Low back-pressure hierarchically structured multichannel microfluidic bioreactors for rapid protein digestion - Proof of concept

A novel, easy-to-fabricate monolithic enzymatic microreactor with a hierarchical, torturous structure of flow-through channels of micrometric sizes and large mesopores was shown to enable rapid and very efficient digestion of proteins at high yields and exceptionally low back-pressures. Four silica monoliths with bi-modal 3D pore structure in micrometer and nanometer size scales were synthesized and characterized for structural and flow properties. The monolith with the highest total pore volume (4cm³/g) and flow-through channels 20-30μm in size, was further functionalized with trypsin to obtain multichannel immobilized enzyme (proteolytic) reactor (IMER). The value of permeability coefficient K evaluated for water (~2.0·10^-11) was found to be two orders of magnitude higher in the novel reactor than reported before for high-performance IMERs, enabling the flow rates of 750mL/cm²min at pressure gradients of 64kPa/cm. Very high practical potentials of the novel microbioreactor were demonstrated in the proteolysis of cytochrome c (Cyt-c) and myoglobin (Myo), without any earlier pretreatment. MALDI-TOF/TOF mass spectrometry analysis of sequence coverage was high: 70% (Cyt-c) and 90% (Myo) for 24min digestion, and 39% (Cyt-c) and 53% (Myo) when the proteolysis time was reduced to 2.4min. The proposed microreactors make full use of all advantages of microfuidic devices and mesoporous biocatalysts, and offer exceptional possibilities for biochemical/proteolytic applications in both large (production) and small (analytical) scales.