Chiodi Elisa, Damin Francesco, Sola Laura, Ferraro Lucia, Brambilla Dario, Ünlü M Selim, Chiari Marcella
Photonics Center, Department of Electrical Engineering, Boston University, Boston, MA 02215, USA.
Istituto di Scienze e Tecnologie Chimiche "G. Natta" SCITEC-Consiglio Nazionale delle Ricerche, 20131 Milano, Italy.
Polymers (Basel). 2021 Jan 21;13(3):340. doi: 10.3390/polym13030340.
The manufacture of a very high-quality microarray support is essential for the adoption of this assay format in clinical routine. In fact, poorly surface-bound probes can affect the diagnostic sensitivity or, in worst cases, lead to false negative results. Here we report on a reliable and easy quality control method for the evaluation of spotted probe properties in a microarray test, based on the Interferometric Reflectance Imaging Sensor (IRIS) system, a high-resolution label free technique able to evaluate the variation of the mass bound to a surface. In particular, we demonstrated that the IRIS analysis of microarray chips immediately after probe immobilization can detect the absence of probes, which recognizably causes a lack of signal when performing a test, with clinical relevance, using fluorescence detection. Moreover, the use of the IRIS technique allowed also to determine the optimal concentration of the probe, that has to be immobilized on the surface, to maximize the target recognition, thus the signal, but to avoid crowding effects. Finally, through this preliminary quality inspection it is possible to highlight differences in the immobilization chemistries. In particular, we have compared NHS ester versus click chemistry reactions using two different surface coatings, demonstrating that, in the diagnostic case used as an example (colorectal cancer) a higher probe density does not reflect a higher binding signal, probably because of a crowding effect.
制造非常高质量的微阵列载体对于在临床常规中采用这种检测形式至关重要。事实上，表面结合不佳的探针会影响诊断灵敏度，在最坏的情况下，会导致假阴性结果。在此，我们报告一种基于干涉反射成像传感器（IRIS）系统的可靠且简便的质量控制方法，用于评估微阵列检测中斑点探针的特性。IRIS系统是一种高分辨率的无标记技术，能够评估结合到表面的物质的质量变化。特别是，我们证明了在探针固定后立即对微阵列芯片进行IRIS分析，可以检测到探针的缺失，在使用荧光检测进行具有临床相关性的测试时，这明显会导致信号缺失。此外，使用IRIS技术还可以确定必须固定在表面的探针的最佳浓度，以最大化目标识别，从而最大化信号，但要避免拥挤效应。最后，通过这种初步质量检查，可以突出固定化学方法的差异。特别是，我们使用两种不同的表面涂层比较了NHS酯与点击化学反应，证明在用作示例的诊断案例（结直肠癌）中，较高的探针密度并不反映较高的结合信号，这可能是由于拥挤效应。
