Highly Sensitive and Multiple Enzyme Activity Assay Using Reagent-release Capillary-Isoelectric Focusing with Rhodamine 110-based Substrates

Abstract

In this study, a simple and highly sensitive enzyme activity assay based on reagent-release capillary-isoelectric focusing is described. Reagent-release capillaries containing a fluorescent substrate, which produces fluorescent products possessing an isoelectric point after reaction with enzymes, provides a simple procedure. This is because it allows to spontaneously inject a sample solution into the capillary by capillary action, mixing reagents, and subsequently concentrating the fluorescent products based on isoelectric focusing. Fluorescent rhodamine 110 and its monoamide derivative, which were generated as a final product and an intermediate, respectively, were then focused and separated by reagent-release capillary-isoelectric focusing. After 30 min of enzyme reactions, two focused fluorescent bands were clearly isolated along the prepared capillaries. Employing the focused band of rhodamine 110 monoamide allowed for highly sensitive detection of enzyme activity in the 10 pg mL -1 order, while that of the conventional assay using a microplate was in the ng mL -1 order. Furthermore, arraying reagent-release capillaries of different substrates on a chip allowed for simultaneous multi-assay of enzyme activity with good sensitivity in the pg mL -1 order for each protein.

A Simple and Easy-to-Use Capillary Isoelectric Focusing Technique Using Reagent-Release Hydrogels

“…In CIEF with RRGs, the profile of the formed pH gradient was almost the same as that obtained using the acid and base solutions. These results shows that the proposed RRGs are applicable to bioassays using CIEF with RRCs [25], which will realize a rapid, simple and easy-to-use bioassay based on RRC-IEF.…”

“…Yang et al [20,21] and Zhu et al [22,23] reported the immobilization of carrier ampholytes (CAs) on a monolithic column in the microchannel, thereby avoiding the addition of carrier ampholytes into the sample solution to prevent current increase. In our previous study, we reported that the use of a "reagent-release capillary" (RRC) facilitated the MIEF procedure simply by introducing a sample solution into a capillary via capillary action, soaking the cathodic and anodic ends of the capillary into basic and acid solutions filled in reservoirs, respectively, and applying an electric field [24,25]. However, this technique still requires a step of manual application of the acid and base solutions to the reservoirs after sample introduction into the RRC.…”

Selective Activation of N,N′‐Diacyl Rhodamine Pro‐fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE)

“…Finally, we found that N , N ′‐dipropionyl rhodamine pro‐fluorophore showed bright, cytoplasmic fluorescence in PLE‐expressing cells thus demonstrating suitability for cellular imaging. Recently, Lavis and others have demonstrated the utility of PLE for activating rhodamine‐based pro‐fluorophores using the tri‐methyl lock approach and recently re‐evaluated prior work with rhodamines released by serine hydrolase with varying peptide pro‐fluorophore scaffolds . Although compound 1 b had been previously prepared testing of 1 b as a substrate for PLE was not reported.…”

Synthetic Fluorophores for Visualizing Biomolecules in Living Systems

“…This property is used in the synthesis of photoactivable rhodamine analogues [ 48 ] and to synthesize fluorogenic substrates when studying the mechanisms of enzyme catalysis. Rhodamine 110 derivatives have been used as substrates to determine the activity of various enzymes [ 49 ]. Hybrid fluorophores consisting of a polypeptide-linked quantum dot and rhodamine that can be cleaved by caspase-1 have been used in apoptosis assays [ 50 ].…”