Researches On Microfluidic Based Rapid Immune Detection System

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).