Given the strong interdisciplinary nature of microfluidic immobilized enzyme reactor (μ-IMER) technology, several branches of science contribute to its successful implementation. A combination of physical, chemical knowledge and engineering skills is often required. The development and application of μ-IMERs in the proteomic community are experiencing increasing importance due to their attractive features of enzyme reusability, shorter digestion times, the ability to handle minute volumes of sample and the prospect of on-line integration into analytical workflows. The aim of this review is to give an account of the current (2017–2021) trends regarding the preparation of microdevices, immobilization strategies, and IMER configurations. The different aspects of microfabrication (designs, fabrication technologies and detectors) and enzyme immobilization (empty and packed channels, and monolithic supports) are surveyed focusing on μ-IMERs developed for proteomic analysis. Based on the advantages and limitations of the published approaches and the different applications, a probable perspective is given.

The goal of this paper was to develop an in-line immobilized enzyme reactor (IMER) integrated into a capillary electrophoresis platform. In our research, we created the IMER by adsorbing trypsin onto the inner surface of a capillary in a short section. Enzyme immobilization was possible due to the electrostatic attraction between the oppositely charged fused silica capillary surface and trypsin. The reactor was formed by simply injecting and removing trypsin solution from the capillary inlet (~1–2 cms). We investigated the factors affecting the efficiency of the reactor. The main advantages of the proposed method are the fast, cheap, and easy formation of an IMER with in-line protein digestion capability. Human tear samples were used to test the efficiency of the digestion in the microreactor.

The Kv1.3 channel has become a therapeutic target for the treatment of various diseases. Several Kv1.3 channel blockers have been characterized from scorpion venom; however, extensive studies require amounts of toxin that cannot be readily obtained directly from venoms. The Pichia pastoris expression system provides a cost-effective approach to overcoming the limitations of chemical synthesis and E. coli recombinant expression. In this work, we developed an efficient system for the production of three potent Kv1.3 channel blockers from different scorpion venoms: Vm24, AnTx, and Ts6. Using the Pichia system, these toxins could be obtained in sufficient quantities (Vm24 1.6 mg/L, AnTx 46 mg/L, and Ts6 7.5 mg/L) to characterize their biological activity. A comparison was made between the activity of tagged and untagged recombinant peptides. Tagged Vm24 and untagged AnTx are nearly equivalent to native toxins in blocking Kv1.3 (Kd = 4.4 pM and Kd = 0.72 nM, respectively), whereas untagged Ts6 exhibits a 53-fold increase in Kd (Kd = 29.1 nM) as compared to the native peptide. The approach described here provides a method that can be optimized for toxin production to develop more selective and effective Kv1.3 blockers with therapeutic potential.

The applicability of capillary electrophoresis (CE) with light-emitting diode-induced fluorescence detection (LEDIF) for the separation of sugars in honey samples was studied. An amount of 25 mM ammonium acetate (pH 4.5) with 0.3% polyethylene oxide (PEO) was found to be optimal for the efficient separation of carbohydrates. 8-aminopyrene-1,3,6-trisulfonic acid (APTS) was used for the labeling of the carbohydrate standards and honey sugars for fluorescence detection. The optimized method was applied in the quantitative analysis of fructose and glucose by direct injection of honey samples. Apart from the labeling reaction, no other sample preparation was performed. The mean values of the fructose/glucose ratio for phacelia honey, acacia honey and honeydew honey were 0.86, 1.61 and 1.42, respectively. The proposed method provides high separation efficiency and sensitive detection within a short analysis time. Apart from the labeling reaction, it enables the injection of honeys without sample pretreatment. This is the first time that fluorescence detection has been applied for the CE analysis of sugars in honeys.