Gutmann Oliver, Kuehlewein Ruben, Reinbold Stefanie, Niekrawietz Remigius, Steinert Chris P, de Heij Bas, Zengerle Roland, Daub Martina
IMTEK-University of Freiburg, Laboratory for MEMS Applications, Georges-Koehler-Allee 103, D-79110 Freiburg, Germany.
Lab Chip. 2005 Jun;5(6):675-81. doi: 10.1039/b418765b. Epub 2005 Apr 27.
In contrast to DNA microarrays, production of protein microarrays is an immense technological challenge due to high complexity and diversity of proteins. In this paper we investigate three essential aspects of protein microarray fabrication based on the highly parallel and non-contact TopSpot technology: evaporation of probes during long lasting production times, optimization of protein immobilization and improvement of protein microarray reproducibility. Evaporation out of the printhead reservoirs was reduced to a minimum by sealing the reservoirs with gas permeable foils or PDMS frames. This led to dramatically lowered setup times through the possibility of long-term, ready-to-print storage of filled printheads. To optimize immobilization efficiency 128 printing buffers were tested by printing two different proteins onto seven different microarray slide types. This way we were able to reduce the CV of spot diameter on the microarray slide below 1.14%. To remarkably increase protein immobilization efficiency on microarray slides the commonly used EDC-NHS system (a laboratory method for immobilization of proteins) was miniaturized by using a new drop-in-drop printing technique. Additionally the very fast UV cross-linking was used to immobilize antibodies. The optimized system was used to produce antibody microarrays and with it microarray ELISA experiments were performed successfully.
与DNA微阵列不同，由于蛋白质的高度复杂性和多样性，蛋白质微阵列的生产是一项巨大的技术挑战。在本文中，我们基于高度并行且非接触式的TopSpot技术，研究了蛋白质微阵列制造的三个重要方面：长时间生产过程中探针的蒸发、蛋白质固定化的优化以及蛋白质微阵列重现性的提高。通过用透气箔片或聚二甲基硅氧烷（PDMS）框架密封喷头储液器，将喷头储液器中的蒸发减少到最低限度。这使得通过对填充喷头进行长期、随时可打印的存储，显著缩短了设置时间。为了优化固定化效率，通过将两种不同的蛋白质打印到七种不同类型的微阵列载玻片上，测试了128种打印缓冲液。通过这种方式，我们能够将微阵列载玻片上斑点直径的变异系数（CV）降低到1.14%以下。为了显著提高蛋白质在微阵列载玻片上的固定化效率，通过使用一种新的逐滴打印技术，将常用的EDC-NHS系统（一种蛋白质固定化的实验室方法）进行了小型化。此外，还使用了非常快速的紫外线交联来固定抗体。优化后的系统用于生产抗体微阵列，并成功地进行了微阵列酶联免疫吸附测定（ELISA）实验。
