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Biocompatible, Fluorescence Enhancing Solvents for Sensitive Fluorophore Mediated Biosensor and Observation of Protein Conformation Change by Atomic Force Microscopy

Kyung K. Kang and Bin Hong. Chemical Engineering, University of Louisville, Ernst Hall 106, Speed School of Engineering, University of Louisville, Louisville, KY 40292

Rapid and accurate quantification of physiological markers in blood plasma is important for disease detection, diagnosis, prognosis, and treatment. Successful sensing tools must provide high specificity and sensitivity. Fluorophore mediated, fiber-optic immuno-biosensor has been recognized as a good tool for detecting various disease representative biomarkers in the physiological samples. A good example is our multi-cardiac marker biosensing system used to quantify four markers in plasma simultaneously for rapid heart attack diagnosis and prognosis. However, two cardiac markers are at only a tens of pico-molar level in plasma, challenging the sensing system. The application of the biocompatible organic solvents for fluorescence enhancement in biosensing realizes an accurate, simultaneous quantification of four markers within 10 minutes at an average signal-to-noise ratio of 20 while not affecting the reusability of the sensors. The fluorescence enhancement mechanism was demonstrated partially from the shifted excitation/emission spectrums of the fluorophore and the isomerization of the trans-cis carbon double-bond in solvent. In addition, since the fluorophore mediated sandwich immunoassay is employed in our biosensors, the conformation change of the protein complex in solvent on the sensor surface could also affect the fluorescent signals. If the complex shrinks, the surface bound fluorophores will get closer to the sensor surface and the sensor will retrieves more fluorescence per bound target biomarker. C-reactive protein, an important prognostic cardiac marker, and its monoclonal antibody were selected for the conformation study due to their relatively large molecular size for easy observation. The proteins were, therefore, immobilized on the gold substrate and imaged by the Atomic force microscopy (AFM) in tapping mode for their three dimensional scales in the solvent as well as the physiological environment. To obtain the actual surface size of the proteins, incompressible nanogold colloid was used as the standard. The average scale of the proteins in the solvent was found to shrink by more than 25%.

Authors acknowledge the partially financial support from National Science Foundation (BES-0330075) on biosensing study. The Institute for Molecular Diversity and Drug Design (IMD3) at the University of Louisville is also acknowledged for Bin Hong's Graduate Fellowship in the study of AFM imaging.