Accelerated micro-sequential injection in lab-on-valve format, applied to enzymatic assays

Abstract

The assay cycle of sequential injection (SI) analysis has been greatly accelerated by simultaneously processing two sample injections within the same manifold. This is achieved through micro-miniaturization of the SI system using the lab-on-valve format (LOV), and by optimizing the assay protocol for stopped-flow reaction rate measurements. The approach has been tested on enzymatic assays of glucose and ethanol, but it is, in principle, applicable to all SI reagent-based assays. The average assay time for a single run has been shortened from 200 s to ≈30 s. Assays were carried out at 22 °C and 37 °C using commercially available reagent kits. For glucose, at 22 °C, the calibration had a linear response (r² = 0.9999) for the concentration range of 100–1000 ppm. At 37 °C, the calibration was linear (r² = 0.9996) for 100–600 ppm glucose, but was a second order polynomial curve (r² = 0.9996) for 100–1000 ppm. For ethanol, at both 22 °C and 37 °C, the calibrations were linear for the concentration range of 50–250 ppm. The r² at two temperatures was 0.9994 and 0.9995, respectively. For both the glucose and ethanol assays, the relative standard deviations were below 3%. Factors affecting sampling frequency are discussed. In this work, sequential injection is shown, for the first time, to achieve sampling frequency comparable to flow injection (FI), while retaining the advantages of small reagent consumption (typically microlitres per assay), minimized waste production, full automation of assay protocols, and zero carryover.