Development of an enzymatic reactor applying spontaneously adsorbed trypsin on the surface of a PDMS microfluidic device

Abstract

Herein, a microfluidic device (MD) containing immobilized trypsin for rapid and efficient proteolysis was described. Trypsin was immobilized via non-specific protein adsorption onto the hydrophobic poly(dimethylsiloxane) (PDMS) channel wall of the MD. Peptide mapping of bovine serum albumin (BSA) samples was carried out to estimate the stability of trypsin adsorbed on PDMS surface. Peptide maps of BSA samples were obtained by capillary zone electrophoresis (CZE), the RSD% for migration times were under 1%. Several proteins (hemoglobin, myoglobin, lysozyme, and BSA) in a wide molecular size range (15-70 kDa) were digested efficiently with ∼50 s contact time. The number of separated peaks correlated well with the expected number of peptides formed in the complete tryptic digestion of the proteins. Peptide mass fingerprinting of BSA and human serum was carried out. Trypsin retained its activity for 2 h; within this period, the MD can be used for multiple digestions. The main properties of this device are simple channel pattern, simple immobilization procedure, regenerability, and disposability; all these features make this MD one of the simplest yet applicable enzymatic microreactors. Graphical abstract Development of microfluidic device including a serpentine channel as an enzyme reactor for protein digestion.

The application of a microfluidic reactor including spontaneously adsorbed trypsin for rapid protein digestion of human tear samples

“…Figure A shows the microfluidic IMER, which is a modified version of the previously developed microreactor , in order to enable eight samples to be simultaneously digested. The disposable chip consists of eight serpentine‐like channels (Fig.…”

“…The channel wall of a PDMS chip can be used directly as solid support for trypsin immobilization, since proteins are readily adsorbed on the hydrophobic, porous surface of PDMS . Recently, we described a serpentine‐like PDMS microfluidic IMER, which uniquely operates with trypsin adsorbed spontaneously on the PDMS channel wall . This IMER was capable of rapid protein digestion of several protein standards and human serum proteins.…”

Advances in enzyme substrate analysis with capillary electrophoresis

“…Recently, a microfluidic device for trypsin digests was developed as an off-line immobilized enzyme reactor prior to capillary electrophoresis mass spectrometry analysis. The device, while only maintaining the original activity for 2 h, exhibited comparable results to the bench top reaction with only 50 s of reaction time required for the immobilized enzyme reaction [96]. …”

Enzyme immobilization on photopatterned temperature‐response poly (N‐isopropylacrylamide) for microfluidic biocatalysis

“…These characteristics are beneficial for many chemical processes, such as separations and reactions. Immobilized enzymes have been applied widely in the fields of chemical engineering and biotechnology, which exhibit the main features of reusability, easy handling, enhanced stability, easy separation from products and substrate specificity . In particular, microchips used as solid supports for enzyme immobilization have been developed to improve yield and selectivity in reaction systems .…”

“…Immobilized enzymes have been applied widely in the fields of chemical engineering and biotechnology, which exhibit the main features of reusability, easy handling, enhanced stability, easy separation from products and substrate specificity. 2 In particular, microchips used as solid supports for enzyme immobilization have been developed to improve yield and selectivity in reaction systems. 3 Different materials such as membranes, monoliths, magnetic microspheres/beads, silica particles and commercially available beads have been integrated into microchips.…”