Enhanced Protein Digestion through the Confinement of Nanozeolite-Assembled Microchip Reactors

Abstract

An on-chip microreactor was proposed toward the acceleration of protein digestion through the construction of a nanozeolite-assembled network. The nanozeolite microstructure was assembled using a layer-by-layer technique based on poly(diallyldimethylammonium chloride) and zeolite nanocrystals. The adsorption of trypsin in the nanozeolite network was theoretically studied based on the Langmuir adsorption isotherm model. It was found that the controlled trypsin-containing nanozeolite networks assembled within a microchannel could act as a stationary phase with a large surface-to-volume ratio for the highly efficient proteolysis of both proteins at low levels and with complex extracts. The maximum proteolytic rate of the adsorbed trypsin was measured to be 350 mM min-1 microg-1, much faster than that in solution. Moreover, due the large surface-to-volume ratio and biocompatible microenvironment provided by the nanozeolite-assembled films as well as the microfluidic confinement effect, the low-level proteins down to 16 fmol per analysis were confidently identified using the as-prepared microreactor within a very short residence time coupled to matrix-assisted laser desorption-time-of-flight mass spectrometry. The on-chip approach was further demonstrated in the identification of the complex extracts from mouse macrophages integrated with two-dimensional liquid chromatography-electrospray ionization-tandem mass spectrometry. This microchip reactor is promising for the development of a facile means for protein identification.

Faecal characteristics as markers of Chelonia mydas feeding

“…The microstructure of faeces responded to dietary treatment. Roughness increases surfaceto-volume ratio, which favours high enzyme loading 36 when hydrolysing the digested foods. This finding is in agreement with roughness improving the in vitro digestion of raw materials 10 .…”

Protein immobilization techniques for microfluidic assays

“…3-D structures have been created by patterning microstructures (e.g., microposts 29,82,83 and micropits 60 ) or through insertion of porous membranes 67,84,85 before assembly of microfluidic chips. In post-assembly approaches, microbeads 54,62,[86][87][88][89][90][91][92][93] can be packed into enclosed channels or various polymers such as hydrogels, [18][19][20][21][22][23][24]57,[94][95][96][97] sol-gels, 64,98,99 polymer monoliths, 61 or membranes 100 can be polymerized in situ. For silica-based 3-D structures such as silica beads 91 and alkoxysilane-based sol-gels, 63 a similar glass/silicon surface immobilization strategy can be used.…”

“…By simply adding proteins to the "sol" before gelation, proteins are encapsulated in optical transparent 3-D nanostructure. Silica sol-gels made from silicon alkoxide colloids 98 are the most common, but titania 64 or zeolite 99 colloids are also used. Immobilization of proteins proceeds under mild conditions, 64 thus proteins can retain near-native activity.…”

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].…”