基于微流体技术分选循环肿瘤细胞的方法研究

循环肿瘤细胞（Circulating Tumor Cells, CTCs）主要来源于肿瘤组织自发脱落的外周血液中,其对恶性肿瘤传播转移具有重要影响,已逐渐被认为是肿瘤远处转移的标志。对外周血中极微量的具有特异性、敏感性的CTCs进行分选、富集及检测,不仅有利于肿瘤的早期诊断、疗效评价及复发转移监控,还可以为后续的CTCs鉴定和下游单细胞基因组和转录组测序提供良好基础,为肿瘤靶向治疗提供新策略,在临床上个性化医疗等领域有重要意义。微流控技术可在微米尺度下整合物理、化学及生物学方法,实现对微量CTCs的高通量、高效率以及低成本分选富集,该技术发展迅速并已被广泛研究应用。本文对微流控芯片进行CTCs分选富集及捕获的最新研究进展进行综述,阐述无标记、有标记分选方法的原理及应用实例,分析各种技术的优缺点,对现阶段该领域存在的问题进行讨论,对微流控CTCs分选芯片的应用前景和未来发展趋势进行展望。     

循环肿瘤细胞的微流控芯片分离及其物理性质测量分析

目的建立一种使用微吸管测量具有良好生理活性的循环肿瘤细胞(circulating tumor cells, CTCs)弹性模量的方法。方法使用商品化的微流控芯片富集血液中具有良好生理活性的CTCs,使用EpCAM抗体确定CTCs,并使用微吸管测量其弹性模量,同时与癌细胞系弹性模量相对比。结果对于癌细胞系的弹性模量,不仅在不同细胞系间存在较大的差异,在同一细胞系间也存在较大的异质性。血液中CTCs相比于同种癌细胞系属于弹性模量较小的癌细胞。结论该方法能够获得生理活性较好的CTCs并测量其弹性模量,为进一步研究CTCs力学特性与癌症诊断和治疗预后之间的相关关系,推进癌细胞物理标志物的发展提供细胞力学数据支持。     

应用微流控芯片富集循环肿瘤细胞的发展现状及展望

循环肿瘤细胞(circulating tumor cell,CTCs)存在于肿瘤患者的外周血中,与恶性肿瘤的转移、预后评价以及临床的个体化治疗均密切相关。相比于组织活检,CTCs的检测仅需抽取静脉血即可进行,因而具有取样简单、可重复性、无创等优点。CTCs的富集方法有很多,近年来兴起的微流控芯片分选技术具有高通量、低消耗、可集成等特点。基于CTCs的物理性质和生物性质所设计的不同芯片在分选速度、效率、纯度以及细胞活性保持方面各有优势,本文将对此加以重点阐述。     

微流控芯片技术在免疫分析中的应用

微流控芯片已广泛用于生物医学、高通量药物合成筛选、环境监测和生物战剂侦检等领域,本文就微流控芯片在免疫分析中的应用做一综述。1微流控芯片技术分析概述微流控芯片技术是通过微细加工技术在芯片上构建由储液池、微反应室、微管道等微功能元件构成的微流路系统,加载生物样品和反应液后,在压力泵或者电场作用下形成微流路,于芯片上进行一种或连续多种的反应,达到对样品高通量快速分析的目的。微流控芯片技术由于具有高度集成性,可在一张芯片上完成采样、稀释、加试剂、反应、分离和检测等多种功能,又被称为微型全分析系统(micro total a…     

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。     

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。     

循环肿瘤细胞检测方法研究进展

循环肿瘤细胞(CTCs)检测在肿瘤发生、发展、早期转移、疗效监测和制定个体化治疗方案等方面都有重要的临床意义。但因其在外周血中数量少,检测难度大,至今没有统一的检测标准。现有的CTCs检测技术包含富集和鉴定两方面。富集方法主要有密度梯度离心法、免疫磁珠分离法、芯片富集技术等。鉴定方法主要有免疫磁珠分离法、聚合酶链反应(PCR)技术法、CTCs芯片检测法等。新技术、新方法在不断的建立和发展中,如微流体芯片技术、CTCs原位检测平台、光控开关流体细胞仪等,CTCs检测的灵敏度、特异度和临床标本的适用性进一步得到提高。文章就其最新研究进展做一综述。     

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。     

基于DM642的嵌入式实时细胞显微分析系统的设计

一种基于数字信号处理器(DSP)TMS320DM642的嵌入式实时细胞显微分析系统,采用高分辨率的CMOS图像传感器,经不同倍数的光学放大透镜,可以实现对血液细胞形态图像的采集.DSP对采集的数据进行处理,通过系统的薄膜液晶显示器(TFT LCD)实时地显示图像及数据处理的结果.本系统克服了目前实验室使用的显微镜装配数码相机对细胞成像的设备庞大、价格昂贵等缺点.本系统结构紧凑,配合微流控芯片系统,可以实现微流控芯片上细胞形态的实时检测,充分发挥了微流控芯片系统检验方便、快捷等优点.     

基于微流控技术的红细胞与VWF损伤规律研究

目的基于微流控技术探讨剪切应力大小和暴露时间长短对血液成分红细胞和血管性血友病因子(VWF)损伤的影响。方法基于微流控芯片搭建血液剪切平台,制备不同剪切应力大小和暴露时间长短下的样品,对血液样品进行游离血红蛋白测定实验,测得不同样品的溶血指数,并通过免疫印迹法和化学发光成像分析不同样品VWF的相对分子质量。结果溶血指数和高相对分子质量VWF降解率与剪切应力和暴露时间之间的定量关系很好地遵循幂函数模型。结论微流控实验平台具有内部微环境精确可控,便于快速检测等优点,可以用于血液损伤规律的定量研究。     

Clinical significance of circulating tumor cells from lung cancer patients using microfluidic chip

Circulating tumor cells (CTCs) exist in the peripheral blood and have an important role in the disease development, tumor metastasis and clinical surveillance, especially in the process of metastasis. However, the technology of detecting CTCs still had a large challenge since they were rare in the peripheral blood. Here, we developed a size-based microfluidic chip, which contained array and filter channel array that could enrich CTCs from blood samples more quickly and conveniently. Combined with clinical specimen, we analyzed CTCs in 200 lung cancer patients by this microfluidic chip. The microfluidic device has high specificity and sensitivity in detecting CTCs (86.0% sensitivity and 98% specificity). Furthermore, the number of CTCs showed a increasing trend according to the stage of the disease (the mean number of I stage 5.0 ± 5.121 versus II stage 8.731 ± 6.36 versus III stage 16.81 ± 9.556 versus IV stage 28.72 ± 17.39 cells/mL, P < 0.05). The number of CTCs was concurrent with the condition of pathological type and metastasis patients. Compared to conventional markers like CEA, CY211, SCC, CTCs showed a higher positive rate in diagnosed patients. The advanced microfluidic device could capture tumor cells without reliance on cell surface expression markers and provide a fast, convenient, economical method in detecting CTCs, thereby offering potential to design effective and individualized cancer therapies.     

Combination of antibody-coated,physical-based microfluidic chip with wave-shaped arrays for isolating circulating tumor cells

Circulating tumor cells (CTCs) are found in the peripheral blood of patients with metastatic cancers, which have critical significance in cancer prognosis and diagnostics. Enumeration is significantly valuable since number of CTCs is strongly correlated to severity of disease. This article is proposed and demonstrated an antibody-coated, size-based microfluidic chip with wave-shaped arrays could efficiently capture CTCs combining two separation methods of both size- and deformability-based and affinity-based segregation. Utilizing immunocapture of capture chemistry of Epithelial Cell Adhension Molecule (EpCAM), tumor cells could be captured by narrow gaps or have a friction with microposts edges to realize both immune-affinity and size capture. This wave-shaped layout of microfluidic chip with varying gaps between adjacent circular microposts can generate perpendicular velocities to the fluidic direction. This oriented fluidic direction will carry cells to next smaller neighboring gap and then be captured gradually. The experiment results indicate capture efficiency is ~90% and viability is ~95% after extracted and cultured 3 days. Furthermore, this chip has been validated for whole blood with cancer cell lines and mimic patient blood. This study demonstrates feasibility using our microfluidic chip for CTCs research, monitoring cancer progress and evaluating therapeutic treatment.     

An efficient method for CTCs screening with excellent operability by integrating Parsortix™-like cell separation chip and selective size amplification

In this article, an attempt for efficient screening of circulating tumor cells (CTCs) with excellent operability on microfluidic chips was reported. A Parsortix?-like cell separation chip was manufactured in our lab. This chip allowed lateral flow of fluid which increased the flow rate of blood. And, an air valve controlled injection pump was manufactured which allowed eight chips working simultaneously. This greatly facilitated the blood treatment process and saved time. As for the mechanism of screening circulating tumor cells, selective size amplification was utilized. By size amplification of cancer cells, both the hardness and the size of CTCs increased which differentiated them from blood cells. And the modification procedure of beads used for size amplification of cancer cells was optimized. Finally, by integrating the commercialized Parsortix?-like cell separation chip and selective size amplification, a practical method for screening circulating tumor cells was established.     

Simultaneous isolation and detection of circulating tumor cells with a microfluidic silicon-nanowire-array integrated with magnetic upconversion nanoprobes

The development of sensitive and convenient methods for detection, enrichment, and analysis of circulating tumor cells (CTCs), which serve as an importance diagnostic indicator for metastatic progression of cancer, has received tremendous attention in recent years. In this work, a new approach characteristic of simultaneous CTC capture and detection is developed by integrating a microfluidic silicon nanowire (SiNW) array with multifunctional magnetic upconversion nanoparticles (MUNPs). The MUNPs were conjugated with anti-EpCAM antibody, thus capable to specifically recognize tumor cells in the blood samples and pull them down under an external magnetic field. The capture efficiency of CTCs was further improved by the integration with a microfluidic SiNW array. Due to the autofluorescence free nature in upconversion luminescence (UCL) imaging, our approach allows for highly sensitive detection of small numbers of tumor cells, which afterward could be collected for further analysis and re-culturing. We have further demonstrated that this approach can be applied to detect CTCs in clinical blood samples from lung cancer patients, and obtained consistent results by analyzing the UCL signals and the clinical outcomes of lung cancer metastasis. Therefore our approach represents a promising platform in CTC capture and detection with potential clinical utilization in cancer diagnosis and prognosis.     

A simple pyramid-shaped microchamber towards highly efficient isolation of circulating tumor cells from breast cancer patients

Isolation and detection of circulating tumor cells (CTCs) has showed a great clinical impact for tumor diagnosis and treatment monitoring. Despite significant progresses of the existing technologies, feasible and cost-effective CTC isolation techniques are more desirable. In this study, a novel method was developed for highly efficient isolation of CTCs from breast cancer patients based on biophysical properties using a pyramid-shaped microchamber. Through optimization tests, the outlet height of 6 μm and the flow rate of 200 μL/min were chosen as the optimal conditions. The capture efficiencies of more than 85% were achieved for cancer cell lines (SKBR3, BGC823, PC3, and H1975) spiked in DMEM and healthy blood samples without clogging issue. In clinic assay, the platform identified CTCs in 13 of 20 breast cancer patients (65%) with an average of 4.25?±?4.96 CTCs/2 mL, whereas only one cell was recognized as CTC in 1 of 15 healthy blood samples. The statistical analyses results demonstrated that both CTC positive rate and CTC counts were positive correlated with TNM stage (p < 0.001; p?=?0.02, respectively). This microfluidic platform successfully demonstrated the clinical feasibility of CTC isolation and would hold great potential of clinical application in predicting and monitoring the prognosis of cancer patients.     

Analytical evaluation for somatic mutation detection in circulating tumor cells isolated using a lateral magnetophoretic microseparator

CTCs are currently in the spotlight because provide comprehensive genetic information that enables monitoring of the evolution of cancer and selection of appropriate therapeutic strategies that cannot be obtained from a single-site tumor biopsy. Despite their importance, current techniques for isolating CTCs are limited in terms of their ability to yield high-quality CTCs from peripheral blood for use in profiling cancer genetic mutations by DNA sequencing technologies. This paper introduces a lateral magnetophoretic microseparator (the ‘CTC-μChip’) for isolating highly pure CTCs from blood, which facilitates the detection of somatic mutations in isolated CTCs. To isolate CTCs from peripheral blood, nucleated cells were first prepared by red blood cell lysis. Then, CTCs were isolated from nucleated cells within 30 min using the CTC-μChip. Analytical evaluation using 5 mL blood samples spiked with 5–50 MCF7 breast cancer cells demonstrated that the average recovery rate of the CTC-μChip was 99.08 %. The average number of residual white blood cells (WBCs) in isolated samples was 53, meaning that the WBC depletion rate is 472,000-fold (5.67 log), assuming that blood contains 5?×?10⁶ WBCs per milliliter. The isolated MCF7 cells had a purity of 6.9???67.9 %, depending on the spiked MCF7 concentration. Using next-generation sequencing technology, heterozygous somatic mutations (PIK3CA and APC) of MCF7 cells were evaluated in the isolated samples. The results showed that somatic mutations could be detected in as few as two MCF7 cells per milliliter of blood, indicating that the CTC-μChip facilitates the detection of somatic variants in CTCs.     

Cross-Talk Problem on a Fluorescence Multi-Channel Microfluidic Chip System

Development of a compact fluorescence-based detection system for use in a micro-analytical system, such as a point-of-care diagnostic system, often requires a multi-channel microfluidic chip system. Since the materials used for microfluidic chips usually are transparent in the visible region and have a refractive indices higher than that of air or the surrounding environment, the fluorescence emission and scattered excitation light can propagate through the chip. We observed that such propagation can cause cross-talk between adjacent channels, and may become the major source of noise in the system and/or photobleach the fluorescent samples in the adjacent channels, particularly for the small distances between the channels found in microfluidic chips, usually in order of several μ m. We monitored this cross-talk using fluorescein as a fluorescent sample and Mylar sheeting as a microfluidic chip material. We then discuss how this cross-talk can be avoided using a simple, inexpensive and effective method.     

Continuous isolation of monocytes using a magnetophoretic-based microfluidic Chip

Monocytes play an important role in the immune system and are responsible for phagocytizing and degrading foreign microorganisms in the body. The isolation of monocytes is important in various immunological applications such as in-vitro culture of dendritic cells. We present a magnetophoretic-based microfluidic chip for rapid isolation of highly purified, untouched monocytes from human blood by a negative selection method. This bioseparation platform integrates several unique features into a microfluidic device, including locally engineered magnetic field gradients and a continuous flow with a buffer switching scheme to improve the performance of the cell separation process. The results indicate high monocyte purity and recovery performances at a volumetric flow rate that is nearly an order of magnitude larger than comparable microfluidic devices reported in literature. In addition, a comprehensive 2-D computational modeling is performed to determine the cell trajectory and trapping length within the microfluidic chip. Furthermore, the effects of channel height, substrate thickness, cell size, number of beads per cell, and sample flow rate on the cell separation performance are studied.     

有核红细胞全血分选微管芯片实验研究

目的 设计微流控芯片分离全血中胎儿有核红细胞（fetal nucleated red blood cell, fNRBCs），实现有核红细胞的快速便捷获取。方法 利用有核细胞在血流作用下的边集效应以及细胞抗原抗体特异性黏附特点,设计微管流控芯片，分离全血中的fNRBCs。以脐血全血为例，通过免疫荧光计数，分析不同剪切率对fNRBCs富集效果的影响。结果 相较于简单静置黏附，增加剪切率可以增加直微管黏附有核红细胞的数量，细胞的富集效果随血流剪切率的增大先增大后减小。结论 利用直微管能够实现fNRBCs的全血快速有效捕获。研究结果 为无创产前诊断的发展以及胎儿细胞转移机制的探究提供实验参考。