Microfluidic Chip Enzyme Reactor

In this thesis, polymer microfluidic chips are usd as the analytical platforms to perform the step of protein digestion in proteomic analysis. Several modification methods have been employed to functionalize the surfaces of polymer microfluidic chips to immobilize/adsorb the protease towards enhancement of proteolysis and facilitation of protein identification.In chapter 1, recent research progress on microfluidic chips, including fabrication techniques, materials, detection system and analytical applications, is described. And also in the chapter, proteomics evolution, the main research goals and updated progress are presented in detail. In the end of this part, the purpose and significance of our study on microfluidic enzymatic reactors are emphasized.In my research, based on polyethylene terephthalate (PET) and poly methyl methacrylate (PMMA) microfluidic chips, three suface modification approaches have been developed, including bioelectrolytes layer-by-layer assembly, nanozeolite assembly, Au nanoparticle assembly. By using these methods, the protease can be effectively immobilized/adsorbed within the microchannels to devise microfluidic enzymatic reactors towards acceleration of protein digestion. Combined with 2D-HPLC/ESI-MS/MS or MALDI-TOF MS, the microchip reactors are capable of performing fast and efficient digestion and identification of the real protein samples extracted from mouse macrophages. Furthermore, the mechanism of enzyme immobilization, immobilized enzyme activity, and the amount of immobilized enzyme are thoroughly explored.The detailed points of this thesis are expanded as follows:1) A microchip reactor has been developed on the basis of a layer-by-layer approach for fast and sensitive digestion of proteins. Natural polysaccharides, positively charged chitosan (CS), and negatively charged hyaluronic acid (HA) were multilayer assembled onto the surface of a poly(ethylene terephthalate) (PET) microfluidic chip to form a microstructured and biocompatible network for enzyme immobilization. The construction of CS/HA assembled multilayers on the PET substrate was characterized by AFM imaging, ATR-IR, and contact angle measurements. The controlled adsorption of trypsin in the multilayer membrane was monitored using a quartz crystal microbalance and an enzymatic activity assay. Combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), the on-chip enzymatic reactor digested and identified several standard proteins with an incubation time of less than 5s. This simple technique may offer a potential solution for low-level protein analysis.2) The influence of the number of CS/HA multilayers on the amount and the bioactivity of immobilized enzyme is further discussed. The controlled assembly of natural polyelectrolytes and the enzyme-adsorption step were monitored by using a quartz-crystal microbalance and atomic force microscopy (AFM). The adsorption behaviour of trypsin within the CS/HA multilayer-assembled PET microchips has been probed using the Langmuir adsorption isotherm model. The efficient on-chip proteolysis of casein extracted from bovine milk and Hepatitis A Virus Vaccine (HAV) was obtained within a few seconds, and the identification of biological samples was feasible.3) The extensive application of nanozeolites in the field of proteomics is studied. Zeolite nanocrystals show great promise as substrates for immobilizing proteins due to their small size, sufficient functional groups for further grafting or attachment, special surface hydrophilic/hydrophobic microregion distribution, stable colloidal properties, and high level of protein adsorption. The enzyme adsorption behavior of three zeolite nanocrystals (LTL, Beta, and Silicalite-1) has been compared with the variation of pH values and trypsin concentrations. Because of the largest surface/volume ratio, nanozeolite LTL exhibits a surprising trypsin adsorption capacity compared to the others. Therefore, LTL has been selected to immobilize enzymes for the fabrication of microreactors with the ability to conduct protein digestion. The detection limit of proteins can easily reach low-femtomole levels and the digestion time can be decreased to a few seconds. The high enzyme loading and bioactivity may result from the large surface area and unique surface characteristics of zeolite nanocrystals.4) A microchip reactor coated with a gold nanoparticle network entrapping trypsin has been designed for the efficient on-line proteolysis of complex extracts originating from the mouse macrophages.The nano-structured surface coating was assembled via a layer-by-layer electrostatic binding of poly(diallyldimethylammonium chloride) and citrate-protected gold nanoparticles (AuNPs). Enzymatic kinetics assay confirmed that trypsin, which was entrapped in the biocompatible AuNPs network with a high loading capacity, preserved its bioactivity. Trace amounts of proteins down to femtomole per analysis at a level of 0．2 ng were confidently digested using the microchip reactor and the resulting tryptic products were identified by MALDI-TOF MS/MS. Totally 497 proteins extracted from the mouse macrophages AMJ2-C8 were efficiently identified by on-line digestion and SCX-RP-HPLC/ESI-MS/MS analysis.