用于单细胞RNA分析的循环肿瘤细胞团捕获和单细胞分离方法的质量评估

循环肿瘤细胞团（Circulating tumor cell cluster, CTC cluster）的单细胞分析对CTC cluster的生物学效应分析极其重要。为了使CTC cluster单细胞分析可以获得最有效的结果,分析前的捕获和单细胞分离的过程必须能够保持CTC cluster的完整性和细胞内部RNA的完整。本研究通过单细胞RNA测序中的逆转录方法逆转录并定量3个看家基因进行RNA质控,同时测量CTC cluster的横向直径用于评估CTC cluster的完整性,从而测定CTC cluster捕获和单细胞分离过程对CTC cluster单细胞分析结果的影响。分别采用胰酶-EDTA和非酶细胞解离溶液（Non-enzymatic cell dissociation solution, NECDS）分离CTC cluster中的单细胞,并考察了单细胞分离过程对胞内RNA的影响。考察了负向富集、正向富集和膜过滤捕获方案中不同操作流程对CTC cluster完整性和单细胞内RNA完整性的影响。结果表明,NECDS可快速分离CTC cluster中的单细胞且不会对细胞内RNA产生影响...     

多功能芯片对合成样本中肝癌细胞HepG2的测试研究

设计并制作了一种集多孔流分离(Multi-orifice flow fractionation,MOFF)技术与磁捕获技术于一体的用于特异性分离和捕获合成样本中肝癌细胞HepG2的多功能微流控细胞芯片.此芯片由玻璃基片和PDMS微通道盖片组成,PDMS盖片上含有3条进样通道、MOFF分离区和六边形腔体的细胞富集检测区.其中,MOFF分离区总长20 mm,由80组长度为0.18 mm、深度为50μm、收缩区域宽度为0.06 mm、扩张区域宽度为0.20 mm的半菱形收缩/扩张重复单元组成,每组收缩/扩张重复单元间的夹角为103.0°.实验以肝癌细胞HepG2-血细胞混悬液为样本;根据磁珠表面修饰c-Met抗体能与肝癌细胞HepG2特异性结合的原理,通过表面羧基化的磁珠、EDC(1 mg/mL)、NHS(1 mg/mL)和c-Met抗体制备了浓度为50μg/mL的免疫磁珠(Anti-MNCs)悬浮液.在样本流速为50μL/min条件下,利用外加磁场实现了血细胞合成样本中微量肝癌细胞HepG2的有效捕获;采用微波加热法以柠檬酸、硫脲为原料制备了用于荧光标记HepG2的碳量子点,在芯片上实现了血液中肝癌细胞HepG2的原位荧光可视化观测.对芯片检测区捕获到的HepG2进行了显微计数分析,对500μL血细胞(107 cell/mL)中含10个HepG2细胞的合成样本,捕获效率达到88.5%±6.7%(n=20).结果表明,所设计的多模式多功能的微流控芯片具有良好的肿瘤细胞分离和检测功能.     

循环肿瘤细胞及细胞外囊泡的纳米检测技术

肿瘤液体活检通过对体液中生物标志物的检测实现疾病的精准诊断,对于恶性肿瘤的早期诊断和动态监测至关重要。循环肿瘤细胞(CTC)是肿瘤组织释放到血液中的肿瘤细胞,细胞外囊泡(EV)是由细胞分泌的膜囊泡,二者都携带肿瘤分子信息,并与肿瘤进展和转移密切相关,是重要的液体活检生物标志物,并且在检测方法和临床意义方面也有很多共性。纳米材料由于其高比表面积、独特的光、电、磁等物理化学特性以及易于功能化修饰等特点,被广泛用于CTC和EV的检测,以提高检测灵敏度和特异性,提供肿瘤形成、进展、转移和治疗反应的信息,具有很好的应用前景。本文回顾了在CTC和EV的特异性识别、高效捕获或分离、目标CTC或EV的鉴定等三方面的纳米技术进展,包括提高分子识别特异性的纳米材料表面识别探针功能化修饰以及捕获、鉴定的纳米材料和纳米技术的最新进展,总结了基于功能化纳米材料的液体活检技术的优势及挑战,为液体活检纳米技术的发展提供信息。     

简易型微流控芯片捕获循环肿瘤细胞的研究

本研究以循环肿瘤细胞(乳腺肿瘤细胞)为研究对象,以聚二甲基硅氧烷(PDMS)、双面粘性薄膜(DSA)、玻片为原材料,采用激光雕刻技术制作微流控芯片,结合巯基-马来酰亚胺基团硅烷化偶联法和免疫荧光技术进行芯片内捕获检测实验,并使用外周血肿瘤细胞来验证此微流控芯片的实用性,使用具有高速摄像功能的荧光显微镜进行镜下观察及拍摄. 成功构建了一种简易型微流控芯片系统,利用此系统可实现对乳腺肿瘤细胞(92±3)%的捕获率,对外周血肿瘤细胞(88±3)%的捕获率,而且芯片的制作工艺简单,对实验仪器要求低,1 min内即可制作完成,简化了制作过程,弥补了传统光刻工艺复杂繁琐的不足,为临床检测疾病的发生与发展提供了新的研究方向.     

阵列式对电极介电电泳芯片及其用于细胞分离富集研究

基于介电电泳原理, 设计并制作了一种新型的能够用于细胞分离和富集的微流控介电电泳芯片. 该芯片由沉积有金电极的石英基片和带有微管道的聚二甲基硅氧烷(PDMS)盖片组成. 通过在管道底部布置间距不同的对电极阵列, 增大了正介电电泳力在管道中的有效作用范围, 能够在降低施加电压的同时, 实现对流动体系中细胞样品的捕获. 在3 V和3 MHz条件下, 该DEP芯片对人血红细胞的捕获效率达到83%; 进一步通过将肝癌细胞捕获在芯片电极上可实现对红细胞和肝癌细胞混合样品的分离, 在5 V和400 kHz条件下对肝癌细胞的捕获效率达到86%.     

基于表面等离子体子共振成像技术研究苏拉明和顺铂对肝癌细胞生长的抑制作用

基于表面等离子体子共振成像(SPRi)技术提出了一种实时、 非标记的新型抗癌药物药效评估方法. 以聚二甲基硅氧烷(PDMS)为材料, 制作了包含微柱结构的微流控芯片作为流通反应池, 配合自行设计组装的SPRi生物传感器完成肿瘤细胞的特异性捕获及检测, 研究了苏拉明和顺铂对肝癌细胞HepG2的生长抑制作用. 同时引入辅助验证实验, 即采用常规八肽胆囊收缩素(简称CCK-8)法测定上述药物对肝癌细胞增殖的抑制作用. SPRi检测结果表明, 苏拉明和顺铂能抑制肿瘤细胞HepG2增殖并呈现剂量、 时间依赖关系.     

基于表面等离子体子共振成像技术研究苏拉明和顺铂对肝癌细胞生长的抑制作用简

基于表面等离子体子共振成像(SPRi)技术提出了一种实时、非标记的新型抗癌药物药效评估方法. 以聚二甲基硅氧烷(PDMS)为材料,制作了包含微柱结构的微流控芯片作为流通反应池,配合自行设计组装的SPRi生物传感器完成肿瘤细胞的特异性捕获及检测,研究了苏拉明和顺铂对肝癌细胞HepG2的生长抑制作用. 同时引入辅助验证实验,即采用常规八肽胆囊收缩素(简称CCK-8)法测定上述药物对肝癌细胞增殖的抑制作用. SPRi检测结果表明,苏拉明和顺铂能抑制肿瘤细胞HepG2增殖并呈现剂量、时间依赖关系.     

基于聚苯胺纳米纤维复合膜界面构建电化学细胞传感器及其对癌细胞的识别检测

合成了聚苯胺纳米纤维,直径在50～70 nm之间;基于静电作用构建聚苯胺纳米纤维-纳米金复合膜界面,并在此界面上层层组装修饰叶酸分子,构建叶酸功能化传感界面,基于叶酸分子与癌细胞表面过量表达的叶酸受体之间的特异性识别作用,将此传感界面应用于对癌细胞的识别和捕获。结果表明:叶酸功能化传感界面能够特异性识别和捕获叶酸受体过量表达的癌细胞。采用电化学阻抗技术,以HeLa细胞为模型,应用于对癌细胞的识别和检测,细胞在1.0×104～6.4×106cells/mL浓度范围内与阻抗变化值ΔRct呈良好的线性关系;检出限为2000 cells/mL。本方法简单、快速灵敏、重现性和稳定性良好;制备的传感器可以再生使用。     

纳米银修饰电极差分脉冲伏安法测定盐酸金霉素

建立了快速测定盐酸金霉素(CTC)的方法。通过NaBH4还原法制备纳米银(AgNPs)溶胶,并利用X射线衍射和紫外-可见光谱进行表征。将制备好的AgNPs滴涂到玻碳电极表面制备修饰电极(AgNPs/GCE),研究了CTC在AgNPs/GCE上的电化学行为及伏安法测定,优化了缓冲溶液和pH等检测条件。结果表明,CTC在pH 3.3的柠檬酸-NaOH-HCl缓冲溶液中检测效果最佳。CTC在AgNPs/GCE上发生2个电子和2个质子的不可逆电化学氧化反应,且反应受吸附控制。最佳条件下,CTC的氧化峰电流与其浓度呈现良好的线性关系,线性范围为0.5~100μmol/L,检出限为0.14μmol/L。该修饰电极可用于河水样品检测。     

基于流体动力单细胞高效捕获的微流控芯片结构研究进展

单细胞分析对于重大疾病的早期诊断及治疗、药物筛选和生理病理过程的研究具有重要意义。微流控芯片能够精确控制单细胞的微环境,实时监测单细胞的行为,已成为单细胞分析的强大工具。单细胞捕获是单细胞分析的重要步骤。目前已报道了多种微流控芯片用于单细胞捕获的方法,其中基于流体动力的微流控芯片单细胞捕获方法具有操作方便、单细胞捕获效率高等优点,受到研究人员的广泛关注及使用。为了全面了解基于流体动力的微流控芯片单细胞捕获方法的研究现状,掌握单细胞高效捕获的微流控芯片结构设计,实现单细胞精准快速分析,本文综述了基于流体动力的单细胞高效捕获（>70%）原理及微流控芯片结构,根据结构设计不同分为微井结构、微柱结构和旁路通道结构,介绍了单细胞高效捕获的微流控芯片优化过程,总结了微流控芯片的材质、结构特点及单细胞捕获效率等,对不同单细胞捕获结构的优势及不足进行了分析。最后,对基于流体动力的微流控芯片单细胞捕获方法的发展趋势进行了展望。     

Graphene-Based Biosensors with High Sensitivity for Detection of Ovarian Cancer Cells

Ovarian cancer has the highest mortality rate in the world. Therefore, it is urgent but still challenging to develop an efficient circulating tumor cell (CTC) detection method to sensitively detect ovarian cancer. To address such issues, herein, for the first time, we present a novel CTC detection method for ovarian cancer cells by designing sensitive and rapid graphene-based biosensors. This graphene-based sensor, consisting of a cell pool and two electrodes, can be prepared by a conventional chip fabrication process. It demonstrates high-sensitivity detection even for several ovarian cancer cells by comparing the electrical signal before and after adding cell solution. Moreover, the graphene-based biosensors can perform rapid detection with good repeatability. This suggests that this novel method is possible to use for the early detection of ovarian cancer with very low CTC cell concentration. This work provides a novel and quick strategy to detect ovarian cancer and further judge or predict the risk of the transfer of ovarian cancer.     

微流控芯片系统在循环肿瘤细胞分离检测中的应用进展

循环肿瘤细胞(CTCs)的分离分析一直是肿瘤相关研究中的热点方向,作为液体活检的重要标志物之一,其在外周血中的含量与癌症病发状况密切相关.然而人体血液中CTCs的含量非常低,通常来说仅有0～10个/mL,因此在开展临床血液样本中CTCs的检测前,往往需要对样本进行前处理,以实现CTCs的分离和富集.微流控芯片技术凭借样...     

Integration of cell phone imaging with microchip ELISA to detect ovarian cancer HE4 biomarker in urine at the point-of-care

Ovarian cancer is asymptomatic in the early stages and most patients present with advanced levels of disease. The lack of cost-effective methods that can achieve frequent, simple and non-invasive testing hinders early detection and causes high mortality in ovarian cancer patients. Here, we report a simple and inexpensive microchip ELISA-based detection module that employs a portable detection system, i.e., a cell phone/charge-coupled device (CCD) to quantify an ovarian cancer biomarker, HE4, in urine. Integration of a mobile application with a cell phone enabled immediate processing of microchip ELISA results, which eliminated the need for a bulky, expensive spectrophotometer. The HE4 level detected by a cell phone or a lensless CCD system was significantly elevated in urine samples from cancer patients (n = 19) than healthy controls (n = 20) (p < 0.001). Receiver operating characteristic (ROC) analyses showed that the microchip ELISA coupled with a cell phone running an automated analysis mobile application had a sensitivity of 89.5% at a specificity of 90%. Under the same specificity, the microchip ELISA coupled with a CCD had a sensitivity of 84.2%. In conclusion, integration of microchip ELISA with cell phone/CCD-based colorimetric measurement technology can be used to detect HE4 biomarker at the point-of-care (POC), paving the way to create bedside technologies for diagnostics and treatment monitoring.     

Diagnostic microchip to assay 3D colony-growth potential of captured circulating tumor cells

Microfluidic technology has been successfully applied to isolate very rare tumor-derived epithelial cells (circulating tumor cells, CTCs) from blood with relatively high yield and purity, opening up exciting prospects for early detection of cancer. However, a major limitation of state-of-the-art CTC-chips is their inability to characterize the behavior and function of captured CTCs, for example to obtain information on proliferative and invasive properties or, ultimately, tumor re-initiating potential. Although CTCs can be efficiently immunostained with markers reporting phenotype or fate (e.g. apoptosis, proliferation), it has not yet been possible to reliably grow captured CTCs over long periods of time and at single cell level. It is challenging to remove CTCs from a microchip after capture, therefore such analyses should ideally be performed directly on-chip. To address this challenge, we merged CTC capture with three-dimensional (3D) tumor cell culture on the same microfluidic platform. PC3 prostate cancer cells were isolated from spiked blood on a transparent PDMS CTC-chip, encapsulated on-chip in a biomimetic hydrogel matrix (QGel?) that was formed in situ, and their clonal 3D spheroid growth potential was assessed by microscopy over one week in culture. The possibility to clonally expand a subset of captured CTCs in a near-physiological in vitro model adds an important element to the expanding CTC-chip toolbox that ultimately should improve prediction of treatment responses and disease progression.     

Poly(methylmethacrylate) and Topas capillary electrophoresis microchip performance with electrochemical detection

A capillary electrophoresis (CE) microchip made of a new and promising polymeric material: Topas (thermoplastic olefin polymer of amorphous structure), a cyclic olefin copolymer with high chemical resistance, has been tested for the first time with analytical purposes, employing an electrochemical detection. A simple end-channel platinum amperometric detector has been designed, checked, and optimized in a poly-(methylmethacrylate) (PMMA) CE microchip. The end-channel design is based on a platinum wire manually aligned at the exit of the separation channel. This is a simple and durable detection in which the working electrode is not pretreated. H(2)O(2) was employed as model analyte to study the performance of the PMMA microchip and the detector. Factors influencing migration and detection processes were examined and optimized. Separation of H(2)O(2) and L-ascorbic acid (AsA) was developed in order to evaluate the efficiency of microchips using different buffer systems. This detection has been checked for the first time with a microchip made of Topas, obtaining a good linear relationship for mixtures of H(2)O(2) and AsA in different buffers.     

Analysis of multiplex PCR fragments with PMMA microchip

Microchip electrophoresis is a promising technique for analysis of bio-molecules. It has the advantages of fast analysis, high sensitivity, high resolution and low-cost of samples. Plastic chip has the potential of mass production for clinical use for its advantages in biocompatibility and low cost. In this work, the method for fabrication of poly(methyl methacrylate) (PMMA) chip was described, and conditions for DNA separation were investigated with the chip. The PMMA microchip was used for detection of multiplex PCR products of 18 and 36 cases with SARS and hepatitis B virus infection under optimized separation conditions. Microchip electrophoresis showed higher sensitivity, higher resolution and less time consumption when compared with gel electrophoresis. The microchip electrophoresis with PMMA chip provided a rapid, sensitive and reliable method for analysis of multiplex PCR products.     

Bioinspired Engineering of a Multivalent Aptamer‐Functionalized Nanointerface to Enhance the Capture and Release of Circulating Tumor Cells

Circulating tumor cell (CTC)‐enrichment by using aptamers has a number of advantages, but the issue of compromised binding affinities and stabilities in real samples hinders its wide applications. Inspired by the high efficiency of the prey mechanism of the octopus, we engineered a deterministic lateral displacement (DLD)‐patterned microfluidic chip modified with multivalent aptamer‐functionalized nanospheres (AP‐Octopus‐Chip) to enhance capture efficiency. The multivalent aptamer–antigen binding efficiency improves 100‐fold and the capture efficiency is enhanced more than 300 % compared with a monovalent aptamer‐modified chip. Moreover, the captured cancer cells can be released through a thiol exchange reaction with up to 80 % efficiency and 96 % viability, which is fully compatible with downstream mutation detection and CTC culture. Using the chip, we were able to find CTCs in all cancer samples analyzed.     

循环肿瘤细胞体内检测技术及其应用研究

从实体瘤脱落进入血液循环系统的肿瘤细胞即循环肿瘤细胞(CTCs)与肿瘤转移密切相关,因此CTCs检测对癌症患者的诊断、治疗监测、病情评估以及肿瘤转移机制研究具有重要意义.由于CTCs在体内含量极少、异质性、分布不均一,通过体外采血发展的CTCs检测技术虽然已取得很大进展,但仍然面临肿瘤细胞损失、失活、失真以及灵敏度低等...     

EQCM监测高氯酸溶液中金电极上苯二酚电荷转移配合物的沉积

用电化学石英晶体微天平(EQCM)研究了高氯酸溶液中电位环扫过程中邻(间、对)苯二酚/醌电荷转移配合物(CTC)的生成和电沉积。结果发现,邻(对)苯二酚可产生CTC并在电极上沉积,而间苯二酚仅发生电聚合反应。以邻苯二酚为例考察了高氯酸和邻苯二酚浓度及电位扫描速度的影响。红外光谱证实了邻苯二酚/醌CTC的生成。结果发现,添加肝素钠对邻苯二酚/醌CTC的沉积有显著影响,肝素钠质量浓度分别低/高于0.05 g/L时,能增强/减弱沉积现象。EQCM技术可作为一种快速、简便、灵敏的工具可望用于CTC制备过程中的实时监测和筛选。