Microfluidic enzymatic-reactors for peptide mapping: strategy, characterization, and performance

Abstract

The design and characterization of two kinds of poly(dimethylsiloxane)(PDMS) microfluidic enzymatic-reactors along with their analytical utility coupled to MALDI TOF and ESI MS were reported. Microfluidic devices integrated with microchannel and stainless steel tubing (SST) was fabricated using a PDMS casting technique, and was used for the preparation of the enzymatic-reactor. The chemical modification was performed by introducing carboxyl groups to PDMS surface based on ultraviolet graft polymerization of acrylic acid. The covalent and physical immobilization of trypsin was carried out with the use of the activation reagents 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide(EDC)/N-hydroxysuccinimide (NHS) and a coupling reagent poly(diallyldimethylammonium chloride)(PDDA), respectively. The properties and success of processes of trypsin immobilization were investigated by measuring contact angle, infrared absorption by attenuated total reflection spectra, AFM imaging and electropherograms. An innovative feature of the microfluidic enzymatic-reactors is the feasibility of performing on-line protein analysis by embedded SST electrode and replaceable tip. The lab-made devices provide an excellent extent of digestion of several model proteins even at the fast flow rate of 3.5 microL min(-1) for the EDC/NHS-made device and 0.8 microL min(-1) for the PDDA-made device, which afford very short residence times of 5 s and 20 s, respectively. In addition, the lab-made devices are less susceptive to memory effect and can be used for at least 50 runs in one week without noticeable loss of activity. Moreover, the degraded PDDA-made device can be regenerated by simple treatment of a HCl solution. These features are the most required for microfluidic devices used for protein analysis.

Microfluidic enzymatic reactors for proteome research

“…Both a high surface-to-volume ratio and microstructured confinement within the microchannel induce order-of-magnitude enhancement of catalytic efficiency. Wu et al [12] explored an on-line digestion protocol to couple the microfluidic microreactor integrated with an SST electrode with a replaceable silica-capillary-nanospray tip attached to ESI MS. The experimental data show that the microreactors are suitable for high-throughput analysis of proteins.…”

Hydrophilic monolith based immobilized enzyme reactors in capillary and on microchip for high-throughput proteomic analysis

“…For IMERs, enzymes can be immobilized on different supports, such as membranes [8], particles [13], the inner walls of capillaries or microchannels [14][15][16][17], and monolithic materials. As a large category of favorable supports for enzyme immobilization, monolithic supports have attracted significant attention due to facile preparation, fast mass transfer and low backpressure [18,19].…”

Silicones for Microfluidic Systems

“…• Immobilization of biomolecules for biomolecular detection using immunoassays [35,75,[177][178][179][180] and enzymatic microreactors [181,182]. • Cell culture under controlled fl ow conditions on surfaces modifi ed with ligands important for cell adhesion and proliferation [35,73,76,105].…”