Developing Peptide-based Fluorescent Probes and New Microarray Immobilization Strategy for Biological Applications

基於多肽的熒光探針設計以及新型微陣列芯片固定化在生物學方面的開發及應用

Student thesis: Doctoral Thesis

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Award date20 Aug 2019

Abstract

The first part of the chapter mainly talks about the peptide and its application. Structures and functions of peptides are also summarized in this chapter. Besides, the potential applications of the peptides are discussed. The peptide applications could be as various as peptide themselves. They can be utilized as biomaterials, drug delivery or drug components. Besides, the chapter also gives a brief introduction of microarray technology, post-translational modifications of histones and fluorescence-based cell membrane trackers. For the microarray technology, as known to all, it has been widely utilized in the biological research and includes DNA microarray, small molecule microarray, peptide microarray and protein microarray. It has some unsurpassable advantages than the other methods. For example, it can contain thousands of samples on one chip and only use little samples in nL level. Also, it can be utilized to select the most active inhibitors or potential drugs. Hence, a perfect immobilization for the microarray chip which should be economical, effective and easy made is quite important. For the post-translational modifications of the histones, there are more than 200 various protein modifications being identified. Each modification plays a significant role in the cellular process such as the cell division, DNA reproduction and even some process related to diseases and cancers. Considering this, many biologists devote themselves to find inhibitors or effective probes which can specifically recognize one modified protein and then track their related functions. As peptides are naturally synthesized and can be degraded in cells without toxic products, we aimed to design effective peptide-based probes for biological applications in this dissertation.

In chapter 1, a brief background of the project has been provided. The peptide application is shown in this chapter. Besides, the microarray technology has been introduced as well as its advantages. Combination of the microarray technology, post-translational modifications of proteins and peptides has been the most important aim of the projects.

In chapter 2, a proximity-induced covalent fluorescent probe for selective detection of bromodomain 4 was designed. As we know, among the different post translational modifications, acetylation is the most common modifications. Besides, bromodomains comprise a family of proteins that selectively interact with acetylated lysine. Herein, we designed a peptide-based probe which can be capable of covalently and selectively reacting with the unique cysteine residue in the bromodomain through proximity effect.

In chapter 3, though microarray technology has advantage of reducing sample usage, enabling the rapid, simultaneous screening of thousands of biological samples, it is still limited to be utilized because of the high cost. In this chapter, we designed a new reversible microarray immobilization based on the thiol-quinone reaction. The thiol-containing molecules can be immobilized on the quinone modified slides and form a thioether linker. However, the thioether linkage can be cleaved with H2O2 treatment and regenerate the quinone group on the surface.

In chapter 4, a fluorescent rotor-based cell membrane probe was designed. Cell membrane is the two-dimensional boundary between cell and its environment. It is a fluid semi-permeable membrane and is in different morphology when the cell is in various biological process. Thus, designing a cell membrane probe to track its activity is quite important. Based on this, we designed a fluorescent rotor-based probe to specifically bind to cell membrane and also it can be utilized to track the process of cell apoptosis.

Finally, chapter 5 provides a summary of the thesis. Also, the future development of microarray technology is discussed. What’s more, the coming work and directions based on the microarray technology are also introduced.