A fluorescence enhanced probe for sulfur dioxide detection and fluorescence imaging in living cellsEI北大核心CSCD

基于香豆素和苯并吡啶基团,构建了用于二氧化硫(SO_(2))高效检测的荧光探针P1,其化学结构通过核磁氢谱(^(1)H NMR)、碳谱(^(13)C NMR)和高分辨质谱(HR-MS)确证。在缓冲溶液体系中,单独的探针P1具有微弱的荧光,识别SO_(2)后荧光发射强度明显增强,能够实现对SO_(2)的专一性裸眼识别,检出限为126 nmol/L。生物应用实验结果表明,该探针具有较低的细胞毒性,可用于生物活细胞中外源性SO_(2)的荧光成像。     

Comparison of two liquid-state NMR methods for the determination of saccharides in carrot (<Emphasis Type="Italic">Daucus carota</Emphasis> L.) roots

In order to determine the saccharide content of plant tissues, we studied a new non-destructive and fast analytical method that we call “direct quantitative proton nuclear magnetic resonance spectroscopy” (d q ¹H NMR): the application of quantitative proton nuclear magnetic resonance spectroscopy (q ¹H NMR) to non modified plant tissues along with capillary tubes containing a reference compound (for quantification) and deuterium oxide (for locking). Using this method, the saccharide content of samples of carrot (Daucus carota L.) roots was compared to the results given from similar samples by the formerly published q ¹H NMR determination of extracts obtained by the O'Donoghue/Davis method. The content in glucose and sucrose is significantly higher with the direct method than when an extraction step is included; the content in fructose is not significantly different. If a possible detection of saccharides included in glycosylated biological compounds is to be excluded, a more complete extraction of saccharides is probably obtained using the direct method.     

Analysis of estrogens in water by magnetic octadecylsilane particles extraction and sweeping micellar electrokinetic chromatography

A method based on magnetic separation was developed for the extraction of several estrogens (including diethylstilbestrol, estrone and estriol) in water followed by sweeping micellar electrokinetic chromatography (MEKC) analysis with UV detection. Novel magnetic octadecylsilane (ODS) particles were prepared using a silanization method with octadecyl trimethoxysilane as the surface modification reagent of magnetic Fe₃O₄ particles. Octadecyl trimethoxysilane was covalently immobilized on the magnetic iron oxide particles. The particles were used as the sorbents in the magnetic separation for the extraction of trace amounts of estrogens from water. The extraction condition and efficiency of the particles for the estrogens were investigated. Combining the magnetic ODS particles extraction and sweeping MEKC with UV detection, the estrogens at concentrations as low as ng/mL in water can be detected without interference from other substances in the sample matrix.     

Quadrupole central transition 17O NMR spectroscopy of biological macromolecules in aqueous solution

We demonstrate a general nuclear magnetic resonance (NMR) spectroscopic approach in obtaining high-resolution (17)O (spin-5/2) NMR spectra for biological macromolecules in aqueous solution. This approach, termed quadrupole central transition (QCT) NMR, is based on the multiexponential relaxation properties of half-integer quadrupolar nuclei in molecules undergoing slow isotropic tumbling motion. Under such a circumstance, Redfield's relaxation theory predicts that the central transition, m(I) = +1/2 ? -1/2, can exhibit relatively long transverse relaxation time constants, thus giving rise to relatively narrow spectral lines. Using three robust protein-ligand complexes of size ranging from 65 to 240 kDa, we have obtained (17)O QCT NMR spectra with unprecedented resolution, allowing the chemical environment around the targeted oxygen atoms to be directly probed for the first time. The new QCT approach increases the size limit of molecular systems previously attainable by solution (17)O NMR by nearly 3 orders of magnitude (1000-fold). We have also shown that, when both quadrupole and shielding anisotropy interactions are operative, (17)O QCT NMR spectra display an analogous transverse relaxation optimized spectroscopy type behavior in that the condition for optimal resolution depends on the applied magnetic field. We conclude that, with the currently available moderate and ultrahigh magnetic fields (14 T and higher), this (17)O QCT NMR approach is applicable to a wide variety of biological macromolecules. The new (17)O NMR parameters so obtained for biological molecules are complementary to those obtained from (1)H, (13)C, and (15)N NMR studies.     

Preparation and application of streptavidin magnetic particles

Two kinds of streptavidin magnetic particles,namely streptavidin GoldMag particles and streptavidin amino terminal particles were prepared by the methods of physical adsorption and covalent interaction respectively.The streptavidin coated on magnetic particle surface,crucial to many applications,was greatly influenced by the choice of the different buffer.Compared with DynalbeadsM-270 streptavidin, the binding capacity for biotin of different streptavidin magnetic particles was determined by enzyme inhibition method,and the coupling capacity and activity of biotinylated oligonucleotide on their sur- face were also analyzed.The results indicated that the streptavidin GoldMag particle prepared by physical adsorption was stable in STE(NaCl-Tris-EDTA)buffer that was frequently used in nucleic acid hybridization and detection.The streptavidin amino terminal particles prepared by covalent interaction could be used both in STE buffer and PBS(phosphate buffered saline)buffer.The biotin binding ca- pacity for 1 mg of streptavidin GoldMag particles and streptavidin amino terminal particles was 4950 and 5115 pmol respectively.The capacity of biotinylated oligonucleotide(24 bp)coupled on 1 mg of GoldMag and amino terminal magnetic particles was 2839 and 2978 pmol separately.These data were about 6-7 times higher than those of DynabeadsM-270 streptavidin.The hybridization results with FITC-labeled complementary probe on magnetic particle surface demonstrated that the oligonucleotide coupled on streptavidin magnetic particles had high biological activity.     

Use of high-performance size-exclusion chromatography for the separation of poliovirus and subviral particles

The size-dependent separation of viral and subviral particles in the range 10(5)-10(7) daltons was undertaken by high-performance liquid chromatography. A combination of Ultrahydrogel 2000 and 1000 size-exclusion columns, equilibrated and developed with Tris buffer (pH 7.4), was used to fractionate extracts of cells infected with radiolabelled poliovirus. Poliovirions (30 nm) and subviral particles (20 nm) were separated according to size with full retention to their biological activities. Procapsids (same size as virions, but devoid of RNA) could not be separated from virions. Sample recoveries as determined with radiolabelled material constantly exceeded 70%. The method was successfully applied to the separation of viral and subviral particles from complex mixtures.     

Signal-enhanced real-time magnetic resonance of enzymatic reactions at millitesla fields

The phenomenon of nuclear magnetic resonance (NMR) is widely applied in biomedical and biological science to study structures and dynamics of proteins and their reactions. Despite its impact, NMR is an inherently insensitive phenomenon and has driven the field to construct spectrometers with increasingly higher magnetic fields leading to more detection sensitivity. Here, we are demonstrating that enzymatic reactions can be followed in real-time at millitesla fields, three orders of magnitude lower than the field of state-of-the-art NMR spectrometers. This requires signal-enhancing samples via hyperpolarization. Within seconds, we have enhanced the signals of 2-¹³C-pyruvate, an important metabolite to probe cancer metabolism, in 22 mM concentrations (up to 10.1% ± 0.1% polarization) and show that such a large signal allows for the real-time detection of enzymatic conversion of pyruvate to lactate at 24 mT. This development paves the pathways for biological studies in portable and affordable NMR systems with a potential for medical diagnostics.

We demonstrate that metabolism can be monitored in real-time with magnetic resonance at milli-tesla fields that are 1000 fold lower than state-of-the-art high field spectrometers.     

Bio-functionalization of monodisperse magnetic nanoparticles and their use as biomolecular labels in a magnetic tunnel junction based sensor

Monodisperse magnetic nanoparticles (NPs) could enable the ultra-sensitive magnetic detection of biological analytes. However, rendering these particles biocompatible has remained a challenge. We report the bio-functionalization and detection of 12-nm manganese ferrite NPs. We have achieved the site-specific binding of biotin-functionalized NPs onto avidin-patterned silicon oxide substrates and DNA-functionalized NPs onto complementary DNA-patterned silicon oxide substrates. Utilizing scanning SQUID microscopy, we show that these substrate-bound NPs retain their magnetic properties. Finally, we demonstrate a novel method of detecting either protein binding or DNA hybridization at room temperature using the NPs and a magnetic tunnel-junction-based biosensor situated in orthogonal magnetic fields.     

Monitoring viral DNA release with capillary electrophoresis

Viral DNA injection into host cells is one of the primary mechanisms of viral propagation. Drug development that targets viral propagation requires fast and sensitive methods for monitoring the release of viral DNA in vitro. Here we demonstrate the use of capillary electrophoresis (CE) for monitoring DNA release from virus particles. As a model for this study, we used T5 bacteriophages that infect the bacterium Escherichia coli K-12 by binding to the outer membrane FhuA receptor and then injecting DNA. DNA release from the T5 phages in vitro was induced by either elevated temperature or by interaction with the purified FhuA receptor. After DNA release, the viral samples were stained with the high affinity fluorescent dye YOYO-1, injected into the capillary and subjected to electrophoresis. YOYO-1-stained DNA generated a well-defined peak, allowing reliable detection of viral DNA from as few as 10(5) viral particles. The staining to track T5 phage DNA release exemplifies the great versatility that CE offers in studying viral systems. This CE-based method can be used to study molecular mechanisms of viral infections and to evaluate anti-viral drug candidates.     

Synthesis and Antifungal Activities of Novel Strobilurin Derivatives Containing Quinolin-2(1H)-one Moiety

To discover novel lead compounds with better antifungal activities, a series of novel strobilurin derivatives containing quinolin-2(1H)-one moiety was designed and synthesized via intermediate derivatization method. Their structures were characterized by means of ¹H nuclear magnetic resonance(¹H NMR), ¹³C NMR and high resolution mass spectrometry(HRMS). The biological assay results indicate that most target compounds exhibit good to excellent fungicidal activities against 10 plant pathogens. Compounds 4d, 5b and 5c possess 94.1%, 83.8% and 80.9% in vitro inhibition respectively against Rhizotonia cereals at the concentration of 50 μg/mL, which are better than that of the control agents. Especially, the inhibition activities of compound 4d against all of the tested fungi approach or exceed those of the controls. The structure-activity relationship was also discussed.     

Photochemical decoration of silver nanoparticles on magnetic microspheres as substrates for the detection of adenine by surface-enhanced Raman scattering

In this work, silver nanoparticles (AgNPs) decorated magnetic microspheres (MMs) are prepared as surface-enhanced Raman scattering (SERS) substrate for the analysis of adenine in aqueous solutions. To prepare these substrates, magnetic particles were first synthesized by coprecipitation of Fe(II) and Fe(III) with ammonium hydroxide. A thin layer of cross-linked polymer was formed on these magnetic particles by polymerization through suspension of magnetic particles into a solution of divinyl benzene/methyl methacrylate. The resulted polymer protected magnetic particles are round in shape with a size of 80 μm in diameter. To form AgNPs on these MMs, photochemical reduction method was employed and the factors in photochemical reduction method were studied and optimized for the preparation of highly sensitive and stable AgNPs on MMs substrates (abbreviated as AgMMs substrates). By dispersing the AgMMs in aqueous samples, cylindrical magnet was used to attract the AgMMs for SERS detections. The observed enhancement factor of AgMMs reached 7 orders in magnitude for detection of adenine with a detection limit approaching to few hundreds of nanomolar.     

多孔磁性硅胶微球的制备及其在基因组脱氧核糖核酸提取中的应用

采用模板法制备了多孔磁性硅胶微球,用于生物样品中基因组脱氧核糖核酸(DNA)的分离纯化。以球形和无定型硅胶为对照,考察了吸附液组成和洗脱时间等实验参数对小牛胸腺基因组DNA在磁性硅胶固相载体上的提取回收率的影响。实验结果表明:20%(W/V)聚乙二醇和2 mol/L氯化钠,洗脱10 m in,DNA的回收率可达80%;采用简单的细胞裂解体系和合适的吸附液组成,磁性微球应用于酿酒酵母中基因组DNA的提取,得到了平均长度约为5 kb、A260/A280大于1.77的高纯度DNA片段。     

Magnetism and application of NiFe@Au nanoparticles

This report describes the synthesis of magnetic NiFe@Au (i.e., NiFe core with Au shell) nanoparticles as functional spectroscopic probes. Both of the magnetic NiFe nanoparticles and its composite NiFe@Au particles were synthesized in aqueous solution. It is more analogous with the biological organism environment. The composite nanoparticles were dispersible in aqueous solution and could be directed by a magnetic field. Such NiFe@Au nanoparticles have been shown to function as magnetic and spectroscopic nanoprobes for surface enhanced Raman scattering (SERS) detection of molecules attached to the surface of the nanoparticles. It shows more potential functional SERS nanoprobes for biomolecular separation and detection.     

An electrochemical immunosensor for carcinoembryonic antigen enhanced by self-assembled nanogold coatings on magnetic particles

A quick and reproducible electrochemical-based immunosensor technique, using magnetic core/shell particles that are coated with self-assembled multilayer of nanogold, has been developed. Magnetic particles that are structured from Au/Fe₃O₄ core-shells were prepared and aminated after a reaction between gold and thiourea, and additional multilayered coatings of gold nanoparticles were assembled on the surface of the core/shell particles. The carcinoembryonic antibody (anti-CEA) was immobilized on the modified magnetic particles, which were then attached on the surface of solid paraffin carbon paste electrode (SPCE) by an external magnetic field. This is an assembly of a novel immuno biosensor for carcinoembryonic antigen (CEA). The sensitivity and response features of this immunoassay are significantly affected by the surface area and the biological compatibility of the multilayered nanogold. The linear range for the detection of CEA was from 0.005 to 50 ng mL⁻¹ and the limit of detection (LOD) was 0.001 ng mL⁻¹. The LOD is approximately 500 times more sensitive than that of the traditional enzyme-linked immunosorbent assay for CEA detection.     

一种香豆素类锌离子荧光探针的合成及性能

通过Pechmann法使间苯二酚和乙酰乙酸乙酯发生脱水缩合反应,合成了4-甲基-7-羟基香豆素,再与六次甲基四胺反应,生成7-羟基-4-甲基-8-甲酰基香豆素,最终与吲哚酰肼发生席夫碱反应合成一种香豆素类荧光探针L。通过核磁共振波谱仪(NMR)、质谱(MS)、傅里叶变换红外光谱仪(FTIR)、荧光发射光谱仪和紫外可见分光光度计等技术手段研究该探针L的结构及其荧光性能。结果表明,探针L与Zn~(2+)的配合比为1∶1,对Zn~(2+)的检测限达到3.6×10~(-8)mol/L,降低了对Zn~(2+)的检测浓度,同时探针L对锌离子具有高选择性,可应用于在生物体内Zn~(2+)的检测。     

Old commercialized magnetic particles new trick: Intrinsic internal standard

Magnetic particles（MPs） are the most widely used commercialized engineering particles,which gained great success in various biological applications.Inspired by their intrinsic Fe isotope composition,we discovered a commercialized MPs-internal standard’s novel function to realize the accurate quantification of biomolecules.The bioassay of carcinoembryonic antigen（CEA） was chosen as a modal system.The Fe isotope in MPs and Au isotope in report probes were simultaneously and sensitively detected by...     

香豆素类铝离子荧光化学传感器的合成及性能

为了方便快捷地检测铝离子,本文以7-羟基香豆素为原料合成了铝离子荧光化学传感器。 通过质谱、核磁共振谱、紫外可见分光光度计等技术手段研究了传感器的结构和性能。 结果表明,该传感器对铝离子有很好的选择性,其检出限为8.5×10^-8 mol/L,其识别过程具有可逆性,同时,Job's plot曲线表明二者形成配位比为1:1的稳定配合物。 该研究对生物体及环境领域中铝离子的实时监测具有潜在的应用价值。     

核磁共振中多量子相干扩散行为的理论表述和计算机模拟

Self-diffusion is one of the most fundamental motions of particles in liquid. Nuclear magnetic resonance （NMR）provides a convenient and noninvasive means for accurately measuring the self-diffusion coefficient of molecules in solution. The theoretical expressions of apparent diffusion rates of MQCs are given and computer simulation based on the method to measure the self-diffusion coefficient by NMR was discussed and the random walk model of particles is used to simulate the apparent diffusion behaviors of intra-molecular and inter-molecular multiple-quantum coherences（MQCs）. The results of computer simulation agree well with theoretical predictions.     

含杂环1,4-戊二烯-3-酮肟酯类化合物的合成及生物活性

为创制具有较高生物活性的绿色农药,以姜黄素为先导,将具有良好生物活性的肟酯基团引入1,4-戊二烯-3-酮的骨架中,设计合成了一系列含杂环1,4-戊二烯-3-酮肟酯类化合物,其结构经红外光谱、核磁共振氢谱及碳谱和高分辨质谱确认。初步的生物活性测试结果表明,在药剂浓度为50mg/L时,化合物5d对小麦赤霉病菌,水稻纹枯病菌和苹果腐烂病菌的抑制活性相对较好,分别为64.75%、52.05%和69.68%;在药剂浓度为500mg/L时,化合物5d、5e和5f对TMV具有较好的治疗和钝化活性,其抑制率分别为56.93%和86.89%、53.08%和83.51%、53.40%和89.01%。以上研究结果表明,1,4-戊二烯-3-酮肟酯类化合物具有一定抑菌和抗病毒活性,在其结构基础上进行适当的改造,有望获得具有较高生物活性的有机分子。     

槲寄生中高圣草素-7-O-β-D-葡萄糖苷的分离及含量测定

对槲寄生中的有效成分高圣草素-7-O-β-D-葡萄糖苷进行了分离和结构鉴定,并对其含量进行了分析。色谱条件：C18柱(200 mm×4.6 mm i.d., 5 μm),流动相为乙腈-0.5%冰醋酸水溶液(体积比为18∶82),流速为1.0 mL/min,柱温为30 ℃,检测波长为284 nm,进样量为10 μL。结果表明,高圣草素-7-O-β-D-葡萄糖苷的峰面积与其质量浓度有良好的线性关系,相关系数r为0.9997;方法的加标回收率为96.0%~100.1%。该方法简便、快速、准确,精密度好,可作为槲寄生质量控制的一个有效方法。