Titania and Alumina Sol−Gel-Derived Microfluidics Enzymatic-Reactors for Peptide Mapping: Design, Characterization, and Performance

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Abstract
The design and characterization of titania-based and alumina-based Poly(dimethylsiloxane) (PDMS) microfluidics enzymatic-reactors along with their analytical features in coupling with MALDI-TOF and ESI−MS were reported. Microfluidics with microchannel and stainless steel tubing (SST) were fabricated using PDMS casting and O2-plasma techniques, and were used for the preparation of an enzymatic-reactor. Plasma oxidation for the PDMS microfluidic system enabled the channel wall of the microfluidics to present a layer of silanol (SiOH) groups. These SiOH groups act as anchors onto the microchannel wall linked covalently with the hydroxyl groups of trypsin-encapsulated sol matrix. As a result, the trypsin-encapsulated gel matrix was anchored onto the wall of the microchannel, and the leakage of gel matrix from the microchannel was effectively prevented. A feature of the microfluidic enzymatic-reactors is the feasibility of performing on-line protein analysis by attached SST electrode and replaceable tip. The success of trypsin encapsulation was investigated by AFM imaging, assay of enzymatic activity, CE detection, and MALDI-TOF and ESI−MS analysis. The lab-made devices provide an excellent extent of digestion even at a fast flow rate of 7.0 μL/min, which affords the very short residence time of ca. 2 s. With the present device, the digestion time was significantly shortened compared to conventional tryptic reaction schemes. In addition, the encapsulated trypsin exhibits increased stability even after continuous use. These features are required for high-throughput protein identification. Keywords: microfluidic enzymatic-reactor • titania • alumina • MALDI-TOF−MS • ESI−MS high-throughput protein identification • proteomics