基于分子信标荧光纳米探针的李斯特菌DNA均相检测方法

基于分子信标(MB)识别和荧光纳米粒子探针技术,建立了均相体系中李斯特菌目标DNA的高灵敏检测新方法.首先以羊抗人免疫球蛋白(IgG)标记的异硫氰酸荧光素(FITC)为核材料,成功制备了FITC-IgG@SiO2核壳荧光纳米粒子,有效防止了传统方法中采用单一FITC制备纳米颗粒时泄露严重的问题.随后以FITC-IgG@SiO2荧光纳米粒子和纳米金分别标记单核细胞增生李斯特菌序列特异性分子信标探针5'端和3'端,成功构建了单核细胞增生李斯特菌序列特异性分子信标荧光纳米探针.在实验优化条件下,α(令α=F/F0,F代表MB和目标DNA杂交以后的荧光强度,F0代表MB完全闭合时的荧光强度)与目标DNA浓度在1～200pmol/L浓度范围内呈良好的线性关系,检出下限为0.3pmol/L,相对标准偏差为2.6%(50pmol/L,n=11).将该方法应用于食品样品中单核细胞增生李斯特菌的检测,结果与国标法一致.     

2能级模型处理长脉冲圆偏振光与钠原子相互作用的合理性

采用密度矩阵方法来处理长脉冲光与钠原子的相互作用,求解24能级钠精细结构的布洛赫方程,给出参与跃迁的每个态密度随时间的演化曲线。对于圆偏振情形,发现在长脉冲(大于100 ns)的持续时间内,最终的原子通过转移只在3S1/2(2,2)到3P/3/2(3,3)之间进行泵浦并且激发态达到稳态。进一步的2能级模型计算与24能级计算结果的对比表明：用2能级近似的速率方程方法取代24能级密度矩阵方法具有合理性,由此简化了模型,节约了后续研究的计算量。     

激光钠信标光斑漂移和光斑半径的数值模拟与分析

激光钠信标的光斑漂移、光斑半径的变化对自适应光学校正有直接的影响。在激光钠信标光斑漂移方差和有效半径理论模型的基础上,采用数值模拟的方法,在三种大气湍流模式下,具体研究了激光钠信标光斑与激光光斑漂移方差和有效半径的差异,计算了不同发射口径时激光钠信标光斑的长曝光与短曝光有效半径的平均值,分析了有再泵浦能量激光对激发钠信标光斑漂移方差和平均有效半径的影响。研究结果表明,大气湍流强度、激光发射口径以及再泵浦激光能量都能够影响激光钠信标光斑漂移和光斑半径。     

无线传感器网络中基于RSSI的质心定位算法研究

无线传感器网络中,由于传统质心算法普遍存在信标节点分布不均与中心化问题,导致定位误差相对较大。针对这些问题,提出了基于RSSI的改进算法。在APIT的基础上,改进算法依靠未知节点接收到不同信标节点的RSSI数值,判断其周围是否存在最佳三角形,若存在则利用最佳三角形进行定位;若不存在则选出一个距其较近的三角形,利用移动信标节点的办法来缩小此三角形的范围进行定位。Matlab平台仿真结果表明,与传统质心算法相比,改进算法减少了定位误差,节点定位精度有所提高。     

Label-free fluorescent molecular beacon based on a small fluorescent molecule non-covalently bound to the intentional gap site in the stem moiety

A label-free fluorescent molecular beacon (MB) based on a fluorescent molecule, 5,6,7-trimethyl-1,8-naphthyridin-2-ylamine (ATMND) which is non-covalently bound to the intentional gap site in the stem moiety of the label-free MB, was developed. In the absence of a cDNA, ATMND fluorescence is significantly quenched because it binds to the unpaired cytosine at the gap site by hydrogen bonding. As a result, the label-free MB shows almost no fluorescence. Upon hybridization with cDNA, the label-free MB undergoes a conformational change to destroy the gap site. This results in an effective fluorescent enhancement because of the release of the ATMND from the gap site to the solution. Fluorescence titration shows that ATMND strongly binds to the cytosine at the gap site (K₁₁ > 10⁶). Circular-dichroism spectroscopy indicates that the binding of ATMND at the gap site of the stem moiety does not induce a significant conformational change to the hairpin DNA. Under optimal conditions, the fluorescent intensity of the label-free MB increases with an increase in cDNA concentration from 50 nM to 1.5 μM. A detection limit of 20 nM cDNA was achieved. A single mismatched target ss-DNA can be effectively discriminated from cDNA. The advantage of the label-free MB is that both its ends can be left free to introduce other useful functionalities. In addition, the label-free MB synthesis introduced in this paper is relatively simple and inexpensive because no label is required.     

无线传感器网络自身定位方法研究与实现

作为一种全新的信息获取和处理平台,无线传感器网络能在应用领域内实现复杂的大范围监测和跟踪任务,因而网络中的节点自身定位就显得尤为重要.而现有节点自身定位算法大多数建立于节点布署于同一平面的理想状态,与实际的被监测区域有一定的差距.因此提出一种改进算法──补偿系数算法,同时在三边测量法中使用了坐标误差最小的最优解──最小二乘解来提高无线传感器网络节点自身定位的准确程度以满足实际应用的需要.     

Probing DNA Hybridization in Homogeneous Solution and at Interfaces via Measurement of the Intrinsic Fluorescence Decay Time of a Single Label

The hybridization of DNA oligomers including molecular beacons can be detected by measurement of either the decay time or the intensity of a single fluorescent label attached to the end of the respective oligonucleotide. The method works both in solution and solid phase and can distinguish between fully complementary and mismatch sequences as demonstrated for a 15-mer oligonucleotide and a 25-mer molecular beacon. The fluorescence lifetime method is advantageous in (a) requiring a single label (and therefore a single labeling step) only; and (b), being based on measurement of a self-referenced magnitude that is hardly affected by parameters such as fluctuations in light intensity that make measurement of intensity more prone to interferences.     

Mechanism of Cellular Uptake to Optimized AuNP Beacon for Tracing mRNA Changes in Living Cells

Molecular beacon is a promising tool for mRNA detection in living cells. But the low detecting efficiency and narrow application range limited its development. In this study, we synthesized a novel gold nanoparticle (AuNP) beacon by optimizing the sequence amount and modified polyethylene glycol (PEG) and cell‐penetrating peptide (CPP) on the gold core. Then, the mechanism of beacon cell uptake was investigated. Lastly, we used the AuNP beacon to study the Akt‐mTOR‐HIF‐1 signaling pathway and the function and mechanism of miR‐7 in breast tumor cells. The results showed that the optimization obviously amplified the fluorescence signal of the AuNP beacon. The mechanism study described the process of AuNP beacon cellular uptake and confirmed amplifying the amount of beacon cellular uptake could obviously enhance the fluorescence signal. Compared to results, the accuracy of the gold nanoparticle beacon is similar to the results of real‐time‐Q‐PCR (RT‐PCR) and western blotting but that the operation is much simpler. Furthermore, in this study, we found that our Akt gold nanoparticle beacon had a similar function to that of the Akt small interfering RNA (siRNA). In summary, the gold nanoparticle beacon may be a promising method for the study of signaling pathways.     

Several concerns about the primer design in the universal molecular beacon real-time PCR assay and its application in HBV DNA detection

A universal hepatitis B virus (HBV) DNA detection kit is appealing for the worldwide diagnosis and monitoring of the treatment of different mutant types of hepatitis B virus. A sensitive and reproducible real-time PCR assay based on the universal molecular beacon (U-MB) technique was developed for the detection of HBV DNA in serum. The U-MB probe used in the assay has no interaction with the HBV DNA sequence. The U-MB technique not only reduced the cost of HBV detection but also had the potential for the development of a universal detection kit for different mutant HBV types and other DNA systems. To demonstrate its clinical utility, 90 serum samples were analyzed using the U-MB real-time PCR method. In the experiments we found that several crucial factors needed to be considered in the primer design, such as the avoidance of formation of severe primer–dimer and primer self-hairpin structure. With the optimized primer sets, satisfactory results were obtained for all the tested samples. We concluded that this assay would be an excellent candidate for a universal HBV DNA detection method. Principle of the U-MB real-time PCR method for HBV DNAdetection     

Fast and sensitive DNA analysis using changes in the FRET signals of molecular beacons in a PDMS microfluidic channel

A new DNA hybridization analytical method using a microfluidic channel and a molecular beacon-based probe (MB-probe) is described. A stem-loop DNA oligonucleotide labeled with two fluorophores at the 5′ and 3′ termini (a donor dye, TET, and an acceptor dye, TAMRA, respectively) was used to carry out a fast and sensitive DNA analysis. The MB-probe utilized the specificity and selectivity of the DNA hairpin-type probe DNA to detect a specific target DNA of interest. The quenching of the fluorescence resonance energy transfer (FRET) signal between the two fluorophores, caused by the sequence-specific hybridization of the MB-probe and the target DNA, was used to detect a DNA hybridization reaction in a poly(dimethylsiloxane) (PDMS) microfluidic channel. The azoospermia gene, DYS 209, was used as the target DNA to demonstrate the applicability of the method. A simple syringe pumping system was used for quick and accurate analysis. The laminar flow along the channel could be easily controlled by the 3-D channel structure and flow speed. By injecting the MB-probe and target DNA solutions into a zigzag-shaped PDMS microfluidic channel, it was possible to detect their sequence-specific hybridization. Surface-enhanced Raman spectroscopy (SERS) was also used to provide complementary evidence of the DNA hybridization. Our data show that this technique is a promising real-time detection method for label-free DNA targets in the solution phase. Figure FRET-based DNA hybridization detection using a molecular beacon in a zigzag-shaped PDMS microfluidic channel