Infrared attenuated total reflection spectroscopy for monitoring biological systems
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Mid-infrared (MIR) spectroscopy has been recognized as an important analytical technique, and is widely applied for qualitative and quantitative analysis of materials with an increasing interest in addressing complex organic or biologic constituents. In the presented thesis, (a) the fundamental principles for IR spectroscopic applications via in vivo catheters in combination with multivariate data analysis technique were developed, and (b) the combination with a second analytical technique ¨C scanning electrochemical microscopy (SECM) - for enhancing the information obtained at complex or frequently changing matrices was demonstrated. The first part of this thesis focused on the combination of different MIR measurment techniques with specific focus on evanescent field absorption spectroscopy along with multivariate data analysis methods, for the discrimination of atherosclerotic and normal rabbit aorta tissues. Atherosclerotic and normal rabbit aorta tissues are characterized by marked differences in chemical composition governed by their water, lipid, and protein content. Strongly overlapping infrared absorption features of the different constituents and the complexity of the tissue matrix render the direct evaluation of molecular spectroscopic characteristics obtained from IR measurements challenging for classification. We have successfully applied multivariate data analysis and classification techniques based on principal component analysis (PCA), partial least squares regression (PLS), and linear discriminant analysis (LDA) to IR spectroscopic data obtained by infrared attenuated total reflectance (IR-ATR) measurements, reflection IR microscopy, and a recently developed IR-ATR catheter prototype for future in vivo diagnostic applications. Training and test data were collected ex vivo at atherosclerotic and normal rabbit aorta samples. The successful classification results at atherosclerotic and normal aorta samples utilizing the developed data evaluation routines reveals the potential of IR spectroscopy combined with multivariate classification strategies for in vitro, and ¨C in future - in vivo applications. The second part of this thesis aimed at the development of a novel multifunctional analytical platform by combining SECM with single-bounce IR-ATR spectroscopy for in situ studies of electrochemically active or electrochemically induced processes at the IR waveguide surface via simultaneous evanescent field absorption spectroscopy. The utility of the developed SECM-IR-ATR platform was demonstrated by spectroscopically monitoring microstructured polymer depositions induced via feedback mode SECM experiments using a 25μm Pt disk ultramicroelectrode (UME). The surface of a ZnSe ATR crystal was coated with a thin layer of 2,5-di-(2-thienyl)-pyrrole (SNS), which was then polymerized in a Ru(bpy) ₃ ² ⁺-mediated feedback mode SECM experiment. The polymerization reaction was simultaneously spectroscopically monitored by recording the absorption intensity changes of specific IR bands characteristic for SNS, thereby providing information on the polymerization progress, mechanism, and level of surface modification. Furthermore, a novel current-independent approach mechanism for positioning the UME in aqueous electrolyte solution was demonstrated by monitoring IR absorption changes of borosilicate glass (BSG) shielding the UME, and of water within the penetration depth of the evanescent field.