A stable and high-resolution isoelectric focusing capillary array device for micropreparative separation of proteins.

A simple capillary array IEF device was developed for high resolution and micropreparative separation of trace amounts of proteins. Based on quasi-chip-scale manufacturing, the specific capillaries (600 µm i.d., 1200 µm o.d. and 20 mm length) were integrated with the miniaturized polymethyl-methacrylate electrode trays. Electroosmotic flow was suppressed effectively by modified cross-linked polyacrylamide coating, and instability of IEF was addressed using the designed concentration of electrolytes via moving reaction boundary theory. As a prototyping, the resolution, reproducibility, throughput, speed and linearity of pH gradient were systemically evaluated with model proteins. The results revealed the following advantages: (i) the reproducibility of array was assessed as RSD values of 0.95% (intra-day) and 2.88% (inter-day); (ii) IEF could be completed in 20 min with up to 400 V/cm electric field; (iii) high resolution separation of model proteins achieved in 20mm length column; (iv) multi-units with 48 micro-columns can be easily integrated to obtain high throughput; and (v) good linearity of pH gradient (R=0.9989). More importantly, utility of the device was tested by using hemoglobins sample from human red blood cell. HbA0 and HbA1c with only ΔpI 0.03 have been successfully separated by the developed method.

Determination of free acidic and alkaline residues of protein via moving reaction boundary titration in microdevice electrophoresis.

As two important physico-chemical parameters, the acidic and alkaline residues of protein are of evident significance for the evaluation of protein properties and the design of relevant separation and analysis. However, there is still no electrophoretic method used for the direct detection of free acidic and alkaline residues of protein. Herein, we developed the concepts of moving reaction boundary (MRB) and MRB titration, relevant MRB titration theory, and the method of microdevice electrophoresis for the determination of free acidic and alkaline residues of protein. In the MRB titration, the boundary was created with acid or alkali and target protein immobilized via highly cross-linked polyacrylamide gel (PAG). It was theoretically revealed that the number of free acidic or alkaline residues of protein was as a function of MRB displacement in the electrophoretic titration system. As a proof of concept, seven model proteins were chosen for the determination of acidic or alkaline residues of protein via MRB titration. The results showed that the numbers of free acidic and alkaline residues of proteins detected were in good agreement with those obtained from the relevant amino sequences in the NCBI database, demonstrating the feasibility of the developed concept, theory and technique. The general methodology of MRB titration has potential application for inexpensive, facilitative and informative protein structure analysis of free acidic or alkaline residues of protein.

A simple chip free-flow electrophoresis for monosaccharide sensing via supermolecule interaction of boronic acid functionalized quencher and fluorescent dye.

Here, a simple micro free-flow electrophoresis (µFFE) was developed for fluorescence sensing of monosaccharide via supermolecule interaction of synthesized boronic acid functionalized benzyl viologen (ο-BBV) and fluorescent dye. The µFFE contained two open electrode cavities and an ion-exchange membrane was sandwiched between two polymethylmethacrylate plates. The experiments demonstrated the following merits of developed µFFE: (i) up to 90.5% of voltage efficiency due to high conductivity of ion-exchange membrane; (ii) a strong ability against influence of bubble produced in two electrodes due to open design of electrode cavities; and (iii) reusable and washable separation chamber (45 mm × 17 mm × 100 µm, 77 µL) avoiding the discard of µFFE due to blockage of solute precipitation in chamber. Remarkably, the µFFE was first designed for the sensing of monosaccharide via the supermolecule interaction of synthesized ο-BBV, fluorescent dye, and monosaccharide. Under the optimized conditions, the minimum concentration of monosaccharide that could be detected was 1 × 10(-11) M. Finally, the developed device was used for the detection of 0.3 mM glucose spiked in human urine. All of the results demonstrated the feasibility of monosaccharide detection via the µFFE.

Simply enhancing throughput of free-flow electrophoresis via organic-aqueous environment for purification of weak polarity solute of phenazine-1-carboxylic acid in fermentation of Pseudomonas sp. M18.

Herein, a simple novel free-flow electrophoresis (FFE) method was developed via introduction of organic solvent into the electrolyte system, increasing the solute solubility and throughput of the sample. As a proof of concept, phenazine-1-carboxylic acid (PCA) from Pseudomonas sp. M18 was selected as a model solute for the demonstration on feasibility of novel FFE method on account of its faint solubility in aqueous circumstance. In the developed method, the organic solvent was added into not only the sample buffer to improve the solubility of the solute, but also the background buffer to construct a uniform aqueous-organic circumstance. These factors of organic solvent percentage and types as well as pH value of background buffer were investigated for the purification of PCA in the FFE device via CE. The experiments revealed that the percentage and the types of organic solvent exerted major influence on the purification of PCA. Under the optimized conditions (30 mM phosphate buffer in 60:40 (v/v) water-methanol at an apparent pH 7.0, 3.26 mL/min background flux, 10-min residence time of injected sample, and 400 V), PCA could be continuously purified from its impurities. The flux of sample injection was 10.05 µL/min, and the recovery was up to 93.7%. An 11.9-fold improvement of throughput was found with a carrier buffer containing 40% (v/v) methanol, compared with the pure aqueous phase. The developed procedure is of evident significance for the purification of weak polarity solute via FFE.

A simple monolithic column electroelution for protein recovery from gel electrophoresis.

Protein recovery from gel electrophoresis plays an important role in functional genomics and proteomics but faces a series of issues (e.g., complex procedure, low recovery, long experimental time). In this study, a monolithic column electroelution (MCE) was developed for protein recovery from gel electrophoresis. With the model proteins of bovine serum albumin (BSA), hemoglobin (Hb), and myoglobin (Mb), the developed device and method were compared with common electroelution procedures in agarose gel electrophoresis (AGE). The comparative experiments revealed that (i) the protein recovery achieved with the developed device was greater than 83%, much higher than the 41% to 50% achieved with the common devices; (ii) the running time to obtain 70% recovery was approximately 15 min, evidently shorter than the 240 min with the common devices; and (iii) the device and procedure were simple and less time-consuming as compared with those of the common devices. It was observed that the serum protein bands cut from polyacrylamide gel electrophoresis could be transferred into solution in 15 to 30 min with 82% yield. The device, along with its relevant procedure, has potential use in protein extraction and proteomics as well as in DNA studies.