Investigation of the pH gradient formation and cathodic drift in microchip isoelectric focusing with imaged UV detection

Xu, Zhongqi; Okabe, Noboru; Arai, Akihiro; Hirokawa, Takatsugu

doi:10.1002/elps.201000395

Cited by 13 publications

(37 citation statements)

References 43 publications

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“…Fig. illustrates that pH 3–10 CAs were compressed near the cathode, a common phenomenon reported previously . In a large slab gel with small reservoirs that allowed electrolytes to diffuse into the gel, the pH gradient was observed to compress due to differing anion and cation migration rates within the gel .…”

Section: Resultssupporting

confidence: 72%

“…The Δp I values of GFP and R‐PE without surfactant were 0.14 and 0.12, but with F‐108 surfactant this reduced to 0.05 and 0.09, respectively. The magnitude of these sIEF results are similar to and slightly smaller than both cIEF (1 × 10 −1 ) and free‐flow/microchip IEF (2 to 4 × 10 −1 ) .…”

Section: Resultssupporting

confidence: 52%

“…Ideally, commercial carrier ampholytes should generate a linear pH gradient that spans the anode to cathode gel length of 300 m. Fig. 3 illustrates that pH 3-10 CAs were compressed near the cathode, a common phenomenon reported previously [6,44,[50][51][52][53][54]. In a large slab gel with small reservoirs that allowed electrolytes to diffuse into the gel, the pH gradient was observed to compress due to differing anion and cation migration rates within the gel [55].…”

Section: Ph Calibration and Ph Gradient Imagingsupporting

confidence: 53%

“…The focusing effectiveness of sIEF was determined, similar to other IEF platforms , by quantifying the minimum difference between isoelectric points, Δp I , and peak capacity, n . These parameters indicate resolvability of proteins by p I as well as the number of protein species that can be distinguished within an IEF gel, respectively . For Δp I , the pH gradient, d(pH)/dx, diffusion coefficient, D, and pH gradient mobility difference, ‐dμ/d(pH), are assumed constant under ideal conditions .…”

Section: Methodsmentioning

confidence: 99%

“…Theoretical and experimental peak capacities agree well. Peak capacity numbers were also comparable to free-flow and microchip IEF, which are between 23 and 48 [12,44]. However, cIEF results analyzed with precision MS or similar more costly analytical tools report peak capacities one to two magnitudes larger [71,72].…”

Section: Sief Resolving Powermentioning

confidence: 99%

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Surface isoelectric focusing (sIEF) with carrier ampholyte pH gradient

2017

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Isoelectric focusing (IEF) is a powerful tool for amphoteric protein separations because of high sensitivity, bio-compatibility, and reduced complexity compared to chromatography or mechanical separation techniques. IEF miniaturization is attractive because it enables rapid analysis, easier adaptation to point of care applications, and smaller sample demands. However, existing small-scale IEF tools have not yet been able to analyze single protein spots from array libraries, which are ubiquitous in many pharmaceutical discovery and screening protocols. Thus, we introduce an in situ, novel, miniaturized protein analysis approach that we have termed surface isoelectric focusing (sIEF). Low volume printed sIEF gels can be run at length scales of ∼300 μm, utilize ∼0.9 ng of protein with voltages below 10 V. Further, the sIEF device platform is so simple that it can be integrated with protein library arrays to reduce cost; devices demonstrate reusability above 50 uses. An acrylamide monomer solution containing broad-range carrier ampholytes was microprinted with a Nano eNabler between micropatterned gold electrodes spaced 300 μm apart on a glass slide. The acrylamide gel was polymerized in situ followed by protein loading via printed diffusional exchange. A pH gradient formed via carrier ampholyte stacking when electrodes were energized; the gradient was verified using ratiometric pH-sensitive FITC/TRITC dyes. Green fluorescent protein (GFP) and R-phycoerythrin (R-PE) were utilized both as pI markers and to test sIEF performance as a function of electric field strength and ampholyte concentration. Factors hampering sIEF included cathodic drift and pH gradient compression, but were reduced by co-printing non-ionic Synperonic® F-108 surfactant to reduce protein-gel interactions. sIEF gels achieved protein separations in <10 min yielding bands < 50 μm wide with peak capacities of ∼8 and minimum pI differences from 0.12 to 0.14. This new sIEF technique demonstrated comparable focusing at ∼100 times smaller dimensions than any previous IEF. Further, sample volumes required were reduced four orders of magnitude from 20 μL for slab gel IEF to 0.002 μL for sIEF. In summary, sIEF advantages include smaller volumes, reduced power consumption, and microchip surface accessibility to focused bands along with equivalent separation resolutions to prior IEF tools. These attributes position this new technology for rapid, in situ protein library analysis in clinical and pharmaceutical settings.

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Section: Resultssupporting

confidence: 72%

Section: Resultssupporting

confidence: 52%

Section: Ph Calibration and Ph Gradient Imagingsupporting

confidence: 53%

Section: Methodsmentioning

confidence: 99%

Section: Sief Resolving Powermentioning

confidence: 99%

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Surface isoelectric focusing (sIEF) with carrier ampholyte pH gradient

2017

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Determination of protein charge variants with (imaged) capillary isoelectric focusing and capillary zone electrophoresis

Kahle

Wätzig

2018

Electrophoresis

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Biopharmaceuticals are increasingly present in pharmaceutical therapy. These highly complex molecules are still challenging for development and quality control. Charge heterogeneity is next to size heterogeneity, glycan analysis, or peptide mapping one main aspect of their thorough characterization. This review focuses on the charge-based analysis of biopharmaceuticals through CE. An overview is given about the three separation techniques cIEF, imaged cIEF and CZE for the analyses of charge variants, emphasizing assays and their precision. Especially the developments in the experimental conditions have been given a high priority: sample preparation, capillary properties, anolytes and catholytes, carrier ampholytes, sacrificing agents, solubility enhancement, isoelectric point marker, mobilization techniques, and detection modes have been discussed. CIEF shows a particularly high heterogeneity in its methods. Therefore, a design space is suggested that states the most important adjustable parameters and their range. This enables an adequate design of experiments for individual sample separations, in order to accelerate and simplify method developments.

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Instabilities of the pH gradient in carrier ampholyte‐based isoelectric focusing: Elucidation of the contributing electrokinetic processes by computer simulation

Thormann

Mosher

2020

Electrophoresis

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Electrokinetic processes that lead to pH gradient instabilities in carrier ampholyte‐based IEF are reviewed. In addition to electroosmosis, there are four of electrophoretic nature, namely (i) the stabilizing phase with the plateau phenomenon, (ii) the gradual isotachophoretic loss of carrier ampholytes at the two column ends in presence of electrode solutions, (iii) the inequality of the mobilities of positively and negatively charged species of ampholytes, and (iv) the continuous penetration of carbonate from the catholyte into the focusing column. The impact of these factors to cathodic and anodic drifts was analyzed by simulation of carrier ampholyte‐based focusing in closed and open columns. Focusing under realistic conditions within a 5 cm long capillary in which three amphoteric low molecular mass dyes were focused in a pH 3–10 gradient formed by 140 carrier ampholytes was investigated. In open columns, electroosmosis displaces the entire gradient toward the cathode or anode whereas the electrophoretic processes act bidirectionally with a transition around pH 4 (drifts for pI > 4 and pI < 4 typically toward the cathode and anode, respectively). The data illustrate that focused zones of carrier ampholytes have an electrophoretic flux and that dynamic simulation can be effectively used to assess the magnitude of each of the electrokinetic destabilizing factors and the resulting drift for a combination of these effects. Predicted drifts of focused marker dyes are compared to those observed experimentally in a setup with coated capillary and whole column optical imaging.

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Investigation of the pH gradient formation and cathodic drift in microchip isoelectric focusing with imaged UV detection

Cited by 13 publications

References 43 publications

Surface isoelectric focusing (sIEF) with carrier ampholyte pH gradient

Surface isoelectric focusing (sIEF) with carrier ampholyte pH gradient

Determination of protein charge variants with (imaged) capillary isoelectric focusing and capillary zone electrophoresis

Instabilities of the pH gradient in carrier ampholyte‐based isoelectric focusing: Elucidation of the contributing electrokinetic processes by computer simulation

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