Rapid prototyping of electrochromatography chips for improved two-photon excited fluorescence detection

In the present study, we introduce two-photon excitation at 532 nm for label-free fluorescence detection in chip electrochromatography. Two-photon excitation at 532 nm offers a promising alternative to one-photon excitation at 266 nm, as it enables the use of economic chip materials instead of fused silica. In order to demonstrate these benefits, one-photon and two-photon induced fluorescence detection are compared in different chip layouts and materials with respect to the achievable sensitivity in the detection of polycyclic aromatic hydrocarbons (PAHs). Customized chromatography chips with cover or bottom slides of different material and thickness are produced by means of a rapid prototyping method based on liquid-phase lithography. The design of thin bottom chips (180 μm) enables the use of high-performance immersion objectives with low working distances, which allows one to exploit the full potential of two-photon excitation for a sensitive detection. The developed method is applied for label-free analysis of PAHs separated on a polymer monolith inside polymer glass sandwich chips made from fused silica or soda-lime glass. The obtained limits of detection range from 40 nM to 1.95 μM, with similar sensitivities in fused silica thin bottom chips for one-photon and two-photon excitation. In deep-UV non- or less-transparent devices two-photon excitation is mandatory for label-free detection of aromatics with high sensitivity.