基于纳米金与纳米银簇间表面等离子增强能量转移效应特异性检测microRNA

microRNAs(miRNAs)的灵敏检测对临床诊断具有十分重要的意义.本研究采用偶联DNA聚合酶和核酸内切酶介导的恒温扩增反应实现靶标循环再生的策略,利用纳米金(AuNPs)与纳米银簇(AgNCs)间表面等离子增强能量转移效应,开发了一种miRNA定量检测方法.在AuNPs表面组装两种探针(Probe a和Probe b)制备响应元件Probe b-Probe a-AuNP,其中Probe a通过3′端巯基共价偶联到AuNPs表面,此外具有靶标miRNA互补序列、核酸内切酶酶切序列和Probe b互补序列,Probe b为荧光AgNCs合成模板.靶标miRNA存在时,启动酶级联恒温扩增反应,导致Probe b脱离AuNPs表面,抑制了Probe b为模板合成的AgNCs与AuNPs间表面等离子增强能量转移效应,使得反应体系荧光信号增强.本方法的检出限为2.5×10-11 mol/L,与miRNAs商业化检测试剂盒相比,避免了逆转录反应,而且操作简单,检测成本低,可应用于生物样本中miRNAs分析.     

Hydrogen evolution assisted deposition of a three-dimensional porous nickel film for the electrocatalytic oxidation of histamine

Microchimica Acta - The authors describe an electrochemical sensor for ultrasensitive voltammetric of histamine (HA) by using a gold electrode that was modified with a film consisting of...     

Enhancement of the sensitivity and selectivity of oxidation of H2O2 on platinum wire at low working potential by platinization and covering of heteropolypyrrole film for amperometric micro-biosensor construction

A microelectrode for glucose determination was constructed by immobilization of glucose oxidase (GOx) on a platinized platinum (Pt) by electrochemical polymerization of a solution containing GOx, pyrrole, and a substituted pyrrole, 4-(3-pyrrolyl)-4-oxobutyric acid. Due to platinization and covering with the polymerized heteropolypyrrole (hPPy) film, the electrode prepared showed high sensitivity to H₂O₂ at a low potential and significantly reduced the response to electroactive compounds, such as ascorbate, urate and 4-acetamidophenol. Working at 200 mV (vs. SCE) the electrode showed a linear response to glucose from 1.6 to 10 mM with a high sensitivity of 1 μA/mM, whereas the response to 1 mM ascorbate, urate, and 4-acetamidophenol was 0.53 μA, 18 nA and 4 nA, respectively, which was about 2.5%, 1.0% and 1.0% of that at a bare electrode. The stability of the electrode was tested at intervals of three or five days, and each test lasted about two hours. After 6 months examination, only 30% of its activity was lost.     

One-step, multiplexed fluorescence detection of microRNAs based on duplex-specific nuclease signal amplification

Traditional molecular beacons, widely applied for detection of nucleic acids, have an intrinsic limitation on sensitivity, as one target molecule converts only one beacon molecule to its fluorescent form. Herein, we take advantage of the duplex-specific nuclease (DSN) to create a new signal-amplifying mechanism, duplex-specific nuclease signal amplification (DSNSA), to increase the detection sensitivity of molecular beacons (Taqman probes). DSN nuclease is employed to recycle the process of target-assisted digestion of Taqman probes, thus, resulting in a significant fluorescence signal amplification through which one target molecule cleaves thousands of probe molecules. We further demonstrate the efficiency of this DSNSA strategy for rapid direct quantification of multiple miRNAs in biological samples. Our experimental results showed a quantitative measurement of sequence-specific miRNAs with the detection limit in the femtomolar range, nearly 5 orders of magnitude lower than that of conventional molecular beacons. This amplification strategy also demonstrated a high selectivity for discriminating differences between miRNA family members. Considering the superior sensitivity and specificity, as well as the multiplex and simple-to-implement features, this method promises a great potential of becoming a routine tool for simultaneously quantitative analysis of multiple miRNAs in tissues or cells, and supplies valuable information for biomedical research and clinical early diagnosis.     

Label-free fluorescent detection of copper(II) using DNA-templated highly luminescent silver nanoclusters

Novel luminescent silver nanoclusters (AgNCs) were synthesized utilizing DNA as templates by a simple, rapid and one-pot procedure. Luminescence studies indicated that these DNA-AgNCs exhibited strong emission with peak maximum at 624 nm. The fluorescence of the DNA-AgNCs was found to be quenched by Cu(2+) enabling its detection with high sensitivity and selectivity.     

Multiplexed analysis of silver(I) and mercury(II) ions using oligonucletide-metal nanoparticle conjugates

A colorimetric assay has been developed for the simultaneous selective detection of silver(I) and mercury(II) ions utilizing metal nanoparticles (NPs) as sensing element based on their unique surface plasmon resonance properties. In this method, sulfhydryl group modified cytosine-(C)-rich ssDNA (SH-C-ssDNA) was self-assembled on gold nanoparticles (AuNPs) to produce the AuNPs-C-ssDNA complex, and sulfhydryl group modified thymine-(T)-rich ssDNA (SH-T-ssDNA) was self-assembled on silver nanoparticles (AgNPs) to produce the AgNPs-T-ssDNA complex. Oligonucleotides (SH-C-ssDNA or SH-T-ssDNA) could enhance the AuNPs or AgNPs against salt-induced aggregation. However, the presence of silver(I) ions (Ag(+)) in the complex of ssDNA-AuNPs would reduce the stability of AuNPs due to the formation of Ag(+) mediated C-Ag(+)-C base pairs accompanied with the AuNPs color change from red to purple or even to dark blue. Moreover, the presence of mercury(II) ions (Hg(2+)) would also reduce the stability of AgNPs due to the formation of Hg(2+) mediated T-Hg(2+)-T base pairs accompanied with the AgNPs color change from yellow to brown, then to dark purple. The presence of both Ag(+) and Hg(2+) will reduce the stability of both AuNPs and AgNPs and cause the visible color change. As a result, Ag(+) and Hg(2+) could be detected qualitatively and quantitatively by the naked eye or by UV-vis spectral measurement. The lowest detectable concentration of a 5 nM mixture of Ag(+) and Hg(2+) in the river water was gotten by the UV-vis spectral measurement.     

Expression and Displaying of β-Glucosidase from Streptomyces Coelicolor A3 in Escherichia coli

Two genes encoding β-glucosidase from Streptomyces coelicolor A3(2) were cloned and expressed in Escherichia coli BL21 (DE3). Two recombinant enzymes (SC1059 and SC7558) were purified and characterized. The molecular mass of the purified SC1059 and SC7558 as determined by SDS-PAGE agrees with the calculated values (51.0 and 52.2 kDa, respectively). Optimal temperature and pH for the two enzymes were both at 35 °C and 6.0. SC7558 exhibited to be much more active than SC1059 under optimal conditions, and it was recombined with ice nucleation protein which could anchor on the surface of the cell. The optimal temperature and pH of the recombinant cells were 55 °C and 8.0, respectively. The resultant cells were to be used as material for immobilized β-glucosidase, which is convenient to catalyze substrates in various complicated conditions.