Non-covalent and covalent binding of proteins on ultrananocrystalline diamond films with different surface terminations

IF 4.3 3区材料科学 Q2 MATERIALS SCIENCE, COATINGS & FILMS

Diamond and Related Materials Pub Date : 2025-05-09 DOI:10.1016/j.diamond.2025.112432

Rezvaneh Ghasemitabesh , Daniel Merker , Jan W. Bröckel , Daniela Bertinetti , Yahya Zakaria , Alexander Welle , Friedrich W. Herberg , Cyril Popov

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引用次数: 0

Abstract

The immobilization of proteins on solid surfaces is a critical aspect of biosensor development, offering enhanced sensitivity and specificity for molecular recognition. Ultrananocrystalline diamond (UNCD) films have emerged as a promising platform for protein immobilization due to their exceptional biocompatibility, chemical stability, and tunable surface properties. In this study, we investigate the non-covalent and covalent binding of green fluorescent protein (GFP) on nanostructured UNCD surfaces employing fluorescence spectroscopy to assess the efficiency of protein immobilization. The non-covalent binding was affected by the surface terminations (hydrogen, oxygen, and fluorine) rendered by different plasma modifications. The oxygen-terminated UNCD surfaces exhibited the highest efficiency attributed to favorable wettability and electrostatic interactions, revealed by zeta potential and contact angle measurements. The covalent immobilization via linker chemistry was studied for both GFP and anti-GFP nanobodies with the variation of the GFP concentration within the range of 10 μM – 10 pM. Milk powder was applied as a blocker to minimize the non-covalent binding of the target proteins. The intensity of the fluorescence signal decreased with the GFP concentration and below 1–10 nM approached that of the buffer control samples. The individual steps of the protein immobilization were investigated by zeta potential measurements, X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Finally, a fluorescent dye-labeled nanobody was constructed as a reporter complex and applied to provide fluorescence signals distinguishable from those of GFP due to the different wavelengths. Such complexes combine the high specificity of the binding proteins with the bright and stable signal of the fluorescent dyes and can be implemented for realization of biosensors.

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蛋白质在不同表面末端的超晶金刚石膜上的非共价和共价结合

将蛋白质固定在固体表面是生物传感器发展的一个关键方面，为分子识别提供了更高的灵敏度和特异性。超微晶金刚石（UNCD）薄膜由于其优异的生物相容性、化学稳定性和可调节的表面特性而成为一种很有前途的蛋白质固定化平台。在这项研究中，我们利用荧光光谱研究了绿色荧光蛋白（GFP）在纳米结构UNCD表面的非共价和共价结合，以评估蛋白质固定的效率。非共价结合受到不同等离子体修饰产生的表面末端（氢、氧和氟）的影响。zeta电位和接触角测量结果显示，氧端接的UNCD表面由于良好的润湿性和静电相互作用而表现出最高的效率。研究了GFP和抗GFP纳米体在10 μM ~ 10 pM范围内的共价固定。奶粉被用作阻滞剂以减少靶蛋白的非共价结合。荧光信号强度随GFP浓度的增加而降低，在1-10 nM以下与缓冲对照样品接近。通过zeta电位测量、x射线光电子能谱（XPS）和飞行时间二次离子质谱（ToF-SIMS）对蛋白固定的各个步骤进行了研究。最后，构建了一个荧光染料标记的纳米体作为报告复合物，并应用于提供与GFP不同波长的荧光信号。这种复合物结合了结合蛋白的高特异性和荧光染料明亮稳定的信号，可以实现生物传感器的实现。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Diamond and Related Materials 工程技术-材料科学：综合

CiteScore

6.00

自引率

14.60%

发文量

702

审稿时长

2.1 months

期刊介绍： DRM is a leading international journal that publishes new fundamental and applied research on all forms of diamond, the integration of diamond with other advanced materials and development of technologies exploiting diamond. The synthesis, characterization and processing of single crystal diamond, polycrystalline films, nanodiamond powders and heterostructures with other advanced materials are encouraged topics for technical and review articles. In addition to diamond, the journal publishes manuscripts on the synthesis, characterization and application of other related materials including diamond-like carbons, carbon nanotubes, graphene, and boron and carbon nitrides. Articles are sought on the chemical functionalization of diamond and related materials as well as their use in electrochemistry, energy storage and conversion, chemical and biological sensing, imaging, thermal management, photonic and quantum applications, electron emission and electronic devices. The International Conference on Diamond and Carbon Materials has evolved into the largest and most well attended forum in the field of diamond, providing a forum to showcase the latest results in the science and technology of diamond and other carbon materials such as carbon nanotubes, graphene, and diamond-like carbon. Run annually in association with Diamond and Related Materials the conference provides junior and established researchers the opportunity to exchange the latest results ranging from fundamental physical and chemical concepts to applied research focusing on the next generation carbon-based devices.