免标记光学纳米生物传感器的研究

利用发光稳定的多孔硅,当蛋白质溶液固定到多孔硅表面时,其发光强度和反射光谱的峰位会随着被测物质量浓度的变化而发生变化的特性。制备免标记光学纳米生物传感器来检测牛血清白蛋白（BSA）,把不同质量浓度的BSA溶液固定到多孔硅上测量反射光谱的峰位和荧光强度的变化。实验结果表明：反射光谱的峰位与牛血清白蛋白的浓度呈线性关系：y=1．4189x＋366．31,R^2=0．9898;荧光强度的降低量与BSA溶液的浓度呈线性关系,检测限为8×10^-9 mol／L。     

基于信号放大技术的适体生物传感器研究进展

信号放大技术因其能实现低浓度分子检测,灵敏度高而在多个研究领域发展非常迅速。而适体作为识别分子已成功应用于多种生物传感器平台,在医疗诊断、环境检测、生化分析中显示出良好的应用前景。近年来,以适体为识别元件的生物传感器越来越受到人们的关注。综述了近3年来基于信号放大技术的适体生物传感器研究新发展。     

基于纳米多孔硅Bragg反射镜免标记生物传感器的研究

构建一种基于多孔硅Bragg反光镜的免标记的纳米生物传感器,通过在纳米多孔硅上固定的适配子的特异性识别能力,检测溶液中四环素浓度。当适配子结合不同浓度的四环素溶液时,引起多孔硅Bragg反光镜内部有效折射率的变化,反射谱峰位进而发生变化。传感器的有效检测范围为1~200μg/L,检测限为1μg/L。     

信号适体的设计策略及应用

信号适体兼具有分子识别和信号转导的功能.从随机寡核苷酸库中筛选出的适体,要经过合理设计和筛选后修饰,才具备信号转导功能.信号适体可分为标记和非标记两大类.本文着重介绍荧光标记信号适体的设计策略,包括基于荧光偏振分析标记一个荧光基团,及基于荧光共振能量转移同时标记荧光基团、淬灭基团,或两个荧光基团的信号适体(包括分子信标适体、结构转换和原位标记信号适体).非标记信号适体的设计,有嵌合法、置换法、光转换复合物法,及适体－多聚物偶联法.此外,亦可直接从体外筛选出信号适体.信号适体的诸多优点利于其用于生物传感器及均相液相中实时蛋白识别与定量分析.     

核酸适体生物传感器快速检测牛奶中抗生素

为了现场快速检测牛奶溶液中四环素类抗生素的含量,对电极进行阳极氧化、氨基化、活化等一系列处理,将四环素核酸适体作为识别分子,共价固定于玻碳电极表面,构建出新型核酸适体生物传感器。传感器可以特异性结合标本中四环素类抗生素,并产生电化学信号,强度与抗生素浓度相关。其最低检测量是1μg/L,检测时间为5 m in。     

荧光纳米生物传感器研究进展

荧光纳米生物传感器检测物质具有灵敏度高、响应迅速、抗干扰性强、无需参比电极等特点而被广泛地运用于生物传感技术领域。本文综述了荧光纳米生物传感器种类和特点,介绍了国内外近期在荧光纳米生物传感器及在生物检测方面的一些研究成果及进展,并作了分析比较。着重讨论了纳米粒子荧光生物传感器和光纤纳米荧光生物传感器的特性及其在生物分析中的应用。     

橙色荧光蛋白的研究进展及其应用

荧光蛋白(Fluorescent protein,FPs)可作为探针用以探究细胞内分子间相互作用,追踪特定代谢物的代谢途径,对活细胞内的各种代谢过程和细胞通路进行详细、准确的描述。目前已有的FPs几乎已经覆盖了从紫外光到远红外光的所有光谱波段,这些FPs借助高分辨率显微技术应用于生命科学的诸多领域,为生物学的发展作出巨大贡献。橙色FPs通常指光谱区间在540–570nm的FPs,近几年来关于橙色FPs的研究进展较快,并且其作为标记蛋白以及荧光共振能量转移技术(Fluorescence resonance energy transfer,FRET)中的荧光受体在生物学及医学领域得到较多的应用。文中综述了近15年橙色FPs领域的相关研究,重点聚焦橙色FPs的发展和应用,为今后橙色FPs的研究提供依据。     

核酸适体的研究进展

近年的研究表明,DNA和RNA不仅起遗传信息储存和传递的作用,还可以藉自身形成的空间结构与其它类型的分子相互作用,以此为基础建立随机寡核苷酸文库,施加选择压力(结合靶目标),淘选与靶目标高度特异结合片段的过程,被称为指数富集的配体系统进化(Systematic evolution of ligand by exponential enrichment,SELEX)技术。筛选出的分子被称为核酸适体(aptamer)。Aptamer源于拉定语     

荧光标记mRNA差异显示技术

目的：应用荧光标记的ｍＢＮＡ差异显示技术。方法：提取未经过／经过ＩＦＮ－ＬＰＳ处理的三组人单核细胞系Ｕ９３７的总ＲＮＡ并以此为模板,采用荧光标记的锚定引物,通过逆转录、差异显示ＰＣＲ反应,经５．６％变性聚丙烯酰胺凝胶电泳分离差异条带,回收后将其再扩增。结果：三组样本的ＤＤ－ＰＣＲ产物电泳显示长３００ｂｐ￣２．０ｋｂ不等的扩增片段,条带清晰、明亮,背景低,各样本相互间的差异不仅呈有无的变化,亦表现出     

荧光标记寡核苷酸探针及其应用

寡核苷酸探针的标记非常重要。近年来 ,用荧光染料对探针进行非放射性标记受到很大重视 ,并取得了迅速发展 ,广泛应用于核酸序列测定、基因检测以及疾病诊断等。以下就寡核苷酸探针的荧光标记及其应用作一简要综述。     

荧光原位杂交技术的研究进展

荧光原位杂交(FISH)是在染色体、间期细胞核和DNA纤维上进行DNA序列定位的一种有效手段。近年来,围绕提高检测的分辨率和灵敏性,不断将免疫染色、量子点和微流控芯片等物理化学技术引入到荧光原位杂交中,促进了它的快速发展。本文主要综述了荧光原位杂交的基本原理和发展历程,重点介绍了免疫染色-荧光原位杂交(immuno-FISH)、量子点-荧光原位杂交(QD-FISH)和微流控芯片-荧光原位杂交(FISH on microchip)等多种新技术及其检测特点,如快速、灵敏、动态、多样化等。随着荧光原位杂交技术的不断完善与发展,将在细胞遗传学、表观遗传学及分子生物学等领域发挥更加重要的作用。     

Recent Advances in Super-Resolution Fluorescence Imaging and Its Applications in Biology

Fluorescence microscopy has become an essential tool for biological research because it can be minimally invasive, acquire data rapidly, and target molecules of interest with specific labeling strategies. However, the diffraction-limited spatial resolution, which is classically limited to about 200 nm in the lateral direction and about 500 nm in the axial direction, hampers its application to identify delicate details of subcellular structure. Extensive efforts have been made to break diffraction limit for obtaining high-resolution imaging of a biological specimen. Various methods capable of obtaining super-resolution images with a resolution of tens of nanometers are currently available. These super-resolution techniques can be generally divided into three primary classes： （1） patterned illumination- based super-resolution imaging, which employs spatially and temporally modulated illumination light to reconstruct sub-diffraction structures; （2） single-molecule localization-based super-resolution imaging, which localizes the profile center of each individual fluo- rophore at subdiffraction precision; （3） bleaching/blinking-based super-resolution imaging. These super-resolution techniques have been utilized in different biological fields and provide novel insights into several new aspects of life science. Given unique technical merits and commercial availability of super-resolution fluorescence microscope, increasing applications of this powerful technique in life science can be expected.     

Label-free quantification in clinical proteomics

Nowadays, proteomic studies no longer focus only on identifying as many proteins as possible in a given sample, but aiming for an accurate quantification of them. Especially in clinical proteomics, the investigation of variable protein expression profiles can yield useful information on pathological pathways or biomarkers and drug targets related to a particular disease. Over the time, many quantitative proteomic approaches have been established allowing researchers in the field of proteomics to refer to a comprehensive toolbox of different methodologies. In this review we will give an overview of different methods of quantitative proteomics with focus on label-free proteomics and its use in clinical proteomics.     

Label-free immunosensor based on gold nanoparticle silver enhancement

A label-free immunosensor for the sensitive detection of human immunoglobulin G (IgG) was prepared based on gold nanoparticle-silver enhancement detection with a simple charge-coupled device (CCD) detector. The gold nanoparticles, which were used as nuclei for the deposit of metallic silver and also for the adsorption of antibodies, were immobilized into wells of a 9-well chip. With the addition of silver enhancement buffer, metallic silver will deposit onto gold nanoparticles, causing darkness that can be optically measured by the CCD camera and quantified using ImageJ software. When antibody was immobilized onto the gold nanoparticles and antigen was captured, the formed immunocomplex resulted in a decrease of the darkness and the intensity of the darkness was in line with IgG concentrations from 0.05 to 10 ng/ml. The CCD detector is simple and portable, and the reported method has many desirable merits such as sensitivity and accuracy, making it a promising technique for protein detection.     

Label-free and amplified quantitation of proteins in complex mixtures using diffractive optics technology

Immunoaffinity approaches remain invaluable tools for characterization and quantitation of biopolymers. Their application in separation science is often limited due to the challenges of immunoassay development. Typical end-point immunoassays require time consuming and labor-intensive approaches for optimization. Real-time label-free analysis using diffractive optics technology (dot^®) helps guide a very effective iterative process for rapid immunoassay development. Both label-free and amplified approaches can be used throughout feasibility testing and ultimately in the final assay, providing a robust platform for biopolymer analysis over a very broad dynamic range. We demonstrate the use of dot in rapidly developing assays for quantitating (1) human IgG in complex media, (2) a fusion protein in production media and (3) protein A contamination in purified immunoglobulin preparations.     

Label-free and homogeneous DNA hybridization detection using gold nanoparticles-based chemiluiminescence system

We find that the catalytic activity of gold nanoparticles (GNPs) on luminol-H₂O₂ chemiluminescence (CL) system is greatly enhanced after it is aggregated by 0.5 M NaCl. We use this observation to design a CL detection of DNA hybridization. It is based on that the single- and double-stranded oligonucleotides have different propensities to adsorb on GNPs in colloidal solution, and the hybridization occurred between the probe DNA and target DNA can result in aggregation of the GNPs, producing strong CL emission. In the assay, no covalent functionalization of the GNPs, the probe, or the target DNA is required. The assay, including hybridization and detection, occurs in homogenous solution. The detection limit of target DNA (3σ) was estimated to be as low as 1.1 fM. The sensitivity was increased more than 6 orders of magnitude over that of GNPs-based colorimetric method. The present CL method for DNA hybridization detection offers the advantages of being simple, cheap, rapid and sensitive.     

Label-free quantitative proteomic analysis of the inhibitory activities of juglone against translation and energy metabolism in Escherichia coli

Plant-derived antimicrobial agents have received increasing attention owing to their potential to control pathogens and excellent efficacy despite the growing prevalence of antibiotic resistance. However, the antibacterial mechanism of juglone, a traditional medicine used to cure skin infections, is still unclear. Therefore, in this study, in order to elucidate the mechanisms underlying the antibacterial activity of juglone, label-free quantitative proteomic technology was applied for analysis of the 417 proteins that were differentially expressed in Escherichia coli after treatment with juglone at one-half of the minimum inhibitory concentration. Gene ontology enrichment analysis of differentially expressed proteins suggested that juglone effectively repressed the expression of dehydrogenase and cytochrome oxidase, indicating that energy generation was blocked. Additionally, juglone induced RNA formation and ribosome assembly, resulting in inhibition of translation. This is the first study to adopt a proteomic approach to investigate the antibacterial mechanism of action of juglone against E. coli.     

Screening of Potential Biomarkers for Gastric Cancer with Diagnostic Value Using Label-free Global Proteome Analysis

Gastric cancer (GC) is known as a top malignant type of tumors worldwide. Despite the recent decrease in mortality rates, the prognosis remains poor. Therefore, it is necessary to find novel biomarkers with early diagnostic value for GC. In this study, we present a large-scale proteomic analysis of 30 GC tissues and 30 matched healthy tissues using label-free global proteome profiling. Our results identified 537 differentially expressed proteins, including 280 upregulated and 257 downregulated proteins. The ingenuity pathway analysis (IPA) results indicated that the sirtuin signaling pathway was the most activated pathway in GC tissues whereas oxidative phosphorylation was the most inhibited. Moreover, the most activated molecular function was cellular movement, including tissue invasion by tumor cell lines. Based on IPA results, 15 hub proteins were screened. Using the receiver operating characteristic curve, most of hub proteins showed a high diagnostic power in distinguishing between tumors and healthy controls. A four-protein (ATP5B-ATP5O-NDUFB4-NDUFB8) diagnostic signature was built using a random forest model. The area under the curve (AUC) values of this model were 0.996 and 0.886 for the training and testing sets, respectively, suggesting that the four-protein signature has a high diagnostic power. This signature was further tested with independent datasets using plasma enzyme-linked immune sorbent assays, resulting in an AUC value of 0.778 for distinguishing GC tissues from healthy controls, and using immunohistochemical tissue microarray analysis, resulting in an AUC value of 0.805. In conclusion, this study identifies potential biomarkers and improves our understanding of the pathogenesis, providing novel therapeutic targets for GC.     

Label-free THz sensing of living body-related molecular binding using a metallic mesh

We have demonstrated label-free THz sensing of living body-related molecular binding using a thin metallic mesh and a polyvinylidene difluoride (PVDF) membrane. Metallic meshes in the THz region are designed for anomalous transmission phenomena derived from a resonant excitation of surface waves. Additionally, they are designed to have a sharp dip in transmittance. The metallic mesh is very sensitive to a change of the refractive index of materials attached to the metallic mesh. In this paper, we report sensing of interactions between lectin and sugar using this technique. We found that the dip frequency shift, transmittance attenuation of the dip frequency, and peak shift of the derivative spectrum of the phase shift depend on the bonding amount of lectin–sugar interactions. We also applied this technique to detect avidin–biotin interactions, leading to the detection of a small amount of biotin (0.17 pg/mm²).