微流控免疫分析芯片的研究进展

在最近十几年中,微流控芯片技术得到了迅速发展,它可以提高分析速度、增加分析效率、减少样本和试剂的消耗。我们对微流控免疫分析芯片的设计、制作以及应用进行了综述。     

微流控肺器官芯片的研究进展

近年来随着微制造技术的快速发展,推动了微流控器官芯片模型的建立。微流控器官芯片作为一种能够模拟器官中的组织和细胞结构及生理环境的微流控设备,得到了广泛的研究和发展。其中微流控肺器官芯片是最早研究的微流控器官芯片之一,与常规体外细胞和动物模型相比,微流控肺器官芯片模型模拟肺生理和病理条件更为精准,对呼吸系统疾病、药物开发及个体化治疗具有重要意义。文章介绍微流控肺器官芯片的原理、制造、模拟呼吸系统疾病和药物的研发及筛选,并总结微流控肺器官芯片发展所面临的挑战。微流控肺器官芯片旨在阐明复杂的病理生理学的肺疾病,并加快药物开发和药物筛选。随着肺部疾病和药物的社会和经济负担的增加,微流控肺器官芯片有望成为一个蓬勃发展的平台,也能减少制药公司和研究人员对传统体外细胞培养和动物模型的依赖。     

基于玻璃基底微流控芯片的制备

介绍一种基于玻璃基底微流控芯片的制作方法,通过光刻工艺在玻璃基底表面刻蚀出微通道。利用氧等离子体对聚二甲基硅氧烷（Polydimethylsiloxane,PDMs）和微通道所在的基底表面进行处理,使PDMS与基底表面性质由疏水性变为亲水性,完成PDMS与玻璃基底的共价键结合。通过实验表明,经氧等离子体处理之后的PDMS与玻璃基底的密封效果较好。鉴于PDMS具有良好的热稳定性,无毒性,以及较好的光学特性等,此微流控芯片可用于生物医学以及化学领域中的化学发光及荧光检测。     

微流控浓度梯度芯片的开发与生物化学应用研究

在生物化学分析中系统研究样本与不同浓度组分间的相互作用是至关重要的。微流控芯片技术能够在微米级的通道内完成精确的液体控制,近年来被普遍应用于生物化学分析领域。微流控浓度梯度芯片是一种能够快速构建稳定生物化学浓度梯度的工具,能够与大多数细胞培养、化学分析等技术相结合,为传统的生化分析提供新平台。本文综述微流控浓度梯度芯片的形成机制及其在生物化学等领域的应用,为拓宽浓度梯度相关应用研究提供新思路。     

微流控技术在过敏原检测领域应用的研究进展

过敏性疾病被认为是当今全球性的健康问题,严重影响着人们的身体健康和生活质量。明确并避免接触过敏原是预防和治疗过敏性疾病最有效的途径。如何快速、精准检测过敏原逐渐成为研究者们日益关注的问题。基于微流体的过敏原检测技术开发的多个快速过敏原检测系统,实现了过敏原即时快速精准检测,由于其具有高灵敏度、高通量以及微型化等特点,对过敏性疾病的预防和诊断起到了至关重要的作用。但由于各种检测方法、技术和材料以及不同过敏原检测的灵敏度、特异性和普及程度存在一定的差异,其广泛的应用前景有待进一步明确,因此,未来还需进一步的研究和优化微流控技术在过敏原检测中的应用,为过敏性疾病的诊断和预防提供新的思路。     

基于微流控芯片模拟体内受精及早期胚胎发育环境的输卵管模型构建

文题释义： 微流控芯片：又被称作芯片实验室,是将传统的化学技术和生物技术结合,并将所有基本操作单元微缩集成在一块芯片上以自动完成全过程的一项新技术,它在生物、化学、医学等领域都有巨大潜力,目前广泛运用于各行各业。 输卵管：女性生殖系统的重要组成部分,体内受精及早期胚胎培养的场所,胚胎在输卵管壶腹部和峡部交界处完成受精过程后,在流动的输卵管液、摆动的输卵管上皮细胞纤毛、收缩的输卵管肌肉等的共同作用下移动至宫腔进行着床,另外输卵管上皮细胞会分泌各种细胞因子辅助胚胎的发育,对胚胎的发育和着床过程非常重要。 背景：胚胎受精和早期胚胎培养是辅助生殖技术中重要的一部分,然而近几十年来胚胎培养技术却基本没有更新,因此胚胎受精和早期胚胎培养的条件成为了限制辅助生殖技术发展的一个瓶颈。 目的：构建基于微流控芯片来模拟体内受精及早期胚胎发育环境的仿真输卵管模型。 方法：采用软光刻法制作微流控芯片,使芯片微通道在形状上符合输卵管的解剖结构;组织消化贴壁法进行小鼠输卵管原代上皮细胞的培养和提纯;用角蛋白免疫荧光法对提纯后的小鼠输卵管原代上皮细胞进行鉴定,并将鉴定后的上皮细胞种植在微流控芯片通道内壁上以模拟输卵管生化环境;将芯片接入自动换液装置以模拟输卵管液流环境。 结果与结论：①这款输卵管模型呈圆柱状,长度为2 cm,直径为1 cm,在形状上与体内输卵管峡部的解剖学特征比较相符合;②角蛋白免疫荧光结果为阳性,提示组织消化贴壁法可分离培养出小鼠输卵管原代上皮细胞;③提纯后的小鼠输卵管原代上皮细胞种植在模型内壁,为胚胎的受精和早期培养提供了与体内微环境类似的生化环境。微流控芯片接入自动换液装置后,通道内的代谢废物能被及时带走,新的营养物质得以补充,实现了对输卵管真实流体环境的模拟;④研究将微流控芯片应用于辅助生殖技术,通过模拟体内受精及早期胚胎发育环境,实现了输卵管解剖学和生化环境的的重建,构建了以输卵管为模型的器官芯片,为进一步改善辅助生殖技术和提高受精率和优胚率奠定基础。 ORCID: 0000-0002-8168-8999(汪萌) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

一种高通量测量单细胞弹性模量的微流控芯片

目的设计微流控芯片以便高效简便地捕获大量单细胞并测量其弹性模量。方法根据流体力学原理,设计微流控阵列及其单细胞捕获单元的通道结构和几何尺寸。培养海拉细胞,制作微流控芯片实物,并采用该芯片进行单细胞捕获实验。采用COMSOL软件对作用在被捕获细胞上的剪切力和压差进行有限元仿真。根据作用在被捕获细胞两侧的压差值和细胞在捕获通道中的伸长长度,计算出细胞的弹性模量。结果所设计的微流控芯片能有效地捕获大量单细胞;计算的单细胞弹性模量为780.7Pa±100.5Pa,与文献中报道的763Pa±93Pa接近。结论本文所提出的微流控芯片可高效捕获单细胞并测量单细胞力学特性。     

基于微流控芯片的姜黄素诱导MCF-7细胞凋亡的研究

目的探讨应用微流控芯片实现高内涵药物筛选（high content screening,HCS）的可行性。方法本文将微流控芯片技术与HCS技术相结合,通过自行设计、制作聚二甲基硅氧烷（polydimelhylsiloxane,PDMS）-玻璃微流控芯片,并在芯片上实现人乳腺癌MCF-7细胞培养、脂质体转染、药物姜黄素刺激等操作,最后通过显微成像技术进行检测。结果姜黄素可以诱导MCF-7细胞凋亡,并呈浓度依赖性,同时获得了细胞在凋亡过程中一些生物信息的改变：随着姜黄素浓度的增加,细胞凋亡比例、EndoG—GFP重定位比例增大,膜通透性增加,细胞核固缩变小。结论上述微流控芯片可以为HCS技术提供良好的研究平台。     

S-BLMs的发展及其在生物传感器中的应用

由于双层类脂膜(bilayer lipid membranes，BLMs)是生物膜极好的实验模型，因此在生物传感器的研制领域显示出广泛的应用前景。在对生物膜结构进化的简短总结后，介绍了有固态基底支撑的BLMs(supported bilayer lipid membranes，S—BLMs)的研究发展状况，并综述了其在生物传感器中的应用研究进展。     

Localized surface plasmon resonance biosensor integrated with microfluidic chip

A sensitive and low-cost microfluidic integrated biosensor is developed based on the localized surface plasmon resonance (LSPR) properties of gold nanoparticles, which allows label-free monitoring of biomolecular interactions in real-time. A novel quadrant detection scheme is introduced which continuously measures the change of the light transmitted through the nanoparticle-coated sensor surface. Using a green light emitting diode (LED) as a light source in combination with the quadrant detection scheme, a resolution of 10⁻⁴ in refractive index units (RIU) is determined. This performance is comparable to conventional LSPR-based biosensors. The biological sensing is demonstrated using an antigen/antibody (biotin/anti-biotin) system with an optimized gold nanoparticle film. The immobilization of biotin on a thiol-based self-assembled monolayer (SAM) and the subsequent affinity binding of anti-biotin are quantitatively detected by the microfluidic integrated biosensor and a detection limit of 270 ng/mL of anti-biotin was achieved. The microfluidic chip is capable of transporting a precise amount of biological samples to the detection areas to achieve highly sensitive and specific biosensing with decreased reaction time and less reagent consumption. The obtained results are compared with those measured by a surface plasmon resonance (SPR)-based Biacore system for the same binding event. This study demonstrates the feasibility of the integration of LSPR-based biosensing with microfluidic technologies, resulting in a low-cost and portable biosensor candidate compared to the larger and more expensive commercial instruments.     

多通道微流控芯片的设计、仿真及细胞迁移应用研究

细胞迁移是指细胞朝着特定的化学浓度梯度发生定向迁移运动,其在胚胎发育、伤口愈合、肿瘤转移中发挥着至关重要的作用。当前研究手段大多通量低,难以综合考虑不同浓度梯度条件对细胞迁移行为的影响。针对上述问题,本文首先设计了一款四通道微流控芯片,其特征如下:借助层流和扩散机制在细胞迁移主通道中建立和维持浓度梯度;可在单一显微镜视野下同时观测四组细胞迁移现象;集成了宽度为20μm的细胞隔离带,可校准细胞初始位置,保证实验结果的准确性。随后,借助Comsol Multiphysics有限元分析软件完成了微流控芯片的仿真分析,证明了芯片上设计S型微通道和水平压力平衡通道有助于在细胞迁移主通道中形成稳定的浓度梯度。最后,采用不同浓度(0、0.2、0.5、1.0μmol·L^-1)与糖尿病及其并发症密切相关的晚期糖基化终末产物(AGEs)孵育中性粒细胞,研究了其在100 nmol·L^-1趋化因子fMLP浓度梯度环境中的迁移行为。结果表明,AGEs抑制了中性粒细胞的迁移能力,证明了四通道微流控芯片的可靠性和实用性。     

Development of a microplate reader compatible microfluidic chip for ELISA

We report a novel microfluidic device use for sandwich enzyme-linked immunoassay assay (ELISA). The related procedures including the introduction of reagents, dilution and distribution of samples, as well as immobilization of enzyme can be readily carried out on a poly (dimethylsiloxane) (PDMS) chip. Particularly, this microfluidic chip comprising of two distinct parallel units, and has an identical dimension as a conventional microtiter plate, which offers access to the directly quantitative detection by the microplate reader. Gradient-concentration reacting solutions at six different concentrations level generated by the microfluidic channel network are simultaneously transported to 24 reaction chambers to form enzymatic products. Alkaline phosphatase (ALP), 4-methylumbelliferyl phosphate (4-MUP) and KH(2)PO(4) are used as enzyme-substrate-inhibitor model, to demonstrate the utility of the developed microchip-based enzyme inhibitor assay. Various conditions such as the surface treatment of chip channels, fluids velocities, substrate concentration, and buffer pH are investigated. The present microfluidic device for ELISA holds several advantages, for instance frugal usage of samples and reagents, less of operating time, favorably integrated configuration, ease of manipulation, and could be explored to a variety of high throughput drug screening.     

Sample concentration and impedance detection on a microfluidic polymer chip

We present an on-chip microfluidic sample concentrator and detection triggering system for microparticles based on a combination of insulator-based dielectrophoresis (iDEP) and electrical impedance measurement. This platform operates by first using iDEP to selectively concentrate microparticles of interest based on their electrical and physiological characteristics in a primary fluidic channel; the concentrated microparticles are then directed into a side channel configured for particle detection using electrical impedance measurements with embedded electrodes. This is the first study showing iDEP concentration with subsequent sample diversion down an analysis channel and is the first to demonstrate iDEP in the presence of pressure driven flow. Experimental results demonstrating the capabilities of this platform were obtained using polystyrene microspheres and Bacillus subtilis spores. The feasibility of selective iDEP trapping and impedance detection of these particles was demonstrated. The system is intended for use as a front-end unit that can be easily paired with multiple biodetection/bioidentification systems. This platform is envisioned to act as a decision-making component to determine if confirmatory downstream identification assays are required. Without a front end component that triggers downstream analysis only when necessary, bio-identification systems (based on current analytical technologies such as PCR and immunoassays) may incur prohibitively high costs to operate due to continuous consumption of expensive reagents.     

浓度与压力梯度可调的三维细胞培养微流控芯片的研制

目的制作化学浓度梯度与压力梯度可调的细胞三维培养微流控芯片,构建可模拟在体细胞生长所处动态微环境的体外模型。方法利用光刻成型技术、模塑法以及等离子键合工艺,制作3通道结构的微流控细胞培养芯片。通过微注射泵控制微通道内溶液流动生成浓度梯度,利用液面高度差生成压力梯度,并通过骨架染色比较二维培养与三维培养下的细胞形态。结果获得了化学浓度梯度与压力梯度可调的微流控细胞培养芯片。在2μL.min-1的流速下,中间通道的浓度梯度3 h后可达到相对稳定。100 Pa的压力差在中间通道生成的视在压力梯度为0.11 Pa/μm,从而驱动三维支架内间隙渗流的生成。并在微流控芯片内实现脐静脉内皮细胞稳定的三维培养。结论该芯片结构简单,制作方便,能灵活调控细胞生长所处的微环境,可进一步用于研究不同的微环境参数对细胞行为的影响。     

A self-contained fully-enclosed microfluidic cartridge for lab on a chip

We describe a self-contained fully-enclosed cartridge for lab-on-a-chip applications where sample and reagents can be applied sequentially as is performed in a heterogeneous immunoassay, or nucleic acid extraction. Both the self-contained and fully-enclosed features of the cartridge are sought to ensure its safe use in the field by unskilled staff. Simplicity in cartridge design and operation is obtained via adopting a valveless concept whereby reagents are stored and used in the form of liquid plugs isolated by air spacers around a fluidic loop. Functional components integrated in the loop include a microfluidic chip specific to the target application, a novel peristaltic pump to displace the liquid plugs, and a pair of removable tubing segments where one is used to introduce biological sample and while the other is to collect eluant. The novel pump is fabricated through soft-lithography technique and works by pinching a planar channel under stainless-steel ball bearings that have been magnetically loaded. The utility of the cartridge is demonstrated for automated extraction and purification of nucleic acids (DNA) from a cell lysate on a battery-operated portable system. The cartridge shown here can be further extended to sample-in-answer-out diagnostic tests.     

体外仿生三维血管生成微流控芯片的构建

目的在微流控芯片上构建模拟人体血管的三维血管管道,实现管道间的物质交换,并形成有自主出芽功能的血管,为血管生成相关的疾病机制研究、药物筛选等提供良好的平台工具。方法利用微流控技术,采用被动进样方式,使人体脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)在微流控芯片上自主贴壁生长形成三维血管管腔,构建体外仿生三维单管道血管和双管道血管芯片。在此基础上,开展对血管响应刺激因子出芽的功能的验证实验。结果体外仿生的双管道芯片的构建成功率达80%以上。在血管管道,内皮细胞无需借助外力支撑,在芯片上自主形成直径300μm±50μm,细胞分布均匀的、类似体内血管的三维血管管腔;物质通过模拟体内的扩散过程达到血管管道,使血管管腔响应物质刺激而出现功能性的血管出芽。结论体外仿生三维血管微流控芯片在体外实现了体内血管的结构与功能,模拟了体内物质扩散对血管生成的影响,可以用于生理过程中血管生成的体外动态观察。     

A microfluidic chip for permeability assays of endothelial monolayer

Endothelial cell monolayer (EM), acting as a barrier between blood and tissue, plays an important role in pathophysiological processes. Here we describe a novel microfluidic chip that is applied for convenient and high throughput in vitro permeability assays of EM. The chip included a gradient generator and an array of cell culture chambers. A microporous membrane as a scaffold component was built between a polydimethylsiloxane (PDMS) layer and a glass substrate to grow EM. Cell culture chambers were separated by microchannels and microvalves. The concentration gradient of compound solutions could be generated automatically and affected EM in different chambers. The permeability of EM at different time with histamine stimulation was in situ measured by the fluorescence detection of the leaked tracer. The existence of continuous flow in the channels allowed EM in a dynamic microenvironment and increased the amount of tracer through the EM, comparing to transwell assays. According to the prototype chip, the chip with a bigger array of cell culture chambers could be achieved easily and applied in the high throughput screening for drugs.     

Drug effects analysis on cells using a high throughput microfluidic chip

Usually cell-based assay is performed using titer plates. Because of the large library of chemical compounds, robust and rapid methods are required to find, refine and test a potential drug candidate in an efficient manner. In this article, the drug effects analysis on human breast cancer cells with a droplet microfluidic chip is reported. Each droplet serves as a nanoliter-volume titer plate and contains a human breast cancer cell MDA-MB-231, Cytochalasin D drug solution and cell viability indicator such as Calcein AM, which emits cytoplasmic green fluorescence. The drug effects on each cell are monitored in real time using a fluorescence microscope and by analyzing the fluorescence image of each cell. Clear change of the cell shape and size has been observed after the drug treatment, which is similar to that of conventional petri dish technique, suggesting this approach is a potential viable technical platform for drug effect analysis and for high throughput drug screen and discovery.