Effect of protein adsorption on electrospun hemoglobin/gelatin-MWCNTs microbelts modified electrode: Toward electrochemical measurement of hydrogen peroxide

https://doi.org/10.1016/j.matchemphys.2020.123827Get rights and content

Highlights

  • Direct electron transfer between the Hb in the microbelts and electrode is achieved after protein adsorption.

  • The microbelts still enable the catalysis of H2O2 after protein adsorption.

  • The microbelts constructed H2O2 sensor possesses high stability and selectivity after protein adsorption.

Abstract

The on-line electrochemical analysis is one of powerful strategies in analytical chemistry and pathophysiology. To achieve high sensitivity and long-term stability of electrochemical biosensor, the bottleneck challenge is the spontaneous proteins adsorption onto the electrode surface within the biological fluids or in vivo environments. In this work, a hemoglobin/gelatin-multiwalled carbon nanotubes microbelts modified electrode (Hb/gelatin-MWCNTs/GC electrode) was successfully fabricated via one-step electrospinning process. The results of atomic force microscopy (AFM), scanning electron microscopy (SEM) and water contact angle test confirmed the electrospun Hb/gelatin-MWCNTs microbelts possessed smooth and hydrophilic surfaces. Furthermore, the electrospun Hb/gelatin-MWCNTs/GC electrode after protein adsorption displayed an excellent electrocatalytic sensitivity toward the reduction of hydrogen peroxide (H2O2). Moreover, the Hb/gelatin-MWCNTs/GC electrode presented very high biological affinity to H2O2 (Kmapp=503.4 ± 2.8 μmol L−1) after 360 min protein adsorption compared to that of the electrode before protein adsorption (Kmapp=298.1 ± 3.1 μmol L−1). The microbelts constructed H2O2 biosensor showed high selectivity, stability and reproducibility after protein adsorption. Therefore, this work provided the proof of the concept that the electrospun Hb/gelatin-MWCNTs/GC electrode displayed excellent sensing performance to H2O2 after protein adsorption, which could enable the implantable electrochemical biosensor for the on-line analysis.

Introduction

The detection and analysis of specific biomolecules are of significance for obtaining chemical and biological information in life processes, explaining the mechanism of life activities and thus aiming for effective diagnosis and treatment of diseases [1]. Hydrogen peroxide (H2O2), generated as a consequence of the incomplete reduction of oxygen in a broad range of biological processes, plays a crucial role in the signal transduction, oxidative pathway of cells and the diagnosis of the aging and diseases [2,3]. So detecting H2O2 in an efficient, fast and sensitive way is the ultimate goal. Electrochemical biosensing technique provides an advanced tool for effective detection of H2O2 with the typically combined advantages of high sensitivity and selectivity, simple instrument and easy access, and low cost [[4], [5], [6], [7]]. However, the current electrochemical biosensing is mainly limited to in vitro detection and off-line analysis for only the pre-collected specimen. To enable the real-time monitoring H2O2 changes under various life activities and biochemical reactions in biological living tissues, the on-line electrochemical sensing and analysis is highly demanded for H2O2 detection [[8], [9], [10], [11], [12]], especially in complicated physiological environment [8,13,14].

In the physiological environment, protein adsorption onto the electrode would occur firstly followed by a series of undesired interactions on the electrode surface with platelets, pathogens and cells in biological fluids [15]. The protein adsorption would reduce the effective electrode surface area and affect the electron transfer process and thus would largely deteriorate the functions and the stability of the biosensor [[16], [17], [18]]. Hence, it is urgent to tackle the protein adsorption on the electrode surface and design the functional electrode surface not affected by protein to maintain the performance of the electrochemical biosensor with high sensitivity, linear range accuracy and long-term stability [19,20].

To the best of our knowledge, hydrophilic materials can effectively reduce protein adsorption [21,22]. Polyethylene glycol (PEG) is known for its super hydrophilicity and biocompatibility and can be adopted to modify the electrode. For example, a DNA sensor constructed via co-immobilization of PEG moieties showed reduced nonspecific protein adsorption on electrode surface [23] and a PEG grafted polyaniline (PANI) nanofiber exhibited antifouling capability in electrochemical biosensing [24]. Self-assembled monolayers (SAMs) has also been employed to reduce the protein adsorption on electrodes. Cui et al. [25] reported a low-fouling alpha-fetoprotein (AFP) aptasensor using the mixed self-assembled aptamers and a novel short-chained zwitterionic peptides, which acted as the recognizing layer and the antifouling layer, respectively. However, these coatings onto electrodes for anti-fouling deteriorated the electron transfer efficiency due to the low electron transfer capability of organic materials [26]. Our group has been focusing on the design of novel and advanced electrochemical biosensors for analysis of H2O2. We took the advantage of the super electronic conductivity of carbon nanotubes (CNTs) and fabricated the composite nanofibers of CNTs with hydrophilic biomolecules via electrospinning technique. Our work [27] proved an excellent direct electron transfer between the Hemoglobin (Hb) from Hb/collagen-CNTs nanofibers and electrode. The coating of the electrospun Hb/collagen-CNTs nanofibers on the electrode surface possessed large specific surface area owing to the formed 3D inner-connected porous structure, which benefited the electron transfer process. Hb plays important role in the biosensor as the electrocatalyst to H2O2 [28]. More remarkably, Hb has good hydrophilicity, which can further reduce the adsorption of protein on the modified electrode surface [29]. On the other hand, gelatin was preferred to increase the dispersion of multiwalled carbon nanotubes (MWCNTs). The gelatin can be functionalized onto the MWCNTs via hydrophobic-hydrophobic interaction with hydrophobic alkyl chain and the hydrophilic groups of gelatin can increase the dispersibility of the MWCNTs in the electrospinning solution. Meanwhile, the great biocompatibility of gelatin would provide a favorable microenvironment for Hb to preserve the bioactivity of Hb [30]. Therefore, we in this work adopted Hb, gelatin and MWCNTs as the coating materials to modify the electrode by electrospinning technology, and then systematically investigated the protein adsorption effect on the electrochemical sensing performance of the electrospun Hb/gelatin-MWCNTs microbelts modified electrode (denoted as Hb/gelatin-MWCNTs/GC electrode).

Section snippets

Chemicals

Bovine hemoglobin (Hb, Mw = 68,000), bovine serum albumin (BSA), gelatin (Type A from bovine skin, gel strength ~300 g Bloom) and 2,2,2-trifluoroethanol (TFE) were obtained from Sigma. The hydrogen peroxide (H2O2, 30%) was obtained from Tianjin Bodi Chemical Co. Ltd. The diameter (10–20 nm) and the length (1–50 μm) of multiwall carbon nanotubes (MWCNTs) were obtained from Shenzhen Nanotech Port Co. Ltd. The MWCNTs were treated with 4 mol L−1 nitric acid for 3 h in ultrasonic condition prior to

Characterizations on the electrospun Hb/gelatin-MWCNTs microbelts

The micrographs in Fig. 2 display the surface morphology of the electrospun Hb/gelatin-MWCNTs microbelts after immersed in PBS with BSA for different time. Before immersion, it can be seen from Fig. 2a that the electrospun Hb/gelatin-MWCNTs microbelts were uniform, smooth, and bead-free. In addition, a few of the microbelts with flat fracture were found. A possible reason was that the MWCNTs improved the conductive capability and increased the electric field force of the electrospun solution,

Conclusions

The biosensor was constructed using the electrospun Hb/gelatin-MWCNTs/GC electrode aiming for H2O2 detection. The direct electron transfer between the Hb and the modified electrode was still achieved after protein adsorption. The electrochemical sensing performance of the constructed biosensor, including sensitivity, selectivity and stability, was well maintained with the presence of the BSA. Much low detection limits to H2O2, 0.0467 μmol L−1 and 0.0471 μmol L−1 for the biosensor without and

Authorship contribution statement

Z.X. Deng: prepared the manuscript and designed the experiments, carried out the experiments of electrospun membranes, carried out the experiments of electrospun membranes modified electrode. J.W. Tao: carried out the acidification of multiwalled carbon nanotubes, carried out the experiments of electrospun membranes. W. Zhang: were responsible for adjustment of experimental parameters of electrostatic spinning, carried out the experiments of electrospun membranes modified electrode. H.J. Mu:

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.

Acknowledgement

This work was supported by the National Natural Science Foundation of China (No. 21275037 and 81300864).

References (42)

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