探针的部分序列即可实现对不同目标DNA的检测.对掺杂于血清中的目标DNA的检测结果验证了本方法在实际样品检测中的应用潜力.     

基于外源荧光探针的脱氧核酶切割RNA反应动力学参数测定方法的改进

对以EB作为外源荧光探针测定脱氧核酶切割RNA反应的动力学参数的方法进行了改进。实验结果表明：对于目前应用最为广泛的10-23脱氧核酶和8-17脱氧核酶,在生理条件下(150mmol／L KCl,2mmol／L MgCL2,pH7．5,37℃)采用改进方法测得的反应动力学参数(kobs 10-23=0．12min^-1,kobs 8-17=0．08min^-1)与原方法相比更接近于放射性标记方法的测定结果(k2 10-23=0．13min^-1,k2 8-17=0．08min^-1)。     

基于DNAzyme的自驱动DNA分子机器检测有机磷农药

通过范德华力作用,将脱氧核酶(DNAzyme)和6-羧基荧光素(FAM)标记的底物链共修饰的金纳米粒子(AuNPs)负载于MnO2纳米片上,构建了一种自驱动DNA分子机器.乙酰胆碱酯酶(AChE)将其底物乙酰硫代胆碱(ATCh)转化为硫代胆碱(TCh)后,MnO2纳米片被降解为Mn2+,激活DNAzyme切割底物链的循...     

基于链置换循环无标记检测端立酶RNA的荧光法

以氧化石墨烯(GO)作为DNA载体和荧光猝灭剂,SYBRGreen Ⅰ(SGⅠ)为荧光信号探针,发夹核酸探针为分子识别探针,基于目标物启动的发夹核酸探针链置换循环反应,建立了一种利用荧光共振能量转移和链置换循环放大技术检测端粒酶RNA (hTR)的荧光新方法.发夹核酸探针hpDNA1和hpDNA2吸附在GO表面,嵌插在发夹DNA探针茎部的SG Ⅰ的荧光信号被GO猝灭.当人工合成的目标物(T1)存在时,T1与hpDNA1杂交打开hpDNA1的茎-环结构而引发hpDNA2与T1之间的链置换循环反应,由此累积产生大量的hpDNA1/hpDNA2杂交双链.刚性的双链DNA脱离GO表面,导致所嵌插的SG Ⅰ产生较强的荧光信号.基于荧光信号的变化,可定量检测0.2～50 nmoL/L的T1,检出限为90 pmol/L.该方法为端粒酶RNA检测提供了一种高灵敏、高特异性且无需标记的荧光新途径.     

脱氧核酶传感器研究进展

脱氧核酶是利用体外分子进化技术获得的一类具有高效催化活性和结构识别能力的单链DNA片段。本文主要介绍了两类脱氧核酶,即RNA切割型脱氧核酶和G-四聚体型脱氧核酶在生物传感体系中的发展和应用。     

罗丹明类荧光探针的合成及对铜离子的检测

合成了罗丹明类Cu2+荧光增强型分子探针3',6'-双(二乙氨基)-2-(N-乙叉基氨基)螺[异吲哚-1,9'-占吨]-3-酮（RA）,并研究了它的光谱性能及对铜离子的识别作用.在乙腈/水（体积比1/1）的介质中,当加入Cu2+后探针RA显玫瑰红色,最大吸收波长为548 nm,最大发射波长为571 nm,且荧光强度显著增强,但是,其它常见离子如Na+, K+, Mg2+, Ca2+, Mn2+, Cd2+, Cr3+, Co2+, Ni2+, Ag+, Pb2+, Zn2+, Fe3+, Hg2+不引起或引起很小的紫外/可见或荧光光谱变化.RA的选择性荧光增强主要是由于Cu2+诱导分子中的酰胺闭环结构发生开环,导致分子结构的共轭程度增大.在6.5×10-8～2.9×10-6 mol?L-1范围内RA可以有效检测Cu2+,检测限为5.0×10-8 mol?L-1.RA对Cu2+的识别不可逆,而且探针RA对pH值不敏感,可以在比较宽的范围内（pH=4.1～10.5）高灵敏、高选择性检测Cu2+.     

利用无标记的分子信标及核酸染料Hoechst 33258检测特定序列核酸

利用无标记的分子信标及核酸染料Hoechst 33258建立了一种高灵敏、高选择性的特定序列核酸检测方法,并以野生型乙型肝炎病毒的一段寡核苷酸序列为目标DNA,对这种方法进行了验证。在此体系中,分子信标的茎完全设计成C/G碱基对。在没有目标DNA时,Hoechst 33258与分子信标作用较弱,其荧光信号很弱;当有目标DNA存在时,分子信标与目标DNA杂交形成双链,Hoechst 33258与双链DNA作用后荧光信号显著增强。在优化条件下,目标DNA浓度在2×10-10~2×10-8mol/L范围内时,Hoechst 33258的荧光强度(ΔI)与目标DNA的浓度(C)之间具有良好的线性关系,回归方程为ΔI=3.3439C+18.6949(R2=0.9982),方法检出限(3σ)为9×10-11mol/L。此方法操作简单、检测速度快、灵敏度高、重现性好、检出限低。     

Gold nanorods-based FRET assay for sensitive detection of Pb2+ using 8-17DNAzyme

In this paper, we have reported a sensitive assay for fluorescence "turn-on" detection of Pb(2+) in aqueous solutions based on FRET between gold nanorods (GNRs) and the FAM-labeled substrate strand of 8-17DNAzyme. The fluorescence of the FAM-labeled substrate strand is quenched when 8-17DNAzyme is adsorbed on GNRs surface through electrostatic interaction. In the presence of lead ions, the fluorescence is restored due to the decrease of FRET efficiency caused by the specific cleavage of the FAM-labeled substrate strand by the enzyme, which weakens the electrostatic interaction between the GNRs and short FAM-labeled DNA fragment. The interference of eleven common metal ions has been tested, indicating that Pb(2+) can be selectively detected. This method exhibits a high sensitivity for Pb(2+) with a detection limit of 61.8 pM and a linear range from 0.1 nM to 100 nM. It is a simple, sensitive, and selective method for Pb(2+) detection. Moreover, this sensing system obtained satisfying results for Pb(2+) detection in tap water samples.     

Zn(2+)-ligation DNAzyme-driven enzymatic and nonenzymatic cascades for the amplified detection of DNA

A generic fluorescence sensing platform for analyzing DNA by the Zn(2+)-dependent ligation DNAzyme as amplifying biocatalyst is presented. The platform is based on the target DNA induced ligation of two substrate subunits and the subsequent opening of a beacon hairpin probe by the ligated product. The strand displacement of the ligated product by the beacon hairpin is, however, of limited efficiency. Two strategies are implemented to overcome this limitation. By one method, a "helper" nucleic acid sequence is introduced into the system, and this hybridizes with the DNAzyme components and releases the ligated product for opening of the hairpin. By the second method, a nicking enzyme (Nt.BspQI) is added to the system, and this nicks the duplex between the beacon and ligated product while recycling the free ligation product. By combining the two coadded components ("helper" sequence and nicking enzyme), the sensitive detection of the analyte is demonstrated (detection limit, 20 pM). The enzyme-free amplified fluorescence detection of the target DNA is further presented by the Zn(2+)-dependent ligation DNAzyme-driven activation of the Mg(2+)-dependent DNAzyme. According to this method, the Mg(2+)-dependent DNAzyme subunits displace the ligated product, and the resulting assembled DNAzyme cleaves a fluorophore/quencher-modified substrate to yield fluorescence. The method enabled the detection of the target DNA with a detection limit corresponding to 10 pM. The different sensing platforms are implemented to detect the Tay-Sachs genetic disorder mutant.     

Amplified fluorescence detection of mercury(II) ions (Hg2+) using target-induced DNAzyme cascade with catalytic and molecular beacons

In this work, a fluorescent sensing strategy was developed for the detection of mercury(II) ions (Hg(2+)) in aqueous solution with excellent sensitivity and selectivity using a target-induced DNAzyme cascade with catalytic and molecular beacons (CAMB). In order to construct the biosensor, a Mg(2+)-dependent DNAzyme was elaborately designed and artificially split into two separate oligonucleotide fragments. In the presence of Hg(2+), the specific thymine-Hg(2+)-thymine (T-Hg(2+)-T) interaction induced the two fragments to produce the activated Mg(2+)-dependent DNAzyme, which would hybridize with a hairpin-structured MB substrate to form the CAMB system. Eventually, each target-induced activated DNAzyme could catalyze the cleavage of many MB substrates through true enzymatic multiple turnovers. This would significantly enhance the sensitivity of the Hg(2+) sensing system and push the detection limit down to 0.2 nM within a 20 min assay time, much lower than those of most previously reported fluorescence assays. Owning to the strong coordination of Hg(2+) to the T-T mismatched pairs, this proposed sensing system exhibited excellent selectivity for Hg(2+) detection, even in the presence of 100 times of other interferential metal ions. Furthermore, the applicability of the biosensor for Hg(2+) detection in river water samples was demonstrated with satisfactory results. These advantages endow the sensing strategy with a great potential for the simple, rapid, sensitive, and specific detection of Hg(2+) from a wide range of real samples.     

Immobilization of DNAzyme catalytic beacons on PMMA for Pb2+ detection

Due to the numerous toxicological effects of lead, its presence in the environment needs to be effectively monitored. Incorporating a biosensing element within a microfluidic platform enables rapid and reliable determinations of lead at trace levels. A microchip-based lead sensor is described here that employs a lead-specific DNAzyme (also called catalytic DNA or deoxyribozyme) as a recognition element that cleaves its complementary substrate DNA strand only in the presence of cationic lead (Pb(2+)). Fluorescent tags on the DNAzyme translate the cleavage events to measurable, optical signals proportional to Pb(2+) concentration. The DNAzyme responds sensitively and selectively to Pb(2+), and immobilizing DNAzyme in the sensor permits both sensor regeneration and localization of the detection zone. Here, the DNAzyme has been immobilized on a PMMA surface using the highly specific biotin-streptavidin interaction. The strategy includes using streptavidin physisorbed on a PMMA surface to immobilize DNAzyme both on planar PMMA and on the walls of a PMMA microfluidic device. The immobilized DNAzyme retains its Pb(2+) detection activity in the microfluidic device and can be regenerated and reused. The DNAzyme shows no response to other common metal cations and the presence of these contaminants does not interfere with the lead-induced fluorescence signal. While prior work has shown lead-specific catalytic DNA can be used in its solubilized form and while attached to gold substrates to quantitate Pb(2+) in solution, this is the first use of the DNAzyme immobilized within a microfluidic platform for real time Pb(2+) detection.     

Incorporation of a DNAzyme into Au-coated nanocapillary array membranes with an internal standard for Pb(ii) sensing

A Pb(ii)-specific DNAzyme has been successfully incorporated into Au-coated polycarbonate track-etched (PCTE) nanocapillary array membranes (NCAMs) by thiol-gold immobilization. Incorporation of the DNAzyme into the membrane provides a substrate-bound sensor using a novel internal control methodology for fluorescence-based detection of Pb(ii). A non-cleavable substrate strand, identical to the cleavable DNAzyme substrate strand except the RNA-base is replaced by the corresponding DNA-base, is used for ratiometric comparison of intensities. The cleavable substrate strand is labeled with fluorescein, and the non-cleavable strand is labeled with a red fluorophore (Cy5 or Alexa 546) for detection after release from the membrane surface. This internal standard based ratiometric method allows for real-time monitoring of Pb(ii)-induced cleavage, as well as standardizing variations in substrate size, solution detection volume, and monolayer density. The result is a Pb(ii)-sensing structure that can be stored in a prepared state for 30 days, regenerated after reaction, and detect Pb(ii) concentrations as low as 17 nM (3.5 ppb).     

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.     

Nucleic Acid Driven DNA Machineries Synthesizing Mg2+‐Dependent DNAzymes: An Interplay between DNA Sensing and Logic‐Gate Operations

Polymerase/nicking enzymes and nucleic‐acid scaffolds are implemented as DNA machines for the development of amplified DNA‐detection schemes, and for the design of logic gates. The analyte nucleic acid target acts, also, as input for the logic gates. In the presence of two DNA targets, acting as inputs, and appropriate DNA scaffolds, the polymerase‐induced replication of the scaffolds, followed by the nicking of the replication products, are activated, leading to the autonomous synthesis of the Mg²⁺‐dependent DNAzyme or the Mg²⁺‐dependent DNAzyme subunits. These biocatalysts cleave a fluorophore/quencher‐functionalized nucleic‐acid substrate, thus providing fluorescence signals for the sensing events or outputs for the logic gates. The systems are used to develop OR, AND, and Controlled‐AND gates, and the DNA‐analyte targets represent two nucleic acid sequences of the smallpox viral genome.     

Signal-amplification detection of small molecules by use of Mg2+- dependent DNAzyme

Because small molecules can be beneficial or toxic in biology and the environment, specific and sensitive detection of small molecules is one of the most important objectives of the scientific community. In this study, new signal amplification assays for detection of small molecules based on Mg²⁺-dependent DNAzyme were developed. A cleavable DNA substrate containing a ribonucleotide, the ends of which were labeled with black hole quencher (BHQ) and 6-carboxyfluorescein (FAM), was used for fluorescence detection. When the small molecule of interest is added to the assay solution, the Mg²⁺-dependent DNAzyme is activated, facilitating hybridization between the Mg²⁺-dependent DNAzyme and the DNA substrate. Binding of the substrate to the DNAzyme structure results in hydrolytic cleavage of the substrate in the presence of Mg²⁺ ions. The fluorescence signal was amplified by continuous cleavage of the enzyme substrate. Ochratoxin A (OTA) and adenosine triphosphate (ATP) were used as model analytes in these experiments. This method can detect OTA specifically with a detection limit as low as 140 pmol?L^?1 and detect ATP specifically with a detection limit as low as 13 nmol?L^?1. Moreover, this method is potentially extendable to detection of other small molecules which are able to dissociate the aptamer from the DNAzyme, leading to activation of the DNAzyme.     

Ligase-based ultrasensitive detection of DNAzyme cleavage product using molecular beacon

Detection for deoxyribozyme(DNAzyme) cleavage usually needs complex and time-consuming radial labeling,gel electrophoresis and autoradiography.A new approach was reported for detection DNAzyme cleavage product based on molecular beacon (MB).Part of the loop of MB was designed to complementary to DNAzyme cleavage product.MB was employed to monitor ligation process of RNA/DNA complex and to convert directly cleavage product information into fluorescence signal.Detection limit of the assay is 0.02 nmol/L.The cleavage product of 8 -17 DNAzyme against HCV-RNA was detected perfectly based on this assay.The method is fast,simple and ultrasensitive,which might hold great promise in DNAzyme reaction and DNAzyme gene therapy.