Vanjur Luka, Carzaniga Thomas, Casiraghi Luca, Zanchetta Giuliano, Damin Francesco, Sola Laura, Chiari Marcella, Buscaglia Marco
Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, 20054 Segrate, Italy.
Istituto di Scienze e Tecnologie Chimiche, Consiglio Nazionale delle Ricerche (CNR-SCITEC), 20131 Milano, Italy.
Polymers (Basel). 2021 Nov 11;13(22):3897. doi: 10.3390/polym13223897.
The physical-chemical properties of the surface of DNA microarrays and biosensors play a fundamental role in their performance, affecting the signal's amplitude and the strength and kinetics of binding. We studied how the interaction parameters vary for hybridization of complementary 23- DNA, when the probe strands are immobilized on different copolymers, which coat the surface of an optical, label-free biosensor. Copolymers of , -dimethylacrylamide bringing either a different type or density of sites for covalent immobilization of DNA probes, or different backbone charges, were used to functionalize the surface of a Reflective Phantom Interface multispot biosensor made of a glass prism with a silicon dioxide antireflective layer. By analyzing the kinetic hybridization curves at different probe surface densities and target concentrations in solution, we found that all the tested coatings displayed a common association kinetics of about 9 × 10 M·s at small probe density, decreasing by one order of magnitude close to the surface saturation of probes. In contrast, both the yield of hybridization and the dissociation kinetics, and hence the equilibrium constant, depend on the type of copolymer coating. Nearly doubled signal amplitudes, although equilibrium dissociation constant was as large as 4 nM, were obtained by immobilizing the probe via click chemistry, whereas amine-based immobilization combined with passivation with diamine carrying positive charges granted much slower dissociation kinetics, yielding an equilibrium dissociation constant as low as 0.5 nM. These results offer quantitative criteria for an optimal selection of surface copolymer coatings, depending on the application.
DNA微阵列和生物传感器表面的物理化学性质对其性能起着至关重要的作用，影响着信号的幅度以及结合的强度和动力学。我们研究了将探针链固定在不同共聚物上时，互补23聚体DNA杂交的相互作用参数如何变化，这些共聚物覆盖了一种无标记光学生物传感器的表面。使用具有不同类型或密度的用于DNA探针共价固定的位点或不同主链电荷的N，N-二甲基丙烯酰胺共聚物，对由带有二氧化硅抗反射层的玻璃棱镜制成的反射式幻影界面多点生物传感器的表面进行功能化。通过分析不同探针表面密度和溶液中靶标浓度下的动力学杂交曲线，我们发现所有测试涂层在小探针密度下均显示出约9×10⁻⁶M⁻¹·s⁻¹的共同缔合动力学，在接近探针表面饱和时下降一个数量级。相比之下，杂交产率、解离动力学以及平衡常数均取决于共聚物涂层的类型。通过点击化学固定探针，尽管平衡解离常数高达4 nM，但信号幅度几乎翻倍，而基于胺的固定化结合携带正电荷的二胺钝化则产生了慢得多的解离动力学，平衡解离常数低至0.5 nM。这些结果为根据应用情况优化选择表面共聚物涂层提供了定量标准。
