title: A stable and high-resolution isoelectric focusing capillary array device for micropreparative separation of proteins.
abstract: 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.
authors.affiliation: Key Laboratory of Microbial Metabolism of Education Ministry, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Si
authors.initials: S
authors.lastname: Li
authors.forename: Jing-Yu
authors.initials: JY
authors.lastname: Dong
authors.forename: Chen-Gang
authors.initials: CG
authors.lastname: Guo
authors.forename: Yi-Xin
authors.initials: YX
authors.lastname: Wu
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Liu-yin
authors.initials: LY
authors.lastname: Fan
authors.forename: Cheng-Xi
authors.initials: CX
authors.lastname: Cao
authors.forename: Wei-Bing
authors.initials: WB
authors.lastname: Zhang
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry; D004566:Electrodes; D053841:Electroosmosis; D006442:Glycated Hemoglobin / Q000302:isolation & purification*; D006441:Hemoglobin A / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000295:instrumentation* / Q000379:methods; D046210:Microfluidic Analytical Techniques; D019904:Polymethyl Methacrylate / Q000737:chemistry; D015203:Reproducibility of Results; D000099166:Polyacrylamides
chemical_list: D000180:Acrylic Resins; D006442:Glycated Hemoglobin; D006441:Hemoglobin A; D019904:Polymethyl Methacrylate; D000099166:Polyacrylamides; C517652:hemoglobin A1c protein, human; C028881:hemoglobin A(0)
keywords: Capillary Array; Coating; Isoelectric focusing; Repeatability; Resolution