Rapid and efficient proteolysis for proteomic analysis by protease-immobilized microreactor.

Proteolysis is an important part of protein identification in proteomics analysis. The conventional method of in-solution digestion of proteins is time-consuming and has limited sensitivity. In this study, trypsin- or alpha-chymotrypsin-immobilized microreactors prepared by a microfluidics-based enzyme-immobilization technique were studied for rapid sample preparation in proteomic analysis. The kinetic studies for hydrolysis of substrate by microreactors revealed that immobilized proteases had higher hydrolytic efficiency than those performed by in-solution digestion. The performance of the microreactors was evaluated by digesting cytochrome c and BSA. Protein digestion was achieved within a short period of time (approximately 5 min) at 30 degrees C without any complicated reduction and alkylation procedures. The efficiency of digestion by trypsin-immobilized reactor was evaluated by analyzing the sequence coverage, which was 47 and 12% for cytochrome c and BSA, respectively. These values were higher than those performed by in-solution digestion. Besides, because of higher stability against high concentration of denaturant, the microreactors can be useful for immediate digestion of the denaturated protein. In the present study, we propose a protease-immobilized microreactor digestion method, which can utilize as a proteome technique for biological and clinical research.

Simple separation of good quality bovine oocytes using a microfluidic device.

We fabricated a simple microfluidic device for separation of bovine oocytes based on the oocyte quality to improve the conception rate of in vitro fertilization (IVF) by using good quality oocytes. The microfluidic device separates oocytes based on sedimentation rate differences in a sucrose buffer, which is dependent on oocyte quality. The microfluidic device has a 700 µm width, 1 mm height, and 10 mm long separation channel. Oocytes were injected from the upper half of the separation channel, and they flowed while sinking. The outlets of the separation channel were divided into upper and lower chambers. Good quality oocytes settled faster than poor quality oocytes in sucrose buffer; therefore, good quality oocytes were collected from the lower outlet. We performed IVF after the microfluidic separation of oocytes. The developmental rate to blastocysts of oocytes collected from the lower outlet was significantly higher than those collected from the upper outlet (36.0% vs. 14.1%). This result was comparable to that in the BCB staining method performed as a comparison method (BCB+ : 35.7%, BCB-: 15.4%). These findings indicate that our microfluidic device could be applied to oocyte separation and contribute to improvement of in vitro embryo production system.

Refolding of difficult-to-fold proteins by a gradual decrease of denaturant using microfluidic chips.

Protein refolding is an important process to obtain active recombinant proteins from inclusion bodies (protein aggregates). However, the conventional refolding method of dialysis or dilution is a time consuming procedure and often, recovering yields of active proteins are low. In this study, we used controllable diffusion through laminar flow in microchannels to control the denaturant concentration. The performance of the designed microfluidic chips was evaluated by the refolding of difficult-to-fold proteins (citrate synthase and the zeta-associated protein 70-kDa protein kinase domain). We demonstrated this by varying the flow rates of the diluting buffer stream(s) and multi-junctions which could control the different flow rate ratios of the buffer stream(s) and the denatured protein stream. By this strategy, we were able to improve the efficiency of protein refolding. Our method achieved refolding within a short period of time at room temperature without the need of any small molecules or chaperone proteins. Moreover, the efficiency of protein refolding by microfluidic chip was found higher than that prepared by dialysis or dilution. These results suggest that microfluidic chips employing this strategy may provide miniaturized tools for rapid and efficient recovery of active proteins from inclusion bodies.