Spectroscopic measurements of noncovalently labeled human serum albumin with near -infrared dyes.

摘要

In this dissertation, spectroscopic measurements of near-infrared (NIR) dyes were evaluated. Chapter 1 will introduce the basic principles of spectroscopy, including absorption and fluorescence. This section will also provide background information about human serum albumin (HSA) and fluorescent dyes. Near-infrared fluorescence and dye theory will also be explored.;Chapter 2 will explore dye binding to biomolecules in the NIR region (650--1100 nm). Covalent and noncovalent interactions of dyes with biomolecules will be discussed, along with the reason for studying the noncovalent binding of heptamethine cyanine dyes with HSA. In this research, HSA was chosen as the biomolecules of interest because it has been extensively characterized. This globular protein binds to a wide variety of ligands, such as proteins, drugs, and dyes. In addition, the current implementation of NIR dyes for bioanalytical research, clinical studies, and medical diagnosis procedures will be discussed in detail.;The spectroscopic measurements of various heptamethine cyanine dyes will be described in Chapter 3. All dyes used in this chapter exhibit absorption and fluorescence emission in the 770 nm to 820 nm region. Extensive studies were conducted on the spectroscopic properties, i.e. absorbance, fluorescence emission, molar absorptivities, and relative fluorescence quantum yields, of the dyes. Aggregation of the NIR dyes was observed and evaluated in terms of their overall structures, functional groups, and side chains. The binding constants and number of binding sites for HSA with several heptamethine cyanine dyes were calculated.;Chapter 4 contains detailed theory and applications of chiroptical techniques in the NIR region. The chiroptical techniques of circular dichroism (CD) and fluorescence-detected circular dichroism (FDCD) were implemented for the measurement of induced chirality of the optically inactive dyes in the NIR wavelength range when complexed with HSA. In this study, the polymethine cyanine dyes consisted of squaric acid derivatives or a heptamethine backbone. In addition, the CD instrument was modified to measure the total fluorescence in the NIR region. Data obtained was compared to measurements conducted with absorption and fluorescence excitation.