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

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 O(2)-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 microL/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.

Simple and Rapid Proteomic Analysis by Protease-Immobilized Microreactors

“…It is known that the flow rate of substrate can affect the efficiency of the immobilized enzyme activity (Fan & Chen, 2007;Honda et al, 2005;Ma et al, 2008;Nel et al, 2008;Wu et al, 2004;Dulay et al, 2005). Therefore, we studied the effect of different delivery speeds of substrates on hydrolysis activities (Yamaguchi et al, 2009).…”

“…High sequence coverage is important to enhance the probability of identification of the protein and increase the likelihood of detection of structural variants generated by processes such as posttranslational modifications. Several methods for protease immobilization have been reported, wherein the protease, usually trypsin, has been immobilized in microchips by sol-gel encapsulation (Sakai-Kato et al, 2003;Wu et al, 2004), covalently bounded (Lee et al, 2008;Fan & Chen, 2007) and physically adsorbed onto different supports . In addition, trypsinimmobilized magnetic particles have been developted to carry out proteolysis with a short digestion time (Li et al, 2007a;.…”

Protein immobilization techniques for microfluidic assays

“…[53][54][55][56][57][58] Important to proteomics, enzymatic digestion before MALDI-TOF/MS (Matrix-assisted laser desorption-ionization time-of-fly/mass spectrometry) peptide mapping of a protein has been explored in microfluidic devices. [59][60][61][62][63][64][65][66] Compared to conventional in-solution enzyme digestion, which is time consuming and offers limited sensitivity, microfluidic formats have shown high conversion rates, facile replacement of inactivated enzyme, and long-term stability. 54,55,63,67 Enzymatic production of fluorescent and colored products for protein analysis (e.g., alkaline phosphatase (ALP) production of chemiluminescence or colorimetric product) enhances detection limits of immunoassays.…”

Proteolytic Digestion and TiO2Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins

“…By immobilizing the enzyme, high enzyme-to-substrate ratios can be achieved, and undesired autolysis products are eliminated. Various methods have been introduced for the enzymeimmobilization process [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. Landers and coworkers introduced a digestion approach that uses electroosmotic flow (EOF) to pump proteins through a proteolytic digestion system packed with immobilized trypsin gel beads [17].…”

“…Landers and coworkers introduced a digestion approach that uses electroosmotic flow (EOF) to pump proteins through a proteolytic digestion system packed with immobilized trypsin gel beads [17]. Liu and coworkers immobilized trypsin on different substrates, including titania and alumina sol-gel [22], gold nanoparticles [23], and nanozeolites [24]. Wu et al developed a microdevice consisting of an immobilized proteolytic enzyme on the surface of acrylic acid-grafted PDMS channels [25].…”