基于BioMEMS技术的DNA固相萃取芯片的制备

基于BioMEMS技术,制备一种新型的Si-PDMS-玻璃结构的DNA固相萃取微流控芯片。在硅基片上制备4种固相载体,分析不同载体的性质和制备特点,优选多孔氧化硅作为萃取DNA的固相载体。对比研究芯片的封装工艺,优选压制法制备PDMS-玻璃盖片,采用粘接技术封装芯片。芯片成功提取老鼠全血中的基因组DNA,提取效率为23.5×10-9g/μL全血,并成功进行PCR反应,达到试剂盒水平。固相萃取微流控芯片具有与其他样品处理芯片、PCR芯片和电泳芯片相集成的潜力,可实现对复杂生物样品的检测和分析。     

基于3D打印技术的集成核酸提取芯片制备

基于固相萃取原理,设计并制作了一种集成核酸提取功能的微流控芯片,用于人体全血中脱氧核糖核酸(DNA)的提纯。芯片主要包括:混合微通道、裂解腔、DNA提取腔、DNA存储腔。采用3D打印技术制作出芯片模板,通过模板注塑成型、氧等离子体键合等工艺制作出微流控芯片。利用该芯片,可以在20 min内完成血液和试剂的顺序加载、快速混合、血细胞裂解、DNA的提取等操作,且试剂消耗量少。通过对所提DNA链上稳定表达的内参基因甘油醛-3-磷酸脱氢酶(GAPDH)进行PCR扩增,并对扩增产物进行熔解曲线分析,以验证所提取DNA的质量。     

功能离子液体/氧化硅基多孔复合材料*

利用有巨大界面特征和发达孔道结构的氧化硅基多孔材料作为功能离子液体的载体,不仅可实现离子液体的固相化从而解决均相离子液体分离难的瓶颈,而且也可增加离子液体的比表面积进而提高离子液体的使用效率和稳定性;探索结合离子液体和氧化硅基多孔材料双重优点的目标功能材料的制备及应用成为近年来的一个研究热点。本文综述了离子液体/氧化硅基多孔复合材料的最新研究进展,探讨了其合成手段的有效性,并对其应用前景进行了展望。     

表面电性可控磁珠微流控芯片在DNA提取中的应用

制备了表面电性可控的氨基化SiO_2@Fe_3O_4磁性复合微球,采用激光刻蚀和热压键合的方法制作了聚甲基丙烯酸甲酯(PMMA)材质的DNA固相萃取芯片,将磁珠灌注于芯片通道中,借助永磁铁固定并控制磁珠,将磁珠芯片应用于人类全血中的基因组DNA提取,优化了提取实验条件,并对提取产物进行凝胶电泳和PCR分析。实验结果表明,磁珠微流控芯片成功地从全血中提取出纯度较高的基因组DNA,提取效率约35%,提取液的凝胶电泳条带与商品化试剂盒提取的基因组DNA一致,提取液可用于进一步的PCR反应。     

核酸检测的校正在二氧化硅吸附等温线中的应用

利用在高盐环境下氧化硅微球表面对核酸的选择性吸附分离纯化核酸已成为一种较为普遍的核酸固相提取方法,简单准确地测量高盐溶液中的核酸浓度,是研究DNA吸附性能的关键.通过制作标准曲线的方法, 校正含高浓度盐溶液中的DNA浓度,计算得到校正系数k,提出了一种检测SiO2对DNA单位吸附量的简便方法--参比法.与传统方法相比,此方法不须脱盐纯化即可方便地绘制出了SiO2微球对鲑鱼精DNA的吸附等温线,为DNA吸附载体的研究奠定了方法学基础.     

衍生多孔碳材料在固相微萃取中的应用研究进展

固相微萃取是一种集采样、萃取、富集和进样于一体的样品前处理技术,其萃取效果与涂层材料密切相关。多孔碳材料具有比表面积大、多孔结构可控、活性位点多和化学稳定性好等优点,广泛应用于电池、超级电容器、催化、吸附和分离等领域,也是一种热门的用作固相微萃取探针的涂层材料。衍生多孔碳材料因种类丰富、可设计性强被广泛研究,研究主要集中在对衍生多孔碳材料的结构优化方面。但是衍生多孔碳材料在固相微萃取中的应用还存在如下问题:(1)共价有机框架衍生多孔碳材料的制备已取得较大进展,但将其应用于固相微萃取领域的研究仍较少;(2)有待进一步明确制备出的衍生多孔碳材料用作固相微萃取涂层表现出优异提取能力的机理;(3)有待进一步深入研究将衍生多孔碳材料用作固相微萃取涂层以实现对不同物理化学性质污染物的广谱高灵敏度分析。文章综述了近3年衍生多孔碳材料在固相微萃取中的应用研究,并展望了未来衍生多孔碳材料在固相微萃取中的研究前景。引用文献共56篇,主要来源于Elsevier。     

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。     

X射线荧光光谱法测定蛇纹石成分

蛇纹石因其外表分化呈灰白、石红色网纹,似蛇皮而得名,主要用作炼钢熔剂、耐火材料、提取氧化镁和多孔氧化硅等.它的质量优劣直接影响钢铁冶炼时造渣性的优劣.     

一种可绝对定量核酸的数字PCR微流控芯片

构建了一种新型的可进行核酸单分子扩增和核酸绝对定量的数字聚合酶链式反应(数字PCR)微流控芯片. 应用多层软光刻技术, 以聚二甲基硅氧烷(PDMS)作为芯片材料, 盖玻片作为基底制作了具有3层结构以及微阀控制功能的微流控芯片. 芯片的大小与载玻片相当, 可同时检测4个样品, 每个样品通入芯片后平均分配到640个反应小室, 每个小室的体积为6 nL. 以从肺癌细胞A549中提取的18sRNA为样品检测了该芯片的可行性. 将样品稀释数倍后通入芯片, 核酸分子随机分布在640个小室中并扩增. 核酸分子在芯片中的分布符合泊松分布原理, 当样品中待测核酸分子平均拷贝数低于0.5个/小室时, 则每个反应小室包含0个或1个分子. 经过PCR扩增后, 有模板分子的小室检测结果为阳性反应, 而无模板分子的小室为阴性反应, 最后通过计数阳性反应室的个数, 可绝对定量原始待测样品中的目标DNA分子拷贝数. 实验结果表明, 该数字 PCR芯片可实现DNA单分子反应和核酸绝对定量, 具有成本低、 灵敏度高、 节省时间和试剂以及操作简单等优点, 为数字PCR方法在普通实验室的应用提供了一种新途径, 可用于癌症及感染性疾病的早期诊断、 单细胞分析、 产前诊断以及各种细菌病毒的核酸检验等研究.     

集成核酸提取的实时荧光PCR微全分析系统

集成核酸提取的实时荧光PCR微全分析系统将核酸提取、PCR扩增与实时荧光检测进行整合,在同一块微流控芯片上实现了核酸分析过程的全自动和全封闭,具有试剂用量少、分析速度快、操作简便等优点。本研究采用微机械加工技术制作集成核酸提取微流控芯片的阳极模,使用组合模具法和注塑法制作具有3D通道的PDMS基片,与玻璃基底通过等离子体键合封装成集成核酸提取芯片。构建了由微流体速度可调节(0~10 mL/min)的驱动控制装置、温控精度可达0.1℃的TEC温控平台、CCD检测功能模块等组成的微全分析系统。以人类血液裂解液为样品,采用硅胶膜进行芯片上核酸提取。系统根据设置好的时序自动执行,以2 mL/min的流体驱动速度完成20μL裂解液上样、清洗;以1 mL/min的流体驱动速度完成DNA洗脱,抽取PCR试剂与之混合注入到反应腔。提取的基因组DNA以链上内参基因GAPDH为检测对象,并以传统手工提取为对照,在该系统平台上进行PCR扩增和熔解曲线分析实验。片上PCR扩增结果显示,扩增曲线明显,Ct值分别为25.3和26.9。扩增产物进行熔解曲线分析得到的熔解温度一致,均为89.9℃。结果表明,此系统能够自动化、全封闭的在微流控芯片上完成核酸提取、PCR扩增与实时定量分析。     

Sample pretreatment microfluidic chip for DNA extraction from rat peripheral blood

A sample pretreatment microfluidic chip was described based on the principle of solid phase extraction and micro electro mechanical system technology. Oxidized porous silicon with the large surface area as the solid phase matrix for absorption of DNA from a biological sample can greatly improve the DNA yield. The factors that could affect the DNA yield were analyzed and the preparation technology and the experiment procedure were improved. The DNA purification process from the rat peripheral blood can be achieved and the DNA yield is 24 ng/(μL whole blood), which can reach the level of the commercial DNA purification kits. Furthermore, the DNA extracted from the whole blood can be amplified by polymerase chain reaction, which can achieve a high efficiency of the amplification. Translated from Chemical Journal of Chinese Universities, 2006, 27(4) (in Chinese)     

Continuous flow microfluidic device for cell separation, cell lysis and DNA purification

A novel integrated microfluidic device that consisted of microfilter, micromixer, micropillar array, microweir, microchannel, microchamber, and porous matrix was developed to perform sample pre-treatment of whole blood. Cell separation, cell lysis and DNA purification were performed in this miniaturized device during a continuous flow process. Crossflow filtration was proposed to separate blood cells, which could successfully avoid clogging or jamming. After blood cells were lyzed in guanidine buffer, genomic DNA in white blood cells was released and adsorbed on porous matrix fabricated by anodizing silicon in HF/ethanol electrolyte. The flow process of solutions was simulated and optimized. The anodization process of porous matrix was also studied. Using the continuous flow procedure of cell separation, cell lysis and DNA adsorption, average 35.7 ng genomic DNA was purified on the integrated microfluidic device from 1 μL rat whole blood. Comparison with a commercial centrifuge method, the miniaturized device can extract comparable amounts of PCR-amplifiable DNA in 50 min. The greatest potential of this integrated miniaturized device was illustrated by pre-treating whole blood sample, where eventual integration of sample preparation, PCR, and separation on a single device could potentially enable complete detection in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.     

DNA purification using dynamic solid-phase extraction on a rotationally-driven polyethylene-terephthalate microdevice

We report the development of a disposable polyester toner centrifugal device for semi-automated, dynamic solid phase DNA extraction (dSPE) from whole blood samples. The integration of a novel adhesive and hydrophobic valving with a simple and low cost microfabrication method allowed for sequential addition of reagents without the need for external equipment for fluid flow control. The spin-dSPE method yielded an average extraction efficiency of ∼45% from 0.6 μL of whole blood. The device performed single sample extractions or accommodate up to four samples for simultaneous DNA extraction, with PCR-readiness DNA confirmed by effective amplification of a β-globin gene. The purity of the DNA was challenged by a multiplex amplification with 16 targeted amplification sites. Successful multiplexed amplification could routinely be obtained using the purified DNA collected post an on-chip extraction, with the results comparable to those obtained with commercial DNA extraction methods. This proof-of-principle work represents a significant step towards a fully-automated low cost DNA extraction device.     

Detection of C677T mutation in methylenetetrahydrofolate reductase gene by denaturing high performance liquid chromatography

In this paper, we described an assay for the detection of the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene using denaturing high-performance liquid chromatography (DHPLC). The conditions for DHPLC analysis were systematically investigated based on a general HPLC instrument (Prostar VARIAN). A 225 bp DNA fragment covering the 677 site of MTHFR gene was amplified by PCR technology using the purified DNA from whole blood or whole blood as template DNA. PCR products were directly injected without the need for purification. The C677T mutation could be clearly distinguished by DHPLC technology. Our data demonstrated that DHPLC was a powerful and alternative tool for detection of genetic variants and single-nucleotide polymorphisms to electrophoresis technology.     

萝芙木根中萝芙木碱的分离纯化

对萝芙木根中萝芙木碱的提纯与检测技术进行研究.利用萝芙木植物的根和皮为原料,对萝芙木碱的分离纯化过程进行了系统的研究,确立了固一液萃取、液一液萃取、等电点沉淀、甲醇重结晶提取萝芙木碱的优化方案,并用高效液相色谱法检测了萝芙木碱的含量.实验所得样品中萝芙木碱的含量不低于98%.结果表明该分离纯化技术生产成本低,操作简单,...     

On-line cell lysis and DNA extraction on a microfluidic biochip fabricated by microelectromechanical system technology

Integrating cell lysis and DNA purification process into a micrototal analytical system (microTAS) is one critical step for the analysis of nucleic acids. On-chip cell lysis based on a chemical method is realized by sufficient blend of blood sample and the lyzing reagent. In this paper two mixing models, T-type mixing model and sandwich-type mixing model, are proposed and simulation of those models is conducted. Result of simulation shows that the sandwich-type mixing model with coiled channel performs best and this model is further used to construct the microfluidic biochip for on-line cell lysis and DNA extraction. The result of simulation is further verified by experiments. It asserts that more than 80% mixing of blood sample and lyzing reagent which guarantees that completed cell lysis can be achieved near the inlet location when the cell/buffer velocity ratio is less than 1:5. After cell lysis, DNA extraction by means of a solid-phase method is implemented by using porous silicon matrix which is integrated in the biochip. During continuous flow process in the microchip, rapid cell lysis and PCR-amplifiable genomic DNA purification can be achieved within 20 min. The potential of this microfluidic biochip is illustrated by pretreating a whole blood sample, which shows the possibility of integration of sample preparation, PCR, and separation on a single device to work as portable point-of-care medical diagnostic system.     

Solid‐Phase Incorporation of an ATRP Initiator for Polymer–DNA Biohybrids

The combination of polymers with nucleic acids leads to materials with significantly advanced properties. To obviate the necessity and complexity of conjugating two macromolecules, a polymer initiator is described that can be directly covalently linked to DNA during solid‐phase synthesis. Polymer can then be grown from the DNA bound initiator, both in solution after the DNA‐initiator is released from the solid support as well as directly on the solid support, simplifying purification. The resulting polymer‐DNA hybrids were examined by chromatography and fluorescence methods that attested to the integrity of hybrids and the DNA. The ability to use DNA‐based supports expands the range of readily available molecules that can be used with the initiator, as exemplified by direct synthesis of a biotin polymer hybrid on solid‐support. This method expands the accessibility and range of advanced polymer biohybrid materials.     

DNA purification on a lab-on-valve system incorporating a renewable microcolumn with in situ monitoring by laser-induced fluorescence

Bead injection in a lab-on-valve (LOV) system was adopted for DNA purification via micro solid-phase extraction (SPE) with a renewable silica microcolumn packed in a channel of the LOV unit. The complex matrix components in human whole blood, including proteins, were well eliminated by choosing properly the sample loading and elution media. The DNA purification process was monitored on-line by using laser-induced fluorescence in a demountable side part of the LOV unit incorporating optical fibers. The practical applicability of the entire system was demonstrated by separation/purification of λ-DNA in a simulated matrix and human blood genetic DNA by performing SPE, in situ monitoring of the purified products, and postcolumn PCR amplification. When DNAs in a simulated matrix (10.0 ng μl⁻¹ λ-DNA, 50 ng μl⁻¹ bovine serum albumin, 1.0% Triton X-100) were processed in the present system and laser-induced fluorescence was monitored at 610 nm, an overall extraction/collection efficiency of 70% was achieved by employing identical sample loading and an elution flow rate of 0.5 μl s⁻¹, along with a precision of 3.8% relative standard deviation. DNA separation and purification from human whole-blood samples were performed under similar conditions. Figure Lab-on-valve mesofluidic system employed for DNA separation and purification integrating a demountable fluorescence flow cell for in-situ laser induced fluorescence detection     

Optimization of the Extraction and Analysis of Natural Androgen Steroids and Their Metabolites in Urine by GC/MS and GC/FID

A multiresidue method was developed for screening, quantification, and confirmation of nine natural androgen steroids and their metabolites in urine. Steroids were first extracted from urine by solid phase extraction, enzymatically deglucuronated, re-extracted using a liquid/liquid extraction for purification, and finally acetylated for GC/MS and GC/FID analysis. Each step of sample preparation, as well as analysis, was optimized: solid phase extraction, liquid/ liquid extraction, and derivatization reaction … Therefore, a rugged sample preparation procedure was developed leading to extracts of sufficient purity (recoveries >66% and few matrix compounds). The whole methodology allowed reliable detection and quantification of the nine steroids at low concentration levels. Linearity and repeatability were established and were found to be satisfactory (R² > 0.996, RSD < 11%). Finally, the method was applied to quantify compounds of interest in real samples collected from healthy volunteers and patients treated with 4-androstenedione or dehydroepiandrosterone.     

Synthesis of Triazole‐linked Homonucleoside Polymers through Topochemical Azide–Alkyne Cycloaddition

There is a great deal of interest in developing stable modified nucleic acids for application in diverse fields. Phosphate‐modified DNA analogues, in which the phosphodiester group is replaced with a surrogate group, are attractive because of their high stability and resistance to nucleases. However, the scope of conventional solution or solid‐phase DNA synthesis is limited for making DNA analogues with unnatural linkages. Other limitations associated with conventional synthesis include difficulty in making larger polymers, poor yield, incomplete reaction, and difficult purification. To circumvent these problems, a single‐crystal‐to‐single‐crystal (SCSC) synthesis of a 1,5‐triazole‐linked polymeric ssDNA analogue from a modified nucleoside through topochemical azide–alkyne cycloaddition (TAAC) is reported. This is the first solvent‐free, catalyst‐free synthesis of a DNA analogue that proceeds in quantitative yield and does not require any purification.