玻璃微-纳流控芯片的制备及在蛋白质电动富集中的应用

发展了一种以"二次刻蚀"技术制备玻璃微-纳流控芯片的新方法. 首先, 采用紫外光刻和化学湿法刻蚀技术在玻璃基片上加工微米深度的微通道; 去除剩余的光胶后, 在刻有微通道的基片上旋涂一层新的光胶; 再通过二次紫外光刻和湿法刻蚀在该基片上加工深度小于100 nm的纳通道; 最后, 采用室温键合技术, 将带有微纳结构的基片与盖片封合制成玻璃微-纳流控复合芯片. 利用本方法可以在普通化学实验室以简易的设备制得具有微-纳米复合结构的玻璃芯片. 将此玻璃微-纳流控复合芯片成功地应用于以电动离子捕集技术富集荧光素钠异硫氰酸酯(FITC)标记的人血清蛋白(HSA). 结果表明, 对于0.5 mg/mL的FITC-HSA, 30 s内富集倍率可达到200倍以上.     

复合式微流控芯片上阴离子型固相萃取微柱柱性能分析

利用原位聚合法在玻璃微管道内制备阴离子交换型固相萃取(SPE)微柱,以NO2^-为分析对象,针对NaNO2-KI—Luminol发光体系设计微流控芯片,并将SPE微柱与微流控芯片连接起来组建成带有SPE微柱的复合式微流控芯片。分析了SPE微柱对NO2^-的吸附保留与富集作用,在复合式微流控芯片上,实现了NO2^-的进样、分离富集和检测,通过漏点曲线和交换容量两种方法分析了SPE微柱的柱容量。为控制SPE微柱的最大进样体积提供有利保障,并实现了食品中NO2^-的在线分离富集与检测。     

微通道反应器在合成反应中的应用

微流控学(microfluidics)是在微米级结构中操控纳升至皮升体积流体的技术与科学,是近10年来迅速崛起的新交叉学科.流体在微流控芯片微米级通道中,由于尺度效应导致了许多不同于宏观体系的特点,例如分子间扩散距离短、微通道的比表面积大、传热和传质速度快等,促进了微流控芯片在有机合成反应中的发展.本文总结了微通道反应器的特点、微通道反应器中常用的流体驱动技术和微通道中流体的混合技术.通过一系列在微流控芯片中进行的有机合成反应,包括液-液均相反应、催化反应、相转移反应和异常激烈的有机合成反应等,进一步说明了微通道反应器同时具有微量和连续流动的优点.微通道反应器的发展不但在合成路线的优化方面有重要意义,而且有助于相关化学工业过程的改进.     

微流控细胞芯片LED诱导荧光检测微系统

基于微流控细胞芯片分析技术和微机电系统(MEMS)加工技术, 设计制作了阵列式微流控细胞检测芯片, 采用自组装的顶窗型光电倍增管(PMT)和信号采集电路采集芯片微管道内流动细胞的荧光信号, 构建了一种针对低浓度细胞悬浮液的微流控细胞芯片发光二极管(LED)诱导荧光的快速检测微系统, 实现了对低浓度(≤40 Cell/mL)荧光标记的HepG2肝癌细胞悬浮液样本的定量计数和测试, 而且在血液细胞共存的条件下, 仍可以有效地对血液中少量HepG2肝癌细胞进行荧光计数和测试. 整个系统结构简单, 操作方便且检测灵敏度较高.     

基于时间分辨免疫分析的冰毒检测微流控芯片

研制了一种聚二甲基硅氧烷(Polydimethylsiloxane,PDMS)-多聚赖氨酸(Poly-l-lysine,PLL)玻片杂合微流控芯片,每张芯片有24条反应通道,每条反应通道体积仅为2.5μL,实现了基于时间分辨免疫分析技术的高通量冰毒(Methamphetamine,MET)定量检测。此芯片利用PDMS和PLL对蛋白质的吸附特性在反应通道内表面固定冰毒完全抗原(MET-BSA),使其与待测样品中的冰毒抗原(MET-Ag)共同竞争标记有冰毒抗体(MET-Ab)的乳胶微球。乳胶微球表面标记了镧系元素,在紫外光的照射下会发出红色荧光,根据竞争法检测原理,冰毒浓度越高,与反应通道内表面冰毒完全抗原结合的乳胶微球数量越少,所发出荧光强度越低。本芯片的结果读取采用紫外照射荧光成像方法,仅需对芯片拍摄荧光图像,即可实现24个样本的同时检测分析。本方法样品消耗量少,通量大,可以在100~3000 ng/m L浓度范围内实现对冰毒的定量检测,可用于缉毒的初步筛查,具有较好的应用前景。     

小型微流控芯片流式细胞仪的研制

基于芯片正交光路检测模式,搭建了一套小型的微流控芯片流式细胞仪.以532 nm小型半导体激光器作为激发光源,激光束被透镜聚焦于芯片通道中央.采用光电二极管检测芯片通道内流动细胞的前向散射光信号,采用小型光电倍增管检测荧光信号,整套仪器体积为19 cm×15 cm×25 cm(长×宽×高),具有结构简单、体积小、价格低廉等特点.荧光检测系统对四甲基罗丹明异硫氰酸的检出限为4.4×10-8 mol/L,采用6 μm荧光微球作为模型样品考察了仪器的分析性能,同时初步实现了细胞样品的分析.     

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。     

微流控芯片中细胞动态胞吞二氧化硅纳米颗粒的研究

研究了用微流控芯片在体外模拟人体血液流动状态下细胞胞吞二氧化硅纳米粒子的方法和特性. 通过调节储液池的液面差, 使细胞从微通道入口流入并在通道内沉积贴壁生长. 将含有贴壁细胞的微流控芯片放入37 ℃/体积分数5%CO₂的培养箱中, 使细胞培养液连续流过贴壁细胞. 培养24 h后, 在流动的培养液中加入作为荧光标记物的500 nm 粒径的掺杂有异硫氰酸荧光素(FITC)的二氧化硅微球(MSN), 继续培养6 h后, 用荧光显微镜测定细胞胞吞二氧化硅纳米粒子后的荧光强度, 考察了不同流速下细胞对二氧化硅微球摄入量的影响. 结果表明, 在动态条件下, 细胞对二氧化硅微球的吞噬量明显下降, 当流速从0.022 mm/s 增加至0.74 mm/s时, 吞噬量从静态测得值的74.7%下降至7.1%.     

基于复合型微流控芯片的紫外检测毛细管电泳系统

提出了一种基于芯片-毛细管复合装置的紫外检测-微流控芯片毛细管电泳分析系统.采用小死体积的耦合技术实现了石英毛细管与“十”字通道型微流控玻璃芯片的耦合.本系统的紫外检测灵敏度与商品化毛细管电泳仪相当.采用夹流进样方式,达到较高的进样重现性,2mmol/L苯甲酸的峰高相对标准偏差(RSD)为1.5%(n=11).可用于复方磺胺甲唑片剂的两种有效成分的快速分离.     

聚合酶链式反应微流控芯片的准分子激光制备和应用研究

摘要采用价格便宜的聚甲基丙烯酸甲酯(PMMA)代替价格昂贵的硅或玻璃作为聚合酶链式反应(PCR)微流控芯片的基片材料,采用柔性大且自动化程度高的准分子激光微加工方法代替加工工艺复杂的光刻化学腐蚀方法,在19 kV和18 mm/min的优化加工参数下,在48 mm×67 mm×1 mm的PMMA基片上制备出20个循环的PCR微流控芯片. 芯片微通道横截面呈梯形,底面光滑. 微通道宽104 μm,深56 μm,长2 060 mm,加工耗时约110 min. 该芯片和相同尺寸的盖片在160 N和105 ℃条件下通过热压经20 min键合在一起,键合强度为0.85 MPa. 键合后的芯片和温控系统集成在一起,采用比例积分微分(PID)方法得到的控温精度为±0.2 ℃,采用红外热像仪得到的相邻温区间的温度梯度分别为16.5和22.2 ℃,最后利用该芯片在对170 bp的DNA片段实现了体外扩增.     

On-chip integrated multi-thermo-actuated microvalves of poly(N-isopropylacrylamide) for microflow injection analysis

An array of thermo-actuated poly(N-isopropylacrylamide) (PNIPAAm) multivalves was designed and fabricated to perform volume-based sample injection for microflow injection analysis on a glass microfluidic chip. The PNIPAAm monolithic plug valves were prepared inside the vinylized glass channels by photopolymerization in water-ethanol (1:1) medium using 2-hydroxy-2-methyl propiophenone (Darocure-1173) as the initiator and a photo-mask for micropattern transferring. Experimental conditions for the photopolymerization were studied, and the thermo-responsive behavior of the synthesized monolithic plug valves was investigated. To perform active heating and cooling of the on-chip integrated thermo-actuated valves, micro-Peltier devices were used and operation times of 3-s for opening and 7-s for closing were obtained. In the close status, a 2-mm long monolithic plug valve could endure a pressure of no higher than 0.45 MPa. The volume-based sample and reagent injector was composed of two groups of valves (total valve number of 5) and two loops. When the two groups of valves were alternatively opened and closed via thermo-actuation, the sampling loops were able to be switched between loading and injection position without any mechanical moving parts. Cooperating with syringe pumps, the microfluidic chip with the integrated sample injector has been demonstrated for microflow injection chemiluminescence detection of hydrogen peroxide. For a sampling volume of 6 nL, linear response was observed over the H₂O₂ concentration range of 0-2 mmol L⁻¹, and a precision of 0.6% (RSD, n = 11) was achieved for a standard H₂O₂ solution 2 mmol L⁻¹.     

表面温敏聚合物刷的原子力显微镜和石英晶体微天平研究

通过表面引发的原子转移自由基聚合在硅片表面制备了聚（N-异丙基丙烯酰胺）（PNIPAAm）聚合物刷。用原子力显微镜（AFM）分别研究了PNIPAAm的接枝动力学、温度和溶剂性质对厚度的影响以及PNIPAAm链与原子力针尖间的粘附力。结果表明,PNIPAAm链在硅片表面的生长具有很好的可控性。常温下厚度为33nm的PNIPAAm膜在水溶液中的增加到82.4nm;而在甲醇/水（v/v,1:1）溶液中,PNIPAAm分子链处于坍塌收缩状态,厚度降低为45nm;在55℃下干燥所得厚度则仅为22nm。力-距离测量结果表明,在溶液中,PNIPAAm链与原子力针尖之间的粘附力远小于在干态下的粘附力。用石英晶体微天平（QCM-D）对PNIPAAm的可逆相转变进行了研究,结果表明PNIPAAm分子链随温度变化的构象转变是发生在30-34℃之间的连续过程。     

Thermosensitive Poly(N-isopropylacrylamide)-g -Polyamidoamine Dendrimer Derivatives: Preparation and the Drug Release Behaviors

Summary: A series of thermally responsive dendritic core-shell polymers were prepared based upon dendritic polyamidoamine (PAMAM), modified with carboxyl end-capped linear poly(N-isopropylacrylamide) (PNIPAAm-COOH) in different ratios via an esterification process to obtain PNIPAAm-g-PAMAM. The graft ratio of PNIPAAm could be adjusted by changing the feed ratio of PAMAM-OH to PNIPAAm-COOH and was verified by ¹H NMR and gel penetration chromatography (GPC). The lower critical solution temperature (LCST) of PNIPAAm-g-PAMAM evaluated by UV-vis spectrophotometer was about 32 °C. Indomethacin (IMC) as a model drug was loaded in the thermosensitive polymer-grafted dendrimer derivative and its release behavior was studied below and above its LCST (27 °C vs 37 °C). Results showed that the LCST of PNIPAAm-g-PAMAM was around 32 °C compared with that of the pure PNIPAAm. The release behavior of the indomethacin entrapped in the internal cavities of the PNIPAAm-g-PAMAM showed that almost 77% of the drug was cumulatively released at 27 °C after 10 hours, whereas only 20% was released at 37 °C. The release rate of IMC from the IMC/PNIPAAm-g-PAMAM complex at 37 °C is significantly slower than that at 27 °C, which indicates that the PNIPAAm chains grafted on the surface of PAMAM dendrimer could act as a channel switching on-off button through expending or contracting in response to the temperature variation and could control the drug release by varying the surrounding temperature.     

聚(N-异丙基丙烯酰胺)改性硅表面与血浆蛋白质的相互作用

通过表面引发原子转移自由基聚合(ATRP)在硅表面接枝了聚(N-异丙基丙烯酰胺)(PNIPAAm)聚合物刷,并考察了PNIPAAm改性表面在单一蛋白质溶液以及血浆中与血浆蛋白质之间的相互作用.蛋白质吸附测试表明,与未改性的硅表面相比,改性后的表面对纤维蛋白原的吸附量大大降低,特别是在血浆中纤维蛋白原吸附量小于5ng/c...     

聚(N-异丙基丙烯酰胺)/聚丙烯酰胺无机/有机互穿网络水凝胶的制备及表征

通过紫外引发聚合方法制备了无机交联的聚(N-异丙基丙烯酰胺)(PNIPAAm)/有机交联的聚丙烯酰胺(PAAm)互穿网络(IPN)水凝胶.利用FTIR和SEM分别表征了凝胶的化学结构和内部形态;测定了凝胶在高温(50℃)时的退溶胀性能;利用DMA和DSC分别研究了凝胶的储能模量随温度的变化及热相转变行为.研究表明,该IPN凝胶具有温度敏感性;与未互穿的无机交联PNIPAAm凝胶相比,IPN凝胶具有多孔的网络结构和超快的响应速率,如10min内失去90%的水;其储能模量增加了3～4倍,相转变行为变弱,而最低临界溶解温度(LCST)提高了1.4℃.     

Monolithic valves for microfluidic chips based on thermoresponsive polymer gels

The direct preparation of thermoresponsive monolithic copolymers by photopatterning of a liquid phase consisting of an aqueous solution of N-isopropylacrylamide, N-ethylacrylamide, N,N'-methylenebisacrylamide, and 4,4'-azobis(4-cyanovaleric acid) has been studied and the products used as valves within the channels of microfluidic devices. The volume change associated with the polymer phase transition at its lower critical solution temperature (LCST) leads to the rapid swelling and the deswelling of the 2.5% cross-linked monolithic gel thus enabling the polymer to close or open the channel and to function as a nonmechanically actuated valve. The LCST at which the valve switches was easily adjusted within a range of 35 degrees C-74 degrees C by varying the proportions of the monovinyl monomers in the polymerization mixture. The closed valve holds pressures of up to 18 MPa without noticeable dislocation, structural damage, or leakage. In contrast, following deswelling by raising the temperature above LCST the valve offers no appreciable flow resistance since its large, micrometer-size pores are open. Laser-triggered photobleaching of a fluorescent dye contained in the liquid phase enabled monitoring of flow through the device and determination of the times required to open and close the valve. The valves are characterized by very fast actuation times in a range of 1-4 s depending on the type of device. No changes in performance were observed even after repeated open-close cycling of the valves.     

聚氨酯微球的接枝改性及温敏特性

以N-异丙基丙烯酰胺(NIPAAm)为单体, 二苯甲酮(BP)为光敏剂, 过硫酸胺(APS)为自由基引发剂, 采用溶液中光接枝方法制备了具有温度敏感特性的聚氨酯微球(PUS). 傅里叶变换红外光谱(FTIR)和扫描电子显微镜(SEM)结果表明, 在聚氨酯微球表面形成了聚异丙基丙烯酰胺(PNIPAAm)接枝聚合物层. 在接枝过程中, 延长反应时间与增加引发剂浓度均有利于提高接枝率. 常温下, 接枝率随反应时间延长呈线性增长, 当反应时间超过40 min后, 接枝率基本保持稳定; 而引发剂浓度对接枝率的影响存在最佳优化值, 即其浓度为单体质量分数的3%. 采用差示扫描量热法(DSC)对接枝改性前后聚氨酯微球的温敏特性进行分析表征, 证实改性后的微球在35 ℃左右出现低临界互溶温度(LCST), 在此温度附近表现出对温度敏感特性. 接触角测试与溶胀测试结果表明, 在低临界互熔温度以下, 接枝改性的聚氨酯微球具有良好的亲水性.     

Switchable phase transition behavior of thermoresponsive substrates for cell sheet engineering

Recently, there are significant interests in the development of biomaterials with nonlinear response to an external stimulus. Thermoresponsive polymers as a well-known class of stimuli-responsive materials represent reversible hydrophilicity/hydrophobicity characteristics around a critical temperature. This switchable behavior applies for nondestructive cellular detachment from cultivation substrates. In this study, poly (N-isopropylacrylamide) (PNIPAAm)-grafted dishes were made up to harvest retinal pigmented epithelial (RPE) and periodontal ligament cell (PDLC) sheets. Wettability assessments verified that all functionalized surfaces were inverted from hydrophilic to hydrophobic state when the temperature rises from lower critical solution temperature (LCST) at 37 °C. Other physicochemical characteristics such as chemical composition, grafting thickness, and surface topography were investigated through attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and atomic force microscopy (AFM). ATR-FTIR results showed typical peaks of amide group corresponding to successful PNIPAAm polymerization. AFM microscopy results also proved creating a rough PNIPAAm layer with thickness of 29.2 nm after grafting process in the mixture of methanol and water. Cell culture experiments showed an irreversible cellular attachment/detachment from modified surfaces upon temperature changes. These results introduced thermoresponsive TCPS to noninvasively harvest RPE and PDLCs sheets especially for application in scaffold-free tissue engineering decorations. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 1567–1576     

交联聚(N-异丙基丙烯酰胺)/(海藻酸钠/聚(N-异丙基丙烯酰胺))半互穿网络水凝胶的制备及其溶胀性能

将线性聚(N-异丙基丙烯酰胺)(PNIPAAm)和海藻酸钠(SA)分子同时引入到PNIPAAm凝胶中,制备了交联聚(N-异丙基丙烯酰胺)/(海藻酸钠/聚(N-异丙基丙烯酰胺))半互穿网络(Cr-PNIPAAm/(SA/PNIPAAm)semi-IPN)水凝胶。在弱碱性条件下(pH=7.4),改变SA与线性PNIPAAm的质量比对Cr-PNIPAAm/(SA/PNIPAAm)semi-IPN水凝胶的溶胀度没有太大的影响。在酸性条件下(pH=1.0),其溶胀度随着SA与线性PNIPAAm质量比的减小而增大。由于亲水性SA与线性PNIPAAm的协同作用,Cr-PNIPAAm/(SA/PNIPAAm)semi-IPN水凝胶的消溶胀速率得到很大提高。     

Temperature-controlled flow switching in nanocapillary array membranes mediated by poly(N-isopropylacrylamide) polymer brushes grafted by atom transfer radical polymerization

We report actively controlled transport that is thermally switchable and size-selective in a nanocapillary array membrane (NCAM) prepared by grafting poly(N-isopropylacrylamide) (PNIPAAm) brushes onto the exterior surface of a Au-coated polycarbonate track-etched membrane. A smooth Au layer on the membrane surface, which is key to obtaining a uniform polymer film, was prepared by thermal evaporation of approximately 50 nm Au on both exterior surfaces. After evaporation, the inner diameter of the pore is reduced slightly, but the NCAM retains a narrow pore size distribution. PNIPPAm brushes with 10-30 nm (dry film) thickness were grafted onto the Au surface through surface-initiated atom transfer radical polymerization (ATRP) using a disulfide initiator, (BrC(CH3)2COO(CH2)11S)2. Molecular transport through the PNIPAAm polymer brush-modified NCAMs was investigated by real-time fluorescence measurements using fluorescein isothiocyanate (FITC)-labeled dextrans ranging from 4.4 to 282 kDa in membranes with variable initial pore diameters (80, 100, and 200 nm) and different PNIPAAm thicknesses. Manipulating the temperature of the NCAM through the PNIPAAm lower critical solution temperature (LCST) causes large, size-dependent changes in the transport rates. Over specific ranges of probe size, transport is completely blocked below the LCST but strongly allowed above the LCST. The combination of the highly uniform PNIPAAm brush and the monodisperse pore size distribution is critical in producing highly reproducible switching behavior. Furthermore, the reversible nature of the switching raises the possibility of using them as actively controlled filtration devices.