Microfluidic Chip-based Immunosensors And Proteomics Research

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. Their miniaturized architectures provide a number of distinct advantages, such as high sample processing rates, low manufacturing costs, advanced system integration, and reduced volumes of samples and analytes.In chapter 1, recent research progress on microfluidic chips, including fabrication techniques, materials, detection system and analytical applications, is described. In the end of this part, the purpose and significance of our study on microfluidics-based bioanalytical application are emphasized.In my research, by using surface modification approaches, such as PEI, AuNPs nanoparticles assembly, and A12O3 sol-gel. By using these methods, the antibodies, aptamer or protease can be effectively immobilized/adsorbed within the microchannels to devise microfluidic immunoassay or enzymatic reactors combined with electrochemistry method or mass spectrometry; the microchips are capable of performing the analysis of proteins in real samples. The detailed points of this thesis are expanded as follows:1) In this work, we designed an aptamer-based microfluidic chip biosensor and applied it to the detection of low-level human thrombin. Gold nanoparticles are coating on the surface of the modified PMMA microchannel, and sulfhydryl-labeled first-rate aptamer is fixed on AuNPs. Through the specific binding of aptamer and thrombin, the thrombin protein and biotin-labeled second-rate aptamer are connected, and ALP-labeled avidin is linked to biotin. DPV is used to detect the electronic signals from the substrate solution of PAPP and a low detection limit of 1pM has obtained. The biosensor showed high sensitivity, selectivity and broad linear response. The results indicate that the microfluidic chip combined with apatmer-protein interaction has great potential in clinical diagnosis detection.2) A simple and sensitive microchip-based method has been proposed to determine low level of S100B, biomarker of neurological disease, coupled with electrochemical detection system. Firstly, poly(ethyleneimine) (PEI) was applied to modify the poly(methyl methacrylate) (PMMA) microchannels. PEI contained abundant NH2 groups which can covalently immobilize S100B monoclonal antibody in the next step. Afterward, the antigen S100B and polyclonal rabbit anti-S100 were sequentially immobilized through antigen-antibody specific interaction. Finally, the anti-rabbit IgG alkaline phosphatase conjugate (ALP-conjugate) was bound to the polyclonal rabbit anti-S100 in microchannnel. Using three-electrode electrochemical detection system, the microchip-based immunosensor had the detection limit of S100B down to 0.1 pg mL-1, and achieved a detectable linear concentration range of 0.1 pg mL-1～100pg mL-1 by differential pulse voltammetry (DPV). The on-chip immunosensor can not only provide rapid and sensitive detection for target proteins but also be capable of resisting to non-specific adsorption of proteins. The result of the experiment shows that the proposed approach is highly sensitive and specific, which is feasible and has the potential application in clinical analysis and diagnosis.3) An on-chip microreactor for highly efficient proteolysis has been demonstrated using BSA, myoglobin and cytochrome c as model substrates. Alumina sol-gel exhibits a simple surface modification protocol in PET microchannels for enzyme immobilization. The standard proteins were confidently identified with a low femtomole per analysis at a concentration of 0.5ngμL-1 with the digestion time less than few seconds. Our findings also suggest that this on-chip microreactor can be used for the detection of proteins from real biological samples. This versatile system is a good example of proteolytic reactions occurring in the alumina gel-derived microchannels, where the network not only provides a support for encapsulation of enzymes, but also acts as microreactor to facilitate the protein digestion. Such on-chip enzymatic reactor coupled to MALDI-TOF-MS/MS or 2D-LC-ESI-MS/MS systems could be applied to profile the complex protein extracts in proteomic research.