Orthogonal Functionalization of Porous Interfaces in Paper Sheets Through Immobilization of Thermally Stable Peptides

在木质纤维表面固定传感分子，例如酶或抗体，是纸基材料在生物分析诊断中应用的关键要求。尽管主要由纤维素聚合物组成的纸纤维提供了大量的羟基，但上述(生物)功能分子的化学分化和靶向固定化是有限的。因此，对纸纤维或纸进行化学前处理是必要的。目前，在控制纤维修饰的可能性、生物分子在纤维上的负载密度、它们(剩余的)活性以及在通常复杂的生物分析反应中进一步使用这类纸张方面存在许多挑战。特别是，到目前为止，通过常见的化学偶联方法将生物分子定向、正交地固定在高负载密度的纸纤维表面上并不是一件容易的事情。在本项目中，将开发一种将生物活性蛋白质定向偶联到纸纤维上的通用方法，该方法基于以多肽为特定锚定分子的纸纤维的特殊预官能化。与大多数蛋白质不同的是，这种固定在纸纤维上的多肽在热稳定性上非常稳定，因此可以承受高温，通常是在造纸过程中的最终干燥部分。在片层形成后，将在温和的条件下通过酶催化的生物偶联进行生物大分子的定点固定化。这个项目的另一个重要的进一步目标是了解纤维类型和形态以及纤维的改性对所产生的纸张的几何和化学结构的影响，如纸张的孔隙度和纸张密度、疏水性、吸附性能等。该项目将生化/生物分析基础科学与造纸工程和化学的基础科学问题结合起来，从长远发展的角度出发，总体目标是系统地分析可调节的纸张性质如何影响酶和抗体的活性。特别是，将研究哪些用于用肽对纸纤维进行表面修饰的化学偶联方法，以及关于负载密度和生物活性的哪些蛋白质的生物催化偶联策略特别适合于生物分析应用。这项工作的结果将为未来的科学工作提供基础，以便详细了解具有定制生物催化特性的微流控论文。

The immobilization of sensing molecules, e.g. enzymes or antibodies, on the surface of lignocellulosic fibers is a crucial requirement for any application of paper-substrates in bioanalytical diagnostics. Although paper fibers – consisting mainly of cellulose polymers – provide a large number of OH groups, chemical differentiation and targeted immobilization of the aforementioned (bio)functional molecules is limited. Therefore, a chemical pretreatment of paper fibers or papers is necessary. Currently there exist a number of challenges with regard to the possibility of controlling the fiber-modification, loading density of biomolecules on the fiber, their (remaining) activity and the further use of such papers in often complex bioanalytical reactions. In particular, up to date it is not trivial by common chemical conjugation methods to immobilize biomolecules in a directed, orthogonal fashion on the surface of paper fibers with a high loading density. In this project, a general method for such directed conjugation strategy of bioactive proteins to paper fibers will be developed, based on a special pre-functionalization of paper fibers with peptides as specific anchor-molecules. In contrast to most proteins, such peptides immobilized on paper fibers are thermally very stable, and can therefore endure high temperatures, typically present during papermaking at the final drying section. After sheet formation, a site-specific immobilization of biomacromolecules will be carried-out by enzyme-catalyzed bioconjugation under mild conditions. Such a “pre-functionalization” of paper fibers have yet not been addressed in literature.Another important further goal of this project is to understand the influence of the fiber type and morphology as well as the fiber modification on the resulting geometric and chemical structure of the paper sheet, such as the paper's porosity and paper density, hydrophobicity, sorption properties, etc.. The project combines biochemical / bioanalytical fundamental science with fundamental scientific questions along paper engineering and –chemistry.With respect to a long-term development of the project, the overall aim is to systematically analyze how adjustable paper properties affect the activity of enzymes and antibodies. In particular, it will be examined which chemical conjugation methods for surface modification of paper fibers with peptides and which biocatalytic conjugation strategies of the proteins with regard to loading density and biological activity are particularly suitable for bioanalytical applications. The results of this work will provide the basis for future scientific work towards a detailed understanding of microfluidic papers with tailored biocatalytic properties.