Elsevier

Analytica Chimica Acta

Volume 1063, 31 July 2019, Pages 178-186
Analytica Chimica Acta

Isoelectric focusing array with immobilized pH gradient and dynamic scanning imaging for diabetes diagnosis

https://doi.org/10.1016/j.aca.2019.03.014Get rights and content

Highlights

  • A novel IEF system with an array of 24 IPG columns and dynamic scanning imaging (DSI).

  • Merits of fast rehydration of 25 min, quick focusing of 4 min and 2 min DSI for 24 IPG column assay.

  • First report on efficient quantitation of HbA1c in diabetes sample via IEF array but not HPLC.

Abstract

A traditional immobilized pH gradient (IPG) has a high stability for isoelectric focusing (IEF) but suffers from time-consuming rehydration, focusing and staining-imaging as well as complex performance. To address these issues, an IEF system with an array of 24 IPG columns (10 mm × 600 μm × 50 μm) and dynamic scanning imaging (DSI) was firstly designed for protein focusing. Moreover, two IPG columns (pH 4–9 and pH 6.7–7.7 of 10 mm in length) were firstly synthesized for IEF. A series of experiments were carried out based on the IEF array. In contrast to a traditional IPG IEF with more than 10 h rehydration, 5–14 h IEF and ca 10 h stain-imaging, the IEF array had the following merits: 25 min rehydration for sample loading, 4 min IEF, and 2 min dynamics scanning of 24 columns, well addressing the issues of traditional IEF. Furthermore, the IEF array had fair sensitivity (LOD of 60 ng), good recovery (95%), and high stability (1.02% RSD for intra-day and 2.16% for inter-day). Finally, the developed array was successfully used for separation and determination of HbA1c (a key biomarker for diabetes diagnosis) in blood samples. All these results indicated the applicability of the developed IEF array to diabetes diagnosis.

Introduction

As one of the most efficient separation techniques, isoelectric focusing (IEF) has been widely used in 2D separation for the proteomics research [[1], [2], [3], [4]], capillary IEF (cIEF) for the quality control of protein drugs [[5], [6], [7]], free-flow electrophoresis for the fractionation of cells [8,9] and organelles [10,11], and gel slab IEF for the focusing of hemoglobin A1c (HbA1c) [[12], [13], [14], [15], [16], [17], [18]].

Although these IEF techniques based on a natural pH gradient (NPG) had high resolution in the separation of proteins, cells and organelles [[1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]], they always suffered from pH gradient instabilities, such as the drifting thanks to non-equilibrium fluxes of hydrogen and hydroxyl ions [[19], [20], [21]], and the plateau due to water generation by hydrogen and hydroxyl ions neutralization [[19], [20], [21], [22]]. These instabilities greatly limited the relevant applications of NPG-based IEF to proteomics, protein drugs and clinical diagnosis.

To address the NPG-based IEF instabilities, the immobilized pH gradient (IPG) technique was developed for IEF run [21,22]. Particularly, IPG IEF was always used as the first dimension separation for 2-D gel electrophoresis in which a target protein spot could be extracted for direct LC-MS identification [[1], [2], [3], [4],21]. IPG IEF was so successful that it had become a golden standard of top-down proteomic technique [[1], [2], [3], [4]]. However, it always suffered from more than 10 h rehydration for sample loading, 5–14 h IEF run and ca 10 h stain-imaging for band monitoring as well as complex performance [21,22].

To solve these issues, many efforts have been devoted to developing capillary- and chip-IEF. For example, numerous cIEF methods have been reported, such as mobilization of pH gradient [23,24], whole column imaging [[25], [26], [27]], dynamic IEF [28], reagent-release array IEF [29] and immune IEF [30,31] as well as microscale focusing [32]. Various IEF chips have been designed, regarding IEF chip [33,34], pH sensing chip [35], NPG IEF microchip [36], and micro-chamber [37] as well as IPG IEF chip [38,39]. These techniques could greatly simplify sample loading, improve IEF speed and achieve whole column imaging. However, they still faced the challenges of low throughput due to single capillary [[23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35],37,38] and unideal repeatability of IEF induced by non-uniform photo-induced synthesis of IPG [38,39].

In particular, two NPG methods of gel slab IEF [[12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28]] and cIEF [40,41] have been proposed for separation of HbA1c (a key biomarker used for diabetes diagnosis and assessment of long-term blood glucose control [[42], [43], [44]]) in blood sample. Owing to these issues mentioned above, the two methods could not be used to quantify HbA1c for diabetes diagnosis. Instead, high-performance liquid chromatography (HPLC) [44,45], agarose gel electrophoresis (AGE) [46], boronate affinity chromatography [47], POCT [[47], [48], [49]] and tuibidimetric [50] immunoassays were developed for the determination of HbA1c. Among these methods, the last three were able to detect the absolute but not relative content of glycated Hb species and were less used for clinical diagnosis [[47], [48], [49], [50]]. AGE could monitor the relative content of HbA1c but suffered from complex manual performance [46]. HPLC could monitor the relative content of HbA1 with high stability and speed, but faced with incomplete separation of HbA1c from the adjacent Hb species [44].

Herein, an IPG IEF array system was designed for HbA1c assay of diabetes whole blood combining the merits of traditional IPG IEF with the ones of capillary-chip IEF (Fig. 1). The core components were a 24-column array with sharp IPG of pH 6.7–7.7 and a dynamic scanning imaging (DSI). The relevant experiments showed that the IEF array had only 25 min rehydration, 4 min focusing, 2 min DSI of 24 columns and facile performance, obviously addressing the issues of traditional IPG IEF. Moreover, the IEF array showed its high efficiency, good stability and high throughput for HbA1c separation, well solving the shortcomings of HPLC and capillary-/gel-IEF for diabetes diagnosis.

Section snippets

IPG array system

Fig. 1 showed the whole design of IPG IEF array system, including the 24-column array and focusing tray (Panel A and B), DSI (Panel C), integration (Panel D) and whole focusing system with hardware and software (Panel E). The IPG IEF array had a size of 72 mm × 11 mm × 8 mm, it contained 24 IPG columns and support frame (Panel A). The columns were fixed to 24 pairs of micro-poles of support frame, and the space between two adjacent IPG columns was 2.4 mm. This design allowed simultaneous batch

Rehydration

The rehydration speed of IEF array was greatly enhanced in contrast to that of traditional IEF (Table 1). The former could be finished within 25 min for simultaneous rehydration of 24 columns per batch, whereas the later could be completed in more than 10 h rehydration of IPG strips [21,22]. The efficiency of developed rehydration was increased 50 folds at least as compared with the one of traditional IEF (Table 1). Evidently, the efficiency of IEF array could be further increased to 100, 200

Conclusions

Herein, a facile IEF array with 24 columns and DSI was described for protein focusing, particularly for HbA1c assay of diabetes blood samples. The described array exhibited several advantages over classical gel IEF, commercial HPLC, cIEF and chip focusing. Firstly, the array method had more than 50 folds increase of rehydration efficiency as compared with the one of classic gel IEF. Secondly, the separation speed and throughput of IEF array were 75–220 folds and 150–440 folds enhancement in

Contributions of authors

G. Q. Li and C. X. Cao contributed to conceptualization, writing, and analysis. H. G. Li, Y. X. Wang and X. P. Liu contributed to device fabrication and software development. The undergraduate students, F. F. Dong, F. Z. Kong, Q. Zhang, S. Saud and F. Luo contributed to experiment operation and data collection. Y. F. Bi performed the HPLC analyses. H. Xiao and L. Y. Fan contributed to writing. H. J. Lu and Y. Peng performed the MS identification. C. X. Cao and Y. X. Wang supervised the research

Conflict of interest

The authors declared that there was no interest conflict with other researchers.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 31727801), the National Key Development of Scientific Instruments (No. 2011YQ030139).

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