Microfluidic Chip For Enzyme Immobilization And On-line Mass Spectrometry Detection

Glycans offer the potential for the discovery of new biomarkers in cancer diagnosis, it plays an important role in many key biological processes.Before analyzing the compositions and species of glycans,we should hydrolyze the glycoprotein and purify the derivatized glycans, which need too much time. Microfluidic chip platform has the advantages of miniaturization, low cost and high throughput,which provides a powerful platform to hydrolyze the glycoprotein by immobilizing enzymes. Immobilizing enzyme in microfluidic chip has draw much attention since it exhibited much higher efficiency and lower consumption in comparison to the corresponding reactions in solution. It may reduce the reaction time from12 hours to several minutes.Based on the polydimethylsiloxane(PDMS) chip, we designed an immobilized enzyme microreactors. We used ?off-stoichiometry?TE(OSTE) to form the in-chip porous monoliths. The factors that affect the efficiency of enzymatic hydrolysis were optimized. Characterization by scanning electron microscopy showed that there was no significant difference in the appearance of the monoliths based on variations in the stoichiometric composition of the TE monomers. Several factors, such as stirring time, the proportion of methanol or the way to modify the chip, have an impact on the bead size produced. As a demonstration, when the PNGase F enzymatic microreactor was conditioned with the denatured and reduced glycoprotein solution(37?,1.4 ?m/min), ribonuclease B was completely digested. Thus prepared PNGase F microreactors demonstrated good enzymatic activity in the de-glycosylation of ribonuclease B and fetuin assay, respectively.Based on 3D printing technology and the structure of hydrogel, we designed a immobilized enzyme microreactors. When the composition of the acrylamide monomers is 7%-20%, the hydrogel could immobilize enzyme at the acceptable efficiency. It provided a new platform to immobilize enzyme. We also take advantage of two-way valve and capillary to design an interface for ESI-MS. The capillary was integrated on microfluidic chips based on the flexibility of PDMS, using microfabricated channels for sample infusion. This design could reduce dead volume and form a stable spray, which provided a platform for further research on chip-online detection.