Spherical Dichroic Reflector Improves Limit of Detection in Laser-Induced Fluorescence Detection

Abstract

A miniature laser-induced fluorescence (mLIF) detector utilizing a novel spherical dichroic reflector (SDR), an unconventional long working distance high magnification objective, an uncommon broadband emission-matched excitation filter pair, and a silicon-based photodiode detector assembly instead of a photomultiplier tube was developed and evaluated. The detection cell was placed at the spherical center of the SDR instead of the regular focus, yielding a 1.8× signal-to-noise ratio (SNR) improvement. Different from previous works, the use of a 40× objective with a long working distance of 5.38 mm and a broadband BP 527–70 nm emission filter with matched BP 450–30 nm excitation filter improved SNR to 4.6× and 1.9×, respectively. By flow injection analysis (FIA) evaluation, the limit of detection (LOD; 3σ method) for fluorescein sodium was 1.5 × 10–13 M or 8.9 fluorescein molecules in 98 pL detection volume, which was the lowest level of LIFs evaluated by FIA mode. The analysis of three kinds of amino acids with LODs at sub pM to fM level (the lowest levels, hundreds of times lower than previous works using normal capillary) demonstrated the potential of the mLIF in ultratrace analysis of biological and environmental samples, including low copy molecules in a single cell.