www.els-journal.com Page 3 Engineering in Life SciencesThis article is protected by copyright. All rights reserved. AbstractA lifetime-based ratiometric microscale pH sensor system and a fluorescence microscopic setup was developed for the in-line observation of pH in free-flow isoelectric focusing based on the principle of time-domain dual lifetime referencing (t-DLR). The t-DLR method has been developed for pH monitoring and various other applications in the fields of environmental monitoring and biotechnology. Here, we introduce the integration of pH sensor microstructures for t-DLR in microfluidic channels and the application of the t-DLR scheme for pH sensing in miniaturized electrophoretic procedures. The pH sensor was inkjet-printed on glass in rows with a length of 10 mm, a height of 404 ± 18 nm and a width of 371 ± 28 µm, and integrated into a microfluidic chip generated by a lasercutting and lamination technique. It had a working range from pH 4 to 8 with a pK A of 6.10 ± 0.01 and fast response times under 500 ms. The sensor was used for the in-line observation of the pH gradient during isoelectric focussing of the proteins β-lactoglobulin A, conalbumin and myoglobin and their identification by their isoelectric point (pI). The obtained pIs were in good agreement with literature data demonstrating the applicability of the pH sensor in microfluidic continuous separations.