Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm 171 77, Sweden.
CNR-IOM, Istituto Officina dei Materiali-Consiglio Nazionale delle Ricerche, Basovizza, 34149 Trieste, Italy.
ACS Appl Bio Mater. 2022 Jul 18;5(7):3310-3319. doi: 10.1021/acsabm.2c00311. Epub 2022 Jun 25.
The deposition of biomolecules on biosensing surface platforms plays a key role in achieving the required sensitivity and selectivity for biomolecular interactions analysis. Controlling the interaction between the surface and biomolecules is increasingly becoming a crucial design tool to modulate the surface properties needed to improve the performance of the assay and the detection outcome. Carboxymethyl-dextran (CMD) coating can be exploited to promote chemical grafting of proteins, providing a hydrophilic, bioinert, nonfouling surface and a high surface density of immobilized proteins. In the present work, we developed and optimized a technique to produce a cost-effective CMD-based patterned surface for the immobilization of biomolecules to be used on standard protocols optimization. They consist of silicon or glass substrates with patterned bioactive areas able to efficiently confine the sampling solution by simply exploiting hydrophilic/hydrophobic patterning of the surface. The fabrication process involves the use of low-cost instruments and techniques, compatible with large scale production. The devices were validated through a chemiluminescence assay we recently developed for the analysis of binding of DNA nanoassemblies modified with an affinity binder to target proteins immobilized on the bioactive areas. Through this assay we were able to characterize the chemical reactivity of two target proteins toward a dextran matrix on patterned surfaces and to compare it with model CMD-based surface plasmon resonance (SPR) surfaces. We found a high reproducibility and selectivity in molecular recognition, consistent with results obtained on SPR sensor surfaces. The suggested approach is straightforward, cheap, and provides the means to assess patterned functionalized surfaces for bioanalytical platforms.
生物分子在生物传感表面平台上的沉积在实现生物分子相互作用分析所需的灵敏度和选择性方面起着关键作用。控制表面与生物分子之间的相互作用正日益成为一种关键的设计工具，用于调节表面特性，以提高分析物的性能和检测结果。羧甲基葡聚糖 (CMD) 涂层可用于促进蛋白质的化学接枝，提供亲水、生物惰性、抗污染的表面和高浓度固定化蛋白质。在本工作中，我们开发并优化了一种技术，以生产具有成本效益的基于 CMD 的图案化表面，用于标准方案优化中的生物分子固定化。这些表面由硅或玻璃基底组成，具有图案化的生物活性区域，通过简单地利用表面的亲水/疏水图案化，可以有效地限制采样溶液。制造过程涉及使用低成本仪器和技术，与大规模生产兼容。通过我们最近开发的用于分析与亲和配体修饰的 DNA 纳米组装体与固定在生物活性区域的靶蛋白的结合的化学发光测定法对器件进行了验证。通过该测定法，我们能够研究两种靶蛋白对图案化表面上葡聚糖基质的化学反应性，并将其与基于模型 CMD 的表面等离子体共振 (SPR) 表面进行比较。我们发现分子识别具有高重现性和选择性，与 SPR 传感器表面上获得的结果一致。该方法简单、廉价，并为生物分析平台的图案化功能化表面提供了评估手段。
