ConclusionThe present work describes the first application of particle-packing technique to fabricate CE-IMERs for on-line enzyme assay.The approach to fabricate CE-IMERs is very simple and effective,which is accomplished by packing immobilized trypsin beads inthe capillary with silica single particles as the frits. The largepore size of the particles greatly reduces the back pressure dur-ing packing, thus the trypsin beads can be packed in the capillarywith only a syringe and a 5-mm long IMER can be fabricated inonly 5 min. The feasibility and reliability of our packed CE-IMERsfor on-line enzyme assay have been demonstrated by investiga-tion of trypsin digestion/inhibition of BAEE and trypsin digestionof proteins (myoglobin and BSA). Great run-to-run reproducibil-ity (RSD < 4.1% for 100 runs), column-to-column reproducibility(RSD < 5.2%, n = 6), and day-to-day stability (RSD < 3.2%, n = 10) indi-cate that our packed CE-IMERs have excellent robustness forenzyme assay. Using the packed CE-IMERs, accurate assay of on-line trypsin digestion of myoglobin and BSA can be achievedwith 10 min incubation or even no incubation, and the resultsshow almost identical electropherograms and comparable iden-tified peptide products with those using free trypsin with 12 hincubation time required. Our packed CE-IMERs could be a newalternative to open tubular and monolithic IMERs, thus couldexpand the application of CE technique in the enzyme assay for, e.g.peptide mapping and proteomics, especially when MS technique iscoupled to the packed CE-IMERs. It should be pointed out the draw-backs of our packed CE-IMERs are the usage of the 100-m i.d.capillary, which may reduce the CE separation, and that of the 20-m highly porous trypsin beads, which may limit the applicationin proteomics. We believe that with rapid development in partic-ulate material science, single particles with smaller size as well asvarious enzyme-immobilized porous particles could be availablefor fabricate the packed CE-IMERs, the making packed CE-IMER awider application in the fields of on-line enzyme assay.