Reagent-release capillary array-isoelectric focusing device as a rapid screening device for IEF condition optimization

Abstract

This report describes the fabrication and characterization of a simple and disposable capillary isoelectric focusing (cIEF) device containing a reagent-release capillary (RRC) array and poly(dimethylsiloxane) (PDMS) platform, which allows rapid (within 10 min) screening of cIEF conditions by introducing a sample solution into plural RRCs by capillary action followed by electric field application. To prepare the RRC, covalent immobilization of poly(dimethylacrylamide) (PDMA) was conducted to suppress electro-osmotic flow (EOF), followed by physical adsorption of the mixture of carrier ampholyte (CA), surfactant, labeling reagent (LR), and other additives to the PDMA surface to construct a two-layer structure inside a square glass capillary. When the sample solution containing proteins was introduced into the RRC, physically adsorbed CA, surfactant, and LR can be dissolved and released into the sample solution. Then, complexation of LR with proteins, mixing with CA and surfactant, and exposure of the PDMA surface spontaneously occurs for the IEF experiments. Here, three different RRCs that immobilize different CAs were prepared, and simultaneous cIEF experiments involving hemoglobin AFSC mixtures for choosing the best CA demonstrated the proof of concept.

Advancements in Capillary-Assembled Microchip (CAs-CHIP) Development for Multiple Analyte Sensing and Microchip Electrophoresis

“…11). 50 A second application was for the combination of ELISA-cIEF by synthesizing a new fluorescent dye that reacts with the alkaline phosphatase-labeled antibody to produce an organic fluorescent molecule with pI. In this case, the final enzymatic reaction products of capillary ELISA are concentrated by IEF to give highly sensitive signals for proteins.…”

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

“…As model cases, solutions and hydrogels containing 10 -4 M sulforhodamine B (anionic fluorescent dye) were prepared with deionized water and acrylamide monomer solution, respectively. To prevent the electroosmotic flow during the electrokinetic injection, the inner surface of the capillary was modified with poly-(dimethylacrylamide) (PDMA) using the same procedure as reported previously [24]. Both ends of the PDMA-coated capillary filled with a buffer solution were in contact with the solutions or hydrogels.…”

“…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.…”

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

“…To suppress both the fast electroosmotic flow and nonspecific adsorption of proteins, the inner surface of the capillary was modified with polyDMA (PDMA) as previously reported. 31 The reagents for both the enzyme activity assay and CIEF experiments were physically adsorbed onto the surface of the PDMA layer as a thin soluble coating. Typically, a solution containing 0.3% (v/v) Bio-Lyte 3/10, 0.1% (w/v) Tween 20, 0.7% (w/v) glycerol, and 1.0 × 10 -5 M R110-based substrate for trypsin activity assay was introduced into the PDMA-coated square capillary (5.0 cm in length).…”

“…30 In addition, RRCs containing CAs was also developed and combined with CIEF to perform the focusing and separation of proteins with a simple experimental procedure. 31 In this study, we propose to use the fluorescent enzyme substrate, rhodamine 110 (R110) diamide and RRC-IEF for a simple, sensitive, and rapid enzyme activity assay with the consumption of small amounts of samples and reagents. In the R110-based substrate, two amide groups are cleaved and substituted with amino groups by proteases.…”