pH monitoring using dual emission dyes

ph-ratio-measurement in continuous microflow systems

SNARF-4F (10 µM, 5 µM) pH ratio measurements in glass capillaries (inner diameter 400 µm) using different buffers

Dual emission dyes (e.g. SNARF®) fluoresce at two sets of wavelengths in response to a single excitation wavelength. These two wavelengths respond differently to pH – one usually increases on alkalinization whilst the other either remains constant or decreases.


For on-chip monitoring of pH we used the pH-dependent fluorescent dye, carboxy-SNARF-4F (Molecular Probes, Invitrogen) for visualization of the pH change. This dye can be excited at 488 nm and shows an emission spectrum with two maxima at around 580 nm and 640 nm. These two wavelengths respond differently to pH – one usually increases on alkalinization whilst the other either remains constant or decreases. The quotient of these two wavelengths can be used as an indicator for a special pH value.


In our ph-ratio measurement experiments we used the wavelength of excitation at 488 and 561 nm and we measured the emission maxima with different optical filters. 

The corresponding measured values were divided after base line correction and shown in the upper left curve. The experiments shows a clear pH dependency in the range from pH 6 to 9 for both excitation wavelengths. Furthermore, the measured fluorescence intensity curves of the 5 µM solutions are nearly identical to the 10 µM buffer solutions, so the concentration of the sample has no effect on the measurement, which is important requirement for pH monitoring in microfluidics.

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