基于微流控芯片的免疫反应快速检测系统研究
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摘要
本博士学位论文的主要贡献在于：针对生物医学快速检测中所面临的三大缺陷——成本高、分析时间长、检测灵敏度低,开创性的设计制作了以高分子聚合物PDMS芯片为分析平台,通过有效的修饰方法对微流控芯片通道表面进行修饰和改性,成功实现了微流控芯片通道内生物分子的固定,为生物医学中重大问题的快速检测提供了新的方法,实现了对环境藻毒素的高灵敏度检测,为现场应用提供了基础；并且,成功的对疟疾病实现了临床检测,具有快速、灵敏、高效等优点；完成了对肝癌CTC的快速高效检测,为肝癌的个性化治疗提供可靠的指导。
过去的二十年,是微流控技术发展的二十年。从Manz和Widmer等人1990年首次提出微型全分析系统(Miniaturized Total Analysis System, μTAS)的概念,到2002年Quake等以“微流控芯片大规模集成”为题在Science上发表文章,微流控技术作为当前分析科学的重要发展前沿,在研究方面取得了飞速的发展。分析检测技术手段的逐步微型化,以及现场“即时检测”的提出对分析技术提出更高的要求。因此发展针对生物医学中实际重大问题的微流控快速检测系统是微流控芯片研究与应用的重要方面。
首先,环境中有毒物质的快速检测是目前快速检测领域的重点和难点之一。随着有害水藻的爆发,会产生大量的有害藻类毒素。这些毒素通过食物链,进入鱼类、贝类体内,人食用了污染的鱼类、贝类会引发食物中毒,甚至威胁生命健康。国内外现行的藻类毒素检测技术主要包括：生物检测法,化学检测法,细胞毒性检测技术,放射性标定法,以及免疫检测等。其中,生物检测法主要采用喂食小鼠、家蝇等毒素的方法,缺点是周期长,费用高,重复性差,而且需要专门培训的人员来操作。化学检测法,主要通过高效液相色谱,毛细管电泳,色谱质谱联用方法,重现性好,但设备功能单一,体积大和价格昂贵,便携性、选择性差。细胞毒性检测技术,直观方法来观察加入毒素后,细胞的反应来判断毒素种类,灵敏度高,但只能做定性测量,不能测定浓度而且需要良好的细胞培养技术。放射性标定法仪器昂贵,测试费用高,并且适用毒素检测范围窄。免疫检测技术主要利用抗原和抗体专一、特异结合的特点对毒素进行定性,定量检测,现主要有直接竞争检测法,间接法,“三明治”法。这种方法专一性强,灵敏度高,是非常有潜力的一种方法,但操作比较繁琐,商业化试剂盒价格昂贵,主要为手工操作,自动化程度低而且需要专业人员。
其次,针对重大传染病的临床快速诊断是微流控芯片在应用研究的重要方面。近年来,由于疟原虫对药物、传播媒介对杀虫剂抗性的迅速发展,疟疾快速的诊断对控制全球疟疾的作用显得更加重要。迄今为止,显微镜镜检(厚血膜、薄血膜)仍是疟疾诊断的金标准。但是显微镜镜检作为一种诊断方法又存在一些问题,由于疟原虫形体小,形态相似,难以染色和检出,需要有经验的人员才能做到正确的诊断。疟疾流行区由于熟练镜检技术人员及设备缺乏,仅依靠传统厚薄血膜镜检法已越来越不能适应当前疟疾诊断的需要随着免疫学技术的发展,间接荧光抗体和ELISA均很快在疟疾诊断中得到应用,但是此类方法抗体用量相对较大,成本较高。而且,反应时间长,不能满足快速诊断的要求。PCR技术显示了较高的敏感性和特异性可以对疟原虫的感染做出早期快速诊断。但需要更复杂的仪器设备和技术条件作为支持,不适合作为疟疾流行区常规的检测手段,难以在基层推广应用。
最后,针对肝癌循环肿瘤细胞的高灵敏度捕获受到现有技术的制约。采用反转录聚合酶链式反检测HCC患者外周血中甲胎蛋白mRNA从而检测HCC患者CTCs的报道较多。然而,RT-PCR法有其固有局限,如易产生假阳性和假阴性、操作难以标准化、不能准确估算样本中CTC的数目等。此外,更不可忽视的是,RT-PCR法须破坏细胞形态,不能对单个CTC进行观察、分析和计数,而这些数据可提供CTCs恶性程度和侵袭性方面的信息。因此迫切需要建立特异和敏感的循环肝癌细胞分离和检测技术。目前分离CTCs的标准方法是基于肿瘤细胞表面上皮性抗原的磁性激活细胞分选技术(magnetic-activated cell separation, MACS)。由于缺乏针对肝癌细胞表面特异性抗原的单克隆抗体,迄今鲜见MACS用于分离循环肝癌细胞的报道。尽管肝癌细胞属于上皮性细胞,但EpCAM仅在约35%左右的HCC组织标本中表达。因此,现有系统并不适合用于高通量、高灵敏度分离与检测循环肝癌细胞。
针对以上快速检测领域所面临的三大问题,本博士学位论文分为五章节开展研究中作。各章节主要内容如下：
第一章是绪论部分。本章主要讨论微流控芯片的发展概况,生物医学分析领域的发展现状和遇到的挑战,以及微流控芯片技术在生物医学分析方面的应用进展。从而引出本论文工作的研究方向,为设计发展微流控芯片在生物医学分析快速检测领域应用的新方法研究提出理论依据和实际意义。
第二章主要介绍集成化微流控免疫芯片系统用于环境藻类毒素的高灵敏度快速检测分析新方法研究。我们研究建立了用于对代表性藻类毒素微囊藻毒素(microcystins, MCs),石房蛤毒索(Saxitoxin, STX)与柱孢藻毒素(cylindrospermopsin, CNY)三种藻类毒素的现场快速检测微流控芯片。可以对实际样品(水样和鱼类、贝类等食物样品)溶液中MC, STX与CNY进行快速检测的微流控芯片。实验建立了用于对代表性藻类毒素微囊藻类毒素的现场快速检测微流控芯片。芯片包含七个可以同时控制,也可独立操作的免疫亲和反应柱,可以对实际样品(水样和鱼类、贝类等食物样品)溶液中微囊藻毒素进行快速检测的微流控芯片。检测限为0.02ng/ml。实现对多个样品快速,平行的检测。
第三章主要介绍基于抗体检测的微流控芯片用于疟疾快速临床检测的新方法研究。我们研究了针对在条件欠发达地区多发的传染性疾病疟疾(Malaria)其临床快速现场诊断,提出可行的方案。建立了基于间接免疫荧光反应原理的快速检测微流控芯片用于疟疾病(Malaria)的临床诊断研究。实验中通过检验病人血清样品中的MSP1-19与PfF2抗体的检测,实现了疟疾临床快速诊断,免疫反应中得到的不同荧光强度,可以被信号采集模块采集分析,从而提供临床诊断信息。在同一块芯片实现双抗原同时检测。实验证明,微流控芯片免疫系统可以得到可靠的检测结果,并且极大的节约了抗体用量,缩短了反应所需时间,使得检测成本极大的降低。结果指示,研究中采用MSP1-19与PfF2双抗原检测的方法,对疟疾进行快速检测可以提供相对更加可靠的诊断结果。结果显示,研究建立的微流控芯片免疫系统与方法可以有效的用于基于抗体的疟疾血清学检测,为条件欠发达地区的疾病现场检测,实现疾病有效控制提供可行方案。
第四章主要介绍肝癌循环肿瘤细胞(CTC)的微流控芯片分离检测系统新方法研究。我们在实验中建立了一种具有高度特异性和敏感性的肝癌循环肿瘤细胞分离与检测系统。肝细胞癌(hepatocellular carcinoma, HCC)患者体内的循环肿瘤细胞(circulating tumor cells, CTCs),即循环肝癌细胞,是HCC转移和复发的根源。检测循环肝癌细胞对于预测HCC转移复发无疑具有重要临床意义。研究中建立了一种独特的基于ASGPR及其配体相互作用的循环肝癌细胞微流控芯片分选方法,结合基于肝细胞特异性抗体Hep Par1的免疫荧光染色鉴定,发展了一种新的循环肝癌细胞分离与检测系统。实验中用标准细胞加入全血,测定了系统工作的最佳条件。实验证明系统具有很好的特异性和敏感性,且操作方便,易于标准化。分离到的CTCs可进行免疫形态学鉴定和计数。并初步显示了该系统用于临床检测的可行性。
第五章主要介绍高密度结构PDMS芯片制作技术研究。我们讨论了对微流控芯片的制作软光刻技术提出改进,从而得到稳定重复的微结构。为具有高密度阵列微结构的PDMS芯片制作,提供可靠的方法。针对聚合物材料PDMS芯片在复制模塑成型过程中出现的缺陷问题进行了研究,设计了含有50μm高的不同密度微柱阵列芯片。运用实验提出的方案,成功实现了其复制成型,并且保证了模板的多次重复利用。其最低密度共含有14,400个微柱(40mm×18mm),最高密度含有39,760个微柱(40mm×18mm),其对应的最大结构密度为5×104/cm2。本研究为简化复杂PDMS芯片制作处理,提出一步涂层/一步灌注的方法,实现简易操作提供了依据。该研究得出了PDMS芯片制作的具体工艺条件,并进行优化,获得了完整的加工技术方案。
There are three academic values of this doctoral thesis. First, we have developed a new method of monitoring harmful algal blooming (HAB) with a microfluidic device integrated heterogeneous immuno-enzyme assay for rapid and automatic analysis of algal toxins. In one single microfluidic chip, multiple samples were controlled and analyzed in parallel manner. Second, we have designed a simple microfluidic chip to determine malaria, which is suitable for point-of-care applications. An indirect immunofluorescence assay (IFA) was constructed inside the microfluidic device for rapid analysis of the serodiagnosis for malaria. Third, we have established a novel CTC-chip that can efficiently and reproducibly isolate CTCs from the blood of HCC patients. The CTC-chip consists of an array of microposts combined with asialofetuin for CTC capture,which is clinically useful in diagnosis and monitoring of HCC.
Nowadays, microfluidic chips have been widely recognized as a powerful technology that will play an important role in future biological analysis to meet the large-scale and high-throughput requirements. For more than a decade, it has been expected that microfluidic technology would revolutionize the rapid detection industry with distinct advantages, such as high sample processing rates, low manufacturing costs, advanced system integration, and reduced volumes of samples and analyses. To date, however, microfluidics has not yet been able to live up to these expectations. This fact has led to the recent development of new philosophies and methodologies for microfluidic detections.
First of all there has been few reports about utilizing microfluidic device to analyze algal toxins, especially highly automatic integrated microfluidics. Harmful algal blooming (HAB) has become a more and more serious problem around the world with rapid urbanization and consequential eutrophication in our aqueous environment. HAB not only damages the aqueous ecosystem, but also creates various algal toxins, which already affected the living and health of residents. At present, limited number of conventional methods was reported for the quantization of HABs. The general detection methods of microcystins include high performance liquid chromatography (HPLC), mass spectrometry, ELISA, and even mouse bioassay.
Among all the methods, algal toxin analysis in laboratories mainly relies on HPLC methods that require expensive equipment and highly qualified personnel due to the high variability of toxin structures. HPLC also involves lengthy analysis time and pretreatment of the collected samples. Moreover, the bulky size and delicate structure make it impractical for field analysis. Additionally, its detection limit is relatively high, especially for the trace analysis of algal toxins in water environment. One of other methods, conventional enzyme linked immunosorbent assay (ELISA) performed on microwell plates, is usually used in laboratories and involves a step-by-step reagent introduction procedure. The whole process of ELISA may take at least1hour for analysis normally. Above all, a rapid and simple algal toxin analysis method is in great need for field or on-site application.
Second, malaria poses severe problems in regions short of qualified infrastructure. People in those regions could potentially benefit from such low-cost diagnostic methods. So far, there are only few reports about utilizing integrated microfluidic device to analyse malaria. Limited number of malaria diagnosis method was reported. The methods include conventional light microscopy, PCR and detection of antibodies by immunofluorescence or ELISA. Malarial parasites detection by microscopic examination of stained blood smears is still the gold standard method for malaria diagnosis. However, it is time consuming and requires professional technicians to identify the malaria parasites. The PCR-based molecular diagnosis by amplifying ribosomal RNA genes of plasmodium is considered as one of the most sensitive method. But the approach is limited to well-equipped laboratories with trained persons and relatively expensive cost. Antibody detection by immunofluorescence or ELISA is currently used for seroepidemiology of malaria. This is very useful in distinguishing parasite-infected individuals who are undetectable by antigen detection or light microscopy due to low parasite density. However, the method involves step-by-step reagent introduction procedure and takes more than1-hour analysis time at least.
Finally, hepatocellular carcinoma (HCC) is the fifth most frequent cancer in the world. Circulating tumorous cells (CTCs) refer to tumorous cells (invasive or not) spontaneously circulating in the peripheral blood or spread estrogenically into blood vessels, which is conceded as an early step in the cascade of events leading to metastasis. Hematogenous spread is the major route of HCC recurrence, so that detection of CTC has important clinical significance in recurrence prediction and treatment monitoring in HCC patients. In the past decades, along with novel CTC detection methods spoon up, ISET, magnetic automated cell sorter (MACS) and CellSearch are commercially available for CTC detection in the clinical setting. Meanwhile, mcrofluidic chips have been applied in CTC detection area since a so-called CTC-chip was developed to capture rare CTCs as blood flows past EpCAM-coated microposts in2007. All thoes methods depending on EpCam, widely expressed on the surface of epithelial cells and epithelial-derived tumor cells, are not suitable for CTC detection in HCC. Although HCC cells are epithelial cells, only about35%of HCC cases express EpCAM.Thus, a effeivetive methods for HCC-CTC detection is needed.
In this thesis, we introduce our researches in five chapters.
In the first chapter, we give a brief introduction on the microfluidic technology and the challenges of biomedical analysis. Further, we review the microfluidic related technology and their applications in biomedical study. All of the background information provides theoretical and applicable support on the following researches.
In second and third chapters, we introduced new microfluidic platforms based on immune reactions. The core of the platform is one immune-column microfluidic chip, which contains separate immune columns for rapid and easy-to-use immunoassay. We demonstrated its application on microcystin, saxitoxin and cylindrospermopsin analysis. The detection limits of0.02ng/ml for each toxin meet the need for practical water sample analysis including for drinking water. And, with more applicable design of the chip, we use this platform for rapid clinical determination of malaria. In comparison with the conventional ELISA kit, our microfluidic system is now capable to analyze the practical samples with75%time saving and3orders less reagent consumption. The platform, which can be programmed into either manual or automatic mode, requires very little professional knowledge and special skills to be operated. This device opens up the possibility of rapid parallel automatic analysis for multiple samples, which is essential for toxin detection, disease screening and biomarker analysis.
In the fourth chapter, we introduce a novel CTC-chip, to establish a sensitive and specific isolation and enumeration system for circulating tumor cells (CTC) in patients with hepatocellular carcinoma (HCC). The platform can efficiently and reproducibly isolate CTCs from the blood of HCC patients. The CTC-chip consists of an array of microposts combined with asialofetuin for CTC capture and utilizes Hep Par1for identification, which provides an opportunity for enumeration and biological properties characterization of individual isolated CTCs. It is likely clinically useful in diagnosis and monitoring of HCC and may have a role in clinical decision-making.
In the fifth chapter, we introduce a method to improve the standard soft lithography fabrication process to generate highly reduplicate microfabricated patterns. It can also improve the release of poly (dimethylsiloxane)(PDMS) devices from replica molds. This method added one extra step to prepare a single-spin coating of a thin layer of PDMS, compared with traditional microfabrication technique. It was validated with silicon wafers containing arrays of50μm deep SU8microwells with various densities (a maximum density of5×104/cm2).
过去的二十年,是微流控技术发展的二十年。从Manz和Widmer等人1990年首次提出微型全分析系统(Miniaturized Total Analysis System, μTAS)的概念,到2002年Quake等以“微流控芯片大规模集成”为题在Science上发表文章,微流控技术作为当前分析科学的重要发展前沿,在研究方面取得了飞速的发展。分析检测技术手段的逐步微型化,以及现场“即时检测”的提出对分析技术提出更高的要求。因此发展针对生物医学中实际重大问题的微流控快速检测系统是微流控芯片研究与应用的重要方面。
首先,环境中有毒物质的快速检测是目前快速检测领域的重点和难点之一。随着有害水藻的爆发,会产生大量的有害藻类毒素。这些毒素通过食物链,进入鱼类、贝类体内,人食用了污染的鱼类、贝类会引发食物中毒,甚至威胁生命健康。国内外现行的藻类毒素检测技术主要包括：生物检测法,化学检测法,细胞毒性检测技术,放射性标定法,以及免疫检测等。其中,生物检测法主要采用喂食小鼠、家蝇等毒素的方法,缺点是周期长,费用高,重复性差,而且需要专门培训的人员来操作。化学检测法,主要通过高效液相色谱,毛细管电泳,色谱质谱联用方法,重现性好,但设备功能单一,体积大和价格昂贵,便携性、选择性差。细胞毒性检测技术,直观方法来观察加入毒素后,细胞的反应来判断毒素种类,灵敏度高,但只能做定性测量,不能测定浓度而且需要良好的细胞培养技术。放射性标定法仪器昂贵,测试费用高,并且适用毒素检测范围窄。免疫检测技术主要利用抗原和抗体专一、特异结合的特点对毒素进行定性,定量检测,现主要有直接竞争检测法,间接法,“三明治”法。这种方法专一性强,灵敏度高,是非常有潜力的一种方法,但操作比较繁琐,商业化试剂盒价格昂贵,主要为手工操作,自动化程度低而且需要专业人员。
其次,针对重大传染病的临床快速诊断是微流控芯片在应用研究的重要方面。近年来,由于疟原虫对药物、传播媒介对杀虫剂抗性的迅速发展,疟疾快速的诊断对控制全球疟疾的作用显得更加重要。迄今为止,显微镜镜检(厚血膜、薄血膜)仍是疟疾诊断的金标准。但是显微镜镜检作为一种诊断方法又存在一些问题,由于疟原虫形体小,形态相似,难以染色和检出,需要有经验的人员才能做到正确的诊断。疟疾流行区由于熟练镜检技术人员及设备缺乏,仅依靠传统厚薄血膜镜检法已越来越不能适应当前疟疾诊断的需要随着免疫学技术的发展,间接荧光抗体和ELISA均很快在疟疾诊断中得到应用,但是此类方法抗体用量相对较大,成本较高。而且,反应时间长,不能满足快速诊断的要求。PCR技术显示了较高的敏感性和特异性可以对疟原虫的感染做出早期快速诊断。但需要更复杂的仪器设备和技术条件作为支持,不适合作为疟疾流行区常规的检测手段,难以在基层推广应用。
最后,针对肝癌循环肿瘤细胞的高灵敏度捕获受到现有技术的制约。采用反转录聚合酶链式反检测HCC患者外周血中甲胎蛋白mRNA从而检测HCC患者CTCs的报道较多。然而,RT-PCR法有其固有局限,如易产生假阳性和假阴性、操作难以标准化、不能准确估算样本中CTC的数目等。此外,更不可忽视的是,RT-PCR法须破坏细胞形态,不能对单个CTC进行观察、分析和计数,而这些数据可提供CTCs恶性程度和侵袭性方面的信息。因此迫切需要建立特异和敏感的循环肝癌细胞分离和检测技术。目前分离CTCs的标准方法是基于肿瘤细胞表面上皮性抗原的磁性激活细胞分选技术(magnetic-activated cell separation, MACS)。由于缺乏针对肝癌细胞表面特异性抗原的单克隆抗体,迄今鲜见MACS用于分离循环肝癌细胞的报道。尽管肝癌细胞属于上皮性细胞,但EpCAM仅在约35%左右的HCC组织标本中表达。因此,现有系统并不适合用于高通量、高灵敏度分离与检测循环肝癌细胞。
针对以上快速检测领域所面临的三大问题,本博士学位论文分为五章节开展研究中作。各章节主要内容如下：
第一章是绪论部分。本章主要讨论微流控芯片的发展概况,生物医学分析领域的发展现状和遇到的挑战,以及微流控芯片技术在生物医学分析方面的应用进展。从而引出本论文工作的研究方向,为设计发展微流控芯片在生物医学分析快速检测领域应用的新方法研究提出理论依据和实际意义。
第二章主要介绍集成化微流控免疫芯片系统用于环境藻类毒素的高灵敏度快速检测分析新方法研究。我们研究建立了用于对代表性藻类毒素微囊藻毒素(microcystins, MCs),石房蛤毒索(Saxitoxin, STX)与柱孢藻毒素(cylindrospermopsin, CNY)三种藻类毒素的现场快速检测微流控芯片。可以对实际样品(水样和鱼类、贝类等食物样品)溶液中MC, STX与CNY进行快速检测的微流控芯片。实验建立了用于对代表性藻类毒素微囊藻类毒素的现场快速检测微流控芯片。芯片包含七个可以同时控制,也可独立操作的免疫亲和反应柱,可以对实际样品(水样和鱼类、贝类等食物样品)溶液中微囊藻毒素进行快速检测的微流控芯片。检测限为0.02ng/ml。实现对多个样品快速,平行的检测。
第三章主要介绍基于抗体检测的微流控芯片用于疟疾快速临床检测的新方法研究。我们研究了针对在条件欠发达地区多发的传染性疾病疟疾(Malaria)其临床快速现场诊断,提出可行的方案。建立了基于间接免疫荧光反应原理的快速检测微流控芯片用于疟疾病(Malaria)的临床诊断研究。实验中通过检验病人血清样品中的MSP1-19与PfF2抗体的检测,实现了疟疾临床快速诊断,免疫反应中得到的不同荧光强度,可以被信号采集模块采集分析,从而提供临床诊断信息。在同一块芯片实现双抗原同时检测。实验证明,微流控芯片免疫系统可以得到可靠的检测结果,并且极大的节约了抗体用量,缩短了反应所需时间,使得检测成本极大的降低。结果指示,研究中采用MSP1-19与PfF2双抗原检测的方法,对疟疾进行快速检测可以提供相对更加可靠的诊断结果。结果显示,研究建立的微流控芯片免疫系统与方法可以有效的用于基于抗体的疟疾血清学检测,为条件欠发达地区的疾病现场检测,实现疾病有效控制提供可行方案。
第四章主要介绍肝癌循环肿瘤细胞(CTC)的微流控芯片分离检测系统新方法研究。我们在实验中建立了一种具有高度特异性和敏感性的肝癌循环肿瘤细胞分离与检测系统。肝细胞癌(hepatocellular carcinoma, HCC)患者体内的循环肿瘤细胞(circulating tumor cells, CTCs),即循环肝癌细胞,是HCC转移和复发的根源。检测循环肝癌细胞对于预测HCC转移复发无疑具有重要临床意义。研究中建立了一种独特的基于ASGPR及其配体相互作用的循环肝癌细胞微流控芯片分选方法,结合基于肝细胞特异性抗体Hep Par1的免疫荧光染色鉴定,发展了一种新的循环肝癌细胞分离与检测系统。实验中用标准细胞加入全血,测定了系统工作的最佳条件。实验证明系统具有很好的特异性和敏感性,且操作方便,易于标准化。分离到的CTCs可进行免疫形态学鉴定和计数。并初步显示了该系统用于临床检测的可行性。
第五章主要介绍高密度结构PDMS芯片制作技术研究。我们讨论了对微流控芯片的制作软光刻技术提出改进,从而得到稳定重复的微结构。为具有高密度阵列微结构的PDMS芯片制作,提供可靠的方法。针对聚合物材料PDMS芯片在复制模塑成型过程中出现的缺陷问题进行了研究,设计了含有50μm高的不同密度微柱阵列芯片。运用实验提出的方案,成功实现了其复制成型,并且保证了模板的多次重复利用。其最低密度共含有14,400个微柱(40mm×18mm),最高密度含有39,760个微柱(40mm×18mm),其对应的最大结构密度为5×104/cm2。本研究为简化复杂PDMS芯片制作处理,提出一步涂层/一步灌注的方法,实现简易操作提供了依据。该研究得出了PDMS芯片制作的具体工艺条件,并进行优化,获得了完整的加工技术方案。
There are three academic values of this doctoral thesis. First, we have developed a new method of monitoring harmful algal blooming (HAB) with a microfluidic device integrated heterogeneous immuno-enzyme assay for rapid and automatic analysis of algal toxins. In one single microfluidic chip, multiple samples were controlled and analyzed in parallel manner. Second, we have designed a simple microfluidic chip to determine malaria, which is suitable for point-of-care applications. An indirect immunofluorescence assay (IFA) was constructed inside the microfluidic device for rapid analysis of the serodiagnosis for malaria. Third, we have established a novel CTC-chip that can efficiently and reproducibly isolate CTCs from the blood of HCC patients. The CTC-chip consists of an array of microposts combined with asialofetuin for CTC capture,which is clinically useful in diagnosis and monitoring of HCC.
Nowadays, microfluidic chips have been widely recognized as a powerful technology that will play an important role in future biological analysis to meet the large-scale and high-throughput requirements. For more than a decade, it has been expected that microfluidic technology would revolutionize the rapid detection industry with distinct advantages, such as high sample processing rates, low manufacturing costs, advanced system integration, and reduced volumes of samples and analyses. To date, however, microfluidics has not yet been able to live up to these expectations. This fact has led to the recent development of new philosophies and methodologies for microfluidic detections.
First of all there has been few reports about utilizing microfluidic device to analyze algal toxins, especially highly automatic integrated microfluidics. Harmful algal blooming (HAB) has become a more and more serious problem around the world with rapid urbanization and consequential eutrophication in our aqueous environment. HAB not only damages the aqueous ecosystem, but also creates various algal toxins, which already affected the living and health of residents. At present, limited number of conventional methods was reported for the quantization of HABs. The general detection methods of microcystins include high performance liquid chromatography (HPLC), mass spectrometry, ELISA, and even mouse bioassay.
Among all the methods, algal toxin analysis in laboratories mainly relies on HPLC methods that require expensive equipment and highly qualified personnel due to the high variability of toxin structures. HPLC also involves lengthy analysis time and pretreatment of the collected samples. Moreover, the bulky size and delicate structure make it impractical for field analysis. Additionally, its detection limit is relatively high, especially for the trace analysis of algal toxins in water environment. One of other methods, conventional enzyme linked immunosorbent assay (ELISA) performed on microwell plates, is usually used in laboratories and involves a step-by-step reagent introduction procedure. The whole process of ELISA may take at least1hour for analysis normally. Above all, a rapid and simple algal toxin analysis method is in great need for field or on-site application.
Second, malaria poses severe problems in regions short of qualified infrastructure. People in those regions could potentially benefit from such low-cost diagnostic methods. So far, there are only few reports about utilizing integrated microfluidic device to analyse malaria. Limited number of malaria diagnosis method was reported. The methods include conventional light microscopy, PCR and detection of antibodies by immunofluorescence or ELISA. Malarial parasites detection by microscopic examination of stained blood smears is still the gold standard method for malaria diagnosis. However, it is time consuming and requires professional technicians to identify the malaria parasites. The PCR-based molecular diagnosis by amplifying ribosomal RNA genes of plasmodium is considered as one of the most sensitive method. But the approach is limited to well-equipped laboratories with trained persons and relatively expensive cost. Antibody detection by immunofluorescence or ELISA is currently used for seroepidemiology of malaria. This is very useful in distinguishing parasite-infected individuals who are undetectable by antigen detection or light microscopy due to low parasite density. However, the method involves step-by-step reagent introduction procedure and takes more than1-hour analysis time at least.
Finally, hepatocellular carcinoma (HCC) is the fifth most frequent cancer in the world. Circulating tumorous cells (CTCs) refer to tumorous cells (invasive or not) spontaneously circulating in the peripheral blood or spread estrogenically into blood vessels, which is conceded as an early step in the cascade of events leading to metastasis. Hematogenous spread is the major route of HCC recurrence, so that detection of CTC has important clinical significance in recurrence prediction and treatment monitoring in HCC patients. In the past decades, along with novel CTC detection methods spoon up, ISET, magnetic automated cell sorter (MACS) and CellSearch are commercially available for CTC detection in the clinical setting. Meanwhile, mcrofluidic chips have been applied in CTC detection area since a so-called CTC-chip was developed to capture rare CTCs as blood flows past EpCAM-coated microposts in2007. All thoes methods depending on EpCam, widely expressed on the surface of epithelial cells and epithelial-derived tumor cells, are not suitable for CTC detection in HCC. Although HCC cells are epithelial cells, only about35%of HCC cases express EpCAM.Thus, a effeivetive methods for HCC-CTC detection is needed.
In this thesis, we introduce our researches in five chapters.
In the first chapter, we give a brief introduction on the microfluidic technology and the challenges of biomedical analysis. Further, we review the microfluidic related technology and their applications in biomedical study. All of the background information provides theoretical and applicable support on the following researches.
In second and third chapters, we introduced new microfluidic platforms based on immune reactions. The core of the platform is one immune-column microfluidic chip, which contains separate immune columns for rapid and easy-to-use immunoassay. We demonstrated its application on microcystin, saxitoxin and cylindrospermopsin analysis. The detection limits of0.02ng/ml for each toxin meet the need for practical water sample analysis including for drinking water. And, with more applicable design of the chip, we use this platform for rapid clinical determination of malaria. In comparison with the conventional ELISA kit, our microfluidic system is now capable to analyze the practical samples with75%time saving and3orders less reagent consumption. The platform, which can be programmed into either manual or automatic mode, requires very little professional knowledge and special skills to be operated. This device opens up the possibility of rapid parallel automatic analysis for multiple samples, which is essential for toxin detection, disease screening and biomarker analysis.
In the fourth chapter, we introduce a novel CTC-chip, to establish a sensitive and specific isolation and enumeration system for circulating tumor cells (CTC) in patients with hepatocellular carcinoma (HCC). The platform can efficiently and reproducibly isolate CTCs from the blood of HCC patients. The CTC-chip consists of an array of microposts combined with asialofetuin for CTC capture and utilizes Hep Par1for identification, which provides an opportunity for enumeration and biological properties characterization of individual isolated CTCs. It is likely clinically useful in diagnosis and monitoring of HCC and may have a role in clinical decision-making.
In the fifth chapter, we introduce a method to improve the standard soft lithography fabrication process to generate highly reduplicate microfabricated patterns. It can also improve the release of poly (dimethylsiloxane)(PDMS) devices from replica molds. This method added one extra step to prepare a single-spin coating of a thin layer of PDMS, compared with traditional microfabrication technique. It was validated with silicon wafers containing arrays of50μm deep SU8microwells with various densities (a maximum density of5×104/cm2).
引文
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