Exosome enrichment of human serum using multiple cycles of centrifugation

In this work, we compared the use of repeated cycles of centrifugation at conventional speeds for enrichment of exosomes from human serum compared to the use of ultracentrifugation (UC). After removal of cells and cell debris, a speed of 110 000 × g or 40 000 × g was used for the UC or centrifugation enrichment process, respectively. The enriched exosomes were analyzed using the bicinchoninic acid assay, 1D gel separation, transmission electron microscopy, Western blotting, and high‐resolution LC‐MS/MS analysis. It was found that a five‐cycle repetition of UC or centrifugation is necessary for successful removal of nonexosomal proteins in the enrichment of exosomes from human serum. More significantly, 5× centrifugation enrichment was found to provide similar or better performance than 5× UC enrichment in terms of enriched exosome protein amount, Western blot band intensity for detection of CD‐63, and numbers of identified exosome‐related proteins and cluster of differentiation (CD) proteins. A total of 478 proteins were identified in the LC‐MS/MS analyses of exosome proteins obtained from 5× UCs and 5× centrifugations including many important CD membrane proteins. The presence of previously reported exosome‐related proteins including key exosome protein markers demonstrates the utility of this method for analysis of proteins in human serum.     

Higher detectability method for the analysis of nucleosides,putative tumor biomarkers,in blood serum samples by CE‐UV with reversed EOF

The development and validation of methodologies for the analysis of biological samples is of outcome importance in order to obtain trustworthy results. This work reports a novel CE‐UV method for the assessment of nucleosides, putative tumor biomarkers, in blood serum. The separation of seven nucleosides within c.a. 20 min has been achieved with: BGE 30 mmol/L borate at pH 9.90, 50 mmol/L CTAB, and 10% methanol; V = –10 kV; T = 20°C; and capillary dimensions of 56 cm × 50 μm. The sample plug was concentrated by a modified large volume sample stacking strategy that provided better detectability. Validation showed that the method is suitable for bioanalytical purposes and initial applications in serum samples from healthy subjects are also presented. Finally, statistical methods were applied to verify the effect of characteristics such as age, smoking habits, and alcohol consumption on nucleoside concentrations in blood serum. Univariate statistical analysis tests emphasized the need for age matching, which was confirmed by PCA‐DA and PLS‐DA. Cancer history in the nearby family may also interfere in nucleoside levels in blood serum, since adenosine concentrations were statistically higher for volunteers who declared having diseased relatives.     

水溶性阳离子型吡啶基咔咯镓配合物与人血清蛋白的相互作用

利用紫外吸收光谱、荧光光谱、圆二色(CD)光谱和分子对接计算探究了5,10,15-三[4-(N-甲基-吡啶)]咔咯镓配合物(1-Ga)与人血清蛋白(HSA)的相互作用.结果表明,HSA的荧光能被1-Ga静态猝灭,两者的结合常数为2.82×104L/mol,作用距离为3.342 nm.热力学参数显示1-Ga主要通过氢键和疏水作用与HSA结合,位点标记竞争实验表明1-Ga优先结合HSA的布洛芬位点Ⅱ.此外,紫外吸收光谱和CD光谱显示二者的相互作用会导致HSAα-螺旋结构的减少.分子对接计算结果表明1-Ga优先结合在HSA亚结构域ⅢA的位点Ⅱ疏水袋中.     

Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins

Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag–BirA system. Through the high streptavidin (SA)–biotin interaction, the divalent biotinylated APs were clustered in the SA–biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ–AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for the ZZ protein.     

Preparation of poly(3,4-ethylenedioxythiophene) nanofibers modified pencil graphite electrode and investigation of over-oxidation conditions for the selective and sensitive determination of uric acid in body fluids

In this study, we have performed the preparation of over-oxidized poly(3,4-ethylenedioxythiophene) nanofibers modified pencil graphite electrode (Ox-PEDOT-nf/PGE) to develop a selective and sensitive voltammetric uric acid (UA) sensor. It was noted that the over-oxidation potential and time had a prominent effect on the UA response of the Ox-PEDOT-nf/PGE. Characterizations of PEDOT-nf/PGE and Ox-PEDOT-nf/PGE have been performed by cyclic voltammetry, electrochemical impedance spectroscopy, scanning electron microscopy, Fourier transform infrared spectroscopy and Raman spectroscopy. The highest voltammetric response of UA was obtained at pH 2.0. A linear relationship between the concentration of UA and oxidation peak currents was observed in the concentration range of 0.01–20.0 μM. The detection limit (1.3 nM according to S/N = 3) and reproducibility (RSD: 4.6 % for N:10) have also been determined. The effects of different substances on the determination of UA have been investigated. A very high peak separation value of 423 mV was obtained between UA and ascorbic acid which is the major interfering substance for UA. The use of Ox-PEDOT-nf/PGE has been successfully tested in the determination of UA in human blood serum and urine samples for the first time in the literature.     

Carbon dots as a fluorescent probe for label-free detection of physiological potassium level in human serum and red blood cells

A unique photoluminescence carbon dots (CDs) with larger size were prepared by microwave-assisted method. Complex functional groups on the surface of the CDs facilitate the nanoparticles to form affinity with some metal ions. Taking advantage of the effective fluorescence quenching effect of K⁺, a highly sensitive CD-based fluorescence analytical system for label-free detection of K⁺ with limit of detection (LOD) 1.0 × 10⁻¹² M was established. The concentrations of potassium ion in biological samples such as human serum are usually found at millimolar levels or even higher. The proposed method begins with a substantial dilution of the sample to place the K⁺ concentration in the dynamic range for quantification, which covers 3 orders of magnitude. This offers some advantages: the detection of K⁺ only needs very small quantities of biological samples, and the dilution of samples such as serum may effectively eliminate the potential interferences that often originate from the background matrix. The determined potassium levels were satisfactory and closely comparable with the results given by the hospital, indicating that this fluorescent probe is applicable to detection of physiological potassium level with high accuracy. Compared with other relative biosensors requiring modified design, bio-molecular modification or/and sophisticated instruments, this CD-based sensor is very simple, cost-effective and easy detection, suggesting great potential applications for successively monitoring physiological potassium level and the change in biological system.     

刷状结构的阳离子性接枝微粒QPDMAEMA/SiO2对牛血清白蛋白的吸附特性

以微米级硅胶微粒为基质, 通过接枝聚合和大分子反应, 制备了具有刷状结构的阳离子性接枝微粒, 深入研究了其对牛血清白蛋白(BSA)的强吸附能力、吸附机理和吸附热力学. 首先使含叔胺基团的单体甲基丙烯酸二甲基氨基乙酯(DMAEMA)在硅胶微粒表面发生接枝聚合, 制得接枝微粒PDMAEMA/SiO₂, 然后以氯乙胺为试剂, 使接枝大分子PDMAEMA链中的叔胺基团发生季铵化反应, 获得了具有刷状结构的阳离子聚电解质的功能接枝微粒QPDMAEMA/SiO₂. 测定了微粒QPDMAEMA/SiO₂的zeta 电位, 实施了对BSA的等温吸附实验, 考察了介质pH值、离子强度及温度对吸附作用的影响, 研究了吸附热力学. 研究结果表明, 功能接枝微粒QPDMAEMA/SiO₂ 比接枝微粒PDMAEMA/SiO₂ 具有更高的zeta 电位, 在静电相互作用驱动下, 微粒QPDMAEMA/SiO₂对BSA具有很强的吸附能力. 吸附容量随介质pH值的增大呈现先增大后减小的变化趋势,当pH值等于BSA的等电点(pI=4.7)时, 具有最高的吸附容量(高达112 mg·g^-1). 以等电点为界, 离子强度对吸附容量会产生完全相反的影响作用: 当介质pH值小于BSA的等电点时, 电解质浓度增大, 吸附容量增高; 当介质pH值等于BSA的等电点时, 吸附容量几乎不随电解质的浓度发生变化. 吸附过程熵值减小而且放出热量,是一个焓驱动的吸附过程.     

大孔型疏水整体柱的制备及其表征

利用液体致孔剂正庚烷,将双甲基丙烯酸乙二醇酯(EDMA)和甲基丙烯酸缩水甘油酯(GMA)聚合成含有环氧基团的大孔型整体柱,用正丁胺修饰制备成疏水性整体柱。压汞法分析表明,疏水整体柱的孔隙率为60.2%,500nm以上的大孔占疏水整体柱孔隙率的65.7%。在2890cm/h的高流速下,疏水整体柱的背压只有10.9MPa。以牛血清白蛋白(BSA)为研究对象,动态吸附容量为14.4mg/g介质,在一定范围内,分离效果不受操作流速的影响。此外,在1445cm/h的流速下,3min内即可对细胞色素C、RNase A、溶菌酶和鸡卵清蛋白进行基线分离。结果表明,大孔型疏水整体柱可用于蛋白质的快速分离和分析。     

黄芩苷与人血清白蛋白的相互作用研究

利用紫外光谱、荧光光谱、傅立叶红外谱、圆二色谱及分子模型等技术,在生理pH条件下,研究了黄芩苷与人血清白蛋白(HSA)的相互作用,并计算了其结合常数和热力学参数.分子模型研究表明,黄芩苷与HSA在亚结构域ⅡA结合,二者间的作用主要为静电作用和疏水作用,与荧光光谱结果基本一致.红外光谱和圆二色谱显示黄芩苷与HSA结合后未...     

光谱法研究5-羟甲基糠醛与人血清白蛋白的相互作用

在模拟人体生理条件下,用荧光光谱法和紫外光谱法研究5-羟甲基糠醛(5-HMF)和人血清白蛋白(HSA)结合反应的特征,并利用同步荧光法和三维荧光法研究5-HMF与人血清白蛋白作用前后白蛋白的构象变化.研究表明:5-HMF与人血清白蛋白形成复合物,从而猝灭人血清白蛋白的内源性荧光,该过程为静态猝灭过程.根梧Stern-V...