Rapid Prototyping of Electrochromatography Chips for Improved Two-Photon Excited Fluorescence Detection

Abstract

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.

Advances in and prospects of microchip liquid chromatography

“…A polymethacrylate monolith photografted into a PDMS device which was then functionalized to act as a weak anion exchange column (5 mm in length) was used to demonstrate the separation of intact proteins and study different separation parameters using this type of monolith [42]. Liquid phase lithography was used to form a poly(ethylene glycol) diacrylate (PEGDA) channel design sandwiched between two glass slides [43]. A butyl acrylate monolithic column was incorporated into the resulting channel.…”

“…A butyl acrylate monolithic column was incorporated into the resulting channel. Two-photon excitation fluorescence detection was used demonstrating a way to achieve UV fluorescence detection for polyaromatic hydrocarbons (PAHs) with substrates that would usually block such wavelengths [43]. An organic-inorganic hybrid monolith integrating alumina nanoparticles was used for the extraction and separation of 2-amino-4-chlorophenol in a PMMA (poly(methyl methacrylate)) chip [44].…”

High-performance liquid chromatography on glass chips using precisely defined porous polymer monoliths as particle retaining elements

“…1B). For economic reasons we investigated this laser scanning approach utilising selfconstructed PEG-DA sandwich chips [43] rather than expensive glass devices. Here, the microfluidic channels are situated in a photopolymerised polyethylene glycol diacrylate layer sandwiched in between two glass slides.…”

Two-photon fluorescence lifetime for label-free microfluidic droplet sorting

“…For example, a typical 266-nm deep-UV source translates to excitation at 532 nm in the visible range. This is perfectly compatible with common chip materials [31][32][33][34] and optical elements, which also leads to less photostress in living cells [35][36][37].…”