Proteins are of primary importance to the structure and function of all living cells. Study of proteins relies on the ability to separate a complex mixture so that individual proteins can be more easily processed by other techniques. Since protein samples often exist at low concentration in a small volume, the trend in chemical analysis is toward micro total analysis systems (µTAS) or lab-on-a-chip devices. Among µTAS separation methods, the relatively new electric field gradient focusing (EFGF) technique has shown potential. It focuses and separates analytes based on their electrophoretic migration in an opposing hydrodynamic flow. The detection principles that are compatible with µTAS separation may not always scale down. This thesis represents the development of laser-induced two-photon fluorescence detection on a microchip separation device. This detection is based on excitation of native fluorescence of aromatic amino acids by simultaneous absorption of two photons. First, a compact two-photon prototype detector was investigated. Its sensitivity was improved after discovery of the source of the background and subsequent reduction of background levels. Simple CE separation on a square capillary was coupled to this detector to demonstrate its ability for micro-scale detection. However, this detector did not provide a way to view the location illuminated by a laser and was difficult to use for on-microchip detection. A two-photon microscope was constructed on the frame of a commercial Olympus microscope to solve this problem. The eyepiece of this microscope enabled viewing of the detection volume, and the removal of a glass compensator from the trinocular head allowed for UV detection. This detection system was carefully aligned and optimized before coupling to microchip CE. Two microchip substrates including poly (methyl methacrylate) (PMMA) and glass, and two chip layouts were explored for their compatibilities with the microscope detector. It was found that the PMMA chip with conventional chip layout was not suitable for two-photon detection; therefore, a novel chip layout on PMMA was designed. Through testing the new design, it was concluded that precise focusing of the laser was essential to successful detection on microchips. Although the precise focusing of the laser inside microchip channels was not achieved completely in the limited research period, it is believed that this new design should be an appropriate solution to coupling PMMA chips with the two-photon microscope. Finally, glass chips were employed to successfully demonstrate the detection of amino acids.



College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry



Date Submitted


Document Type





two-photon, fluorescence, protein detection