Elsevier

Analytica Chimica Acta

Volume 1139, 1 December 2020, Pages 27-35
Analytica Chimica Acta

Deep eutectic solvents-based polymer monolith incorporated with titanium dioxide nanotubes for specific recognition of proteins

https://doi.org/10.1016/j.aca.2020.09.009Get rights and content

Highlights

  • A TNTs incorporated GMA monolith was first prepared using DESs monomer.

  • TNTs/DESs incorporated monolith can selectively capture proteins by adjusting pH of loading solvent.

  • Enrichment efficiency and adsorption specificity were evaluated by solid phase extraction of different proteins.

  • Enrichment performance was significantly improved by synergistic effect of TNTs and DESs.

  • This monolith was practically applied to the proteins extraction of rat liver.

Abstract

An organic-inorganic hybrid monolith incorporated with titanium dioxide nanotubes (TNTs) and hydrophilic deep eutectic solvents (DESs) was prepared and evaluated by the isolation of proteins using solid phase microextraction. A typical polymerization system was composed of choline chloride/methacrylic acid (ChCl/MAA, DESs monomer), glycidyl methacrylate (GMA), as well as ethylene glycol dimethacrylate (EDMA) in the presence of TNTs. Then the epoxy groups on the surface of the resulting monolith were modified with amino groups. The synergistic effect of TNTs and DESs monomer to improve the enrichment performance of the sorbent significantly was demonstrated. Compared with the corresponding TNTs/DESs-free monolith, the recoveries of BSA and OVA were increased to 98.6% and 92.7% (RSDs < 2.0%), with an improvement of more than 60.0%. With a correlation coefficient of determination (R2) higher than 0.9995, the enrichment factors (EFs) were 21.9–28.3-fold. In addition, the resulting monolith was further applied to specifically capture proteins from rat liver according to their pI value, followed by HPLC-MS/MS analysis. The results indicated that the developed monolith was an effective material to isolate protein species of interest according to the pI value of target proteins.

Introduction

The preparation of protein adsorption nanomaterials with biocompatible surfaces is a significant research field. Kinetic studies have shown that a variety of main driving forces may be involved in the adsorption process, including hydrogen bonding, van der Waals, electrostatic and hydrophobic interactions [1]. Since more structures of proteins are retained on the hydrophilic surface under the condition of electrostatic neutrality, they have been applied to the separation and enrichment of biological macromolecules from complex samples [2,3]. Vergara-Barberan et al. [4] selectively capture the target proteins based on methacrylate monolith modified with gold nanoparticle by adjusting the pH of the solvent. Later, they also developed a similar method for isolating viscotoxins from the European mistletoe extract [5]. Besides, hydrophilic polymers have good capture specificity for proteins in hydrogen-bonding separation technologies. Péikryl et al. [6] have studied a new method for separating biomacromolecules by hydrophilic interaction based on magnetic IDA-modified hydrophilic methacrylate-based polymer.

Polymer monoliths are continuously attracting great attention due to their unique characteristics such as simple polymerization, high permeability, and excellent mass transfer properties [7], which have been applied to life sciences, pharmaceutical science and environmental science [8,9]. Recently, polymeric monoliths have been widely adopted to sample pretreatment [10]. However, polymer monoliths have a limited surface area, resulting in a lower binding capacity. Moreover, the lack of monomers with certain functional groups prevents from desired properties. To overcome these limitations of monoliths, plenty of strategies were investigated, including the introduction of nanomaterials and new monomers as well as the chemical modification of surface groups [11,12].

As a two-dimensional nanomaterial with a high aspect ratio, TNTs have a multi-walled, vortex-shaped, open structure, as well as large internal and external specific surface area. The characteristics above are very advantageous as adsorbents [13]. Due to the tubular structure, TNTs have a larger surface area and higher hydroxyl density than TiO2 nanoparticles, which increases the number of potential reaction points [14]. Therefore, the extraction kinetics is faster, resulting in a higher extraction recovery of the target analyte. It was reported that TiO2 could enhance the hydrophilicity of the monolith and gave rise to an effective capture of hydrophilic proteins [15]. Besides, the high density of hydroxyl groups on TNTs can be used to extract analytes by electrostatic interaction. For example, Huang et al. [16] loaded samples on the isoelectric point (pI) of TNTs to enhance the interaction between the targets and the surface of the nanostructure for adsorption. TNTs exhibited an adjustable surface charge. If the analytes have acidic or basic functional groups, the pH will also affect the ionization state of the analytes. Therefore, the pH of TNTs is important as a variable for protein adsorption. However, there have been no reports on the use of TNTs to enrich non-specifically modified proteins.

Deep eutectic solvents (DESs) are novel ionic liquids analogs that are eutectic mixture of a hydrogen bond acceptor with a hydrogen bond donor. They are widely applied in multiple aspects, including selective identification of biological macromolecules, separation processes and nanomaterials preparation [[17], [18], [19], [20]]. DESs can interact with proteins through hydrogen bonds and then exhibit an adsorption effect on proteins. When the pH value is close to the pI of proteins, the electrostatic interaction between the adsorption material and protein is minimized [4] and the adsorption of proteins tends to occur [3]. Thus, when polymerizable DES is used as a monomer to prepare monoliths, the non-specific adsorption of proteins on the DES-based polymer can be expected to be inhibited effectively.

Due to the reaction activity of epoxy groups, functional monomer glycidyl methacrylate (GMA) is usually used as “reactive support” to prepare polymer materials [21,22]. The surface of GMA-based monoliths is susceptible to chemical functionalization to obtain hydrophilic/hydrophobic, chiral and ion exchange functions for various chromatographic purposes [4,23,24]. For example, amino modification can significantly increase hydrophilicity and hydrogen bonds of GMA-based monoliths. Although the direct use of monomers with amino groups can simplify the polymerization process by one-step polymerization, some certain limitations exist at the same time. For example, a significant proportion of functional groups can be embedded within the dense polymer structure, and their function effect may greatly be reduced [22]. As a result, the post-modification strategy of the monolith is developed in this work to overcome the limitation.

The aim of the study is to develop a novel sorbent that can isolate proteins by adjusting the condition of pH. In view of the fact mentioned above, a TNTs/DESs based monolithic column containing GMA was first synthesized, and then the epoxy groups on the surface of the resulting monolith were modified with amino groups. The enrichment performance of the resulting monolith as a sorbent of solid phase extraction (SPE) was demonstrated by extracting bovine serum albumin (BSA) and ovalbumin (OVA). The specific recognition of proteins was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as the HPLC-MS/MS analysis. To our knowledge, TNTs/DESs organic-inorganic hybrid monolith developed here has not been reported before.

Section snippets

Chemical reagents and materials

Glycidyl methacrylate (GMA) was supplied by Aladdin Industrial Corporation (Shanghai, China, https://www.aladdin-e.com). Ethylene glycol dimethacrylate (EDMA, 98%), 3-Methacryloxypropyltrimethoxysilane (KH570, 98%) and 3-(trimethoxysilyl) propyl methacrylate (γ-MPS, 98%) were obtained from Sigma-Aldrich (St. Louis, Mo, USA, China, http://www.sigmaaldrich.com). Titanium dioxide-B nanotubes (TNTs, 99%) were obtained from Nanjing Xianfeng Nanomaterials Technology Co., Ltd. (Nanjing, China, //www.xfnano.com

Design and fabrication of TNTs-poly(GMA-co-DESs-co-EDMA) monolith

The preparation process of the TNTs-poly (GMA-co-DESs-co-EDMA) column was shown in Scheme 1. The monolithic matrix was synthesized by in situ polymerization using GMA and DESs monomer as functional monomers. The DESs monomer was composed of hydrogen bond acceptor and hydrogen bond donor, thus used as a new kind of hydrophilic monomer to effectively inhibit the non-specific adsorption of proteins and enhance the adsorption capacity of the proteins. Since the epoxy groups were easy to

Conclusion

A novel TNTs/DESs organic-inorganic hybrid monolith has been successfully developed and used for protein separation in SPE. In comparison with different types of monolith containing TNTs or DESs, it was demonstrated that TNTs and DESs played a synergistic role in improving the enrichment capacity of the resulting monolith. Compared with the identification results of untreated rat liver proteins, the number of proteins at the pI close to loading pH identified from the treated sample was

CRediT authorship contribution statement

Xue Zhang: Writing - original draft, Methodology, Investigation. Mei-Hong Chai: Investigation, Data curation. Ze-Hui Wei: Data curation, Writing - review & editing. Wen-Jing Chen: Validation. Zhao-Sheng Liu: Conceptualization. Yan-Ping Huang: Funding acquisition, Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by National Natural Science Foundation of China (Grant No. 21775109).

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