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Class identification of bio-molecules based on multi-color native fluorescence spectroscopy

 

All biological molecules are composed of only a few basic building blocks and, therefore, exhibit similar physical properties. In particular, aromatic amino acids can be found in biological molecules and exhibit native fluorescence. Tryptophan, phenylalanine and tyrosine are some of the most common fluorescent molecules. In addition many enzymes or cofactors, NADH (reduced b-nicotinamide adenine dinucleotide) and Riboflavin being the most prominent examples, exhibit pronounced native fluorescence. Laser induced fluorescence (LIF) is a promising tool for differentiating fluorescing molecules. As the variety of biological molecules is huge compared to the number of basic building blocks, the fluorescence spectra of different analytes are often quite similar. A high spectral resolution is required to reveal differences in the emission spectra. In addition multi-wavelength excitation has to be used to gain more specific information to ease the bio-agent class identification. Native fluorescence from various proteins, bacteria and fungi was excited by a 266 nm and a 355 nm laser source. Emission spectra were collected between 280 and 560 nm with a regular fluorescence setup from different analytes (bovine serum albumin, horse heart cytochrome C, bacillus thuringiensis, yeast, NADH, tryptophan). To extract the distinguishing features for each analyte the detailed spectral information was evaluated by principal component analysis (PCA). PCA a lead to a clear separation among the simulants, and thus class identification was successfully conducted. Future issues to be addressed include randomized testing on unknown analytes in order to explore the identification probability, recording fluorescence spectra for analyte mixtures, evaluating the class identification probability for mixtures of analytes, and examining class identification for simulants in different solutions (e.g., di-ionized and tap water).

 
citation

Bassler, M. ; Kiesel, P. ; Schmidt, O. ; Johnson, N. M. Class identification of bio-molecules based on multi-color native fluorescence spectroscopy. Optics East 2006, Session SA201; 2006 October 1-4; Boston; MA; USA.