Development of a Microfluidic Reverse Flow Injection Analysis (rFIA) Method with Automatic Background Fluorescence Correction for the Detection of Nanomolar Concentrations of Ammonium in Natural Waters

Detection of low concentrations of ammonium in the oligotrophic surface ocean is challenging but essential for understanding primary productivity in the marine ecosystem. Ammonium is the most energetically favorable form of inorganic nitrogen that phytoplankton can use, and its availability often limits phytoplankton biomass. Recently, the majority of methods for quantification of ammonium at nanomolar concentrations have used the reaction between ammonia and o-phthaldialdehyde (OPA) either following gas diffusion of ammonia across a membrane or through direct reaction using sulfite as a reducing agent to form a fluorescent product. One benefit of the separation technique is that natural fluorescence of the samples does not yield a false positive signal, however membrane failure and clogging are challenging analytical problems. The direct reaction between the analyte and OPA, although devoid of problems associated with membranes, requires a correction for background fluorescence. This work presents the development of a micro-fluidic based reverse flow injection analysis method with automatic background fluorescence correction with a detection limit approximately 1000 times more sensitive. The method employs a sulfite-formaldehyde reagent mixed with the sample into which an o-phthaldialdehyde (OPA) reagent is injected and heated to activate fluorescence. Fluorescence of the sample is measured before the injection of OPA and at the peak of OPA injection, allowing for differentiation of the background fluorescence from the signal proportional to analyte. The detection limit and the limit of quantitation for this technique are 2.2 nM and 7.5 nM, respectively, and the coefficient of variation is 0.554 %. The benefits of this rFIA technique include low detection limits, automatic background correction for natural fluorescence, and ease of incorporation into in situ analyzers.