The monitoring of pH in microplates can be used for a variety of applications amongst others:

• Determination of enzyme activity (e.g. hydrolases)
• Monitoring of Cell Proliferation
• Starter Cultures

HydroPlate^® can monitor enzymatically catalyzed reactions if a pH change is involved, for example during hydrolysis of an ester. From the pH-change the enzymatic
activity can be calculated. Also different cell lines or dairy starter cultures can be compared using the HydroPlate^®.
Monitoring of cell growth can be done by detecting the pH change as one of the metabolic key parameters.

For pH monitoring in microplates the HydroPlate^® (PreSens) can be used and measured with the Berthold Technologies multimode reader Mithras LB 940. The sensors are thin, hydrophilic, and almost calibration-free: Only 16 wells of the first plate have to be calibrated by an easy six-point calibration. All other sensors of the same batch have the same calibration values. Thus, later comparison of 96 different compounds without losing wells by using standards in every plate can be done. 12 wells of one HydroPlate^® per batch are needed for this calibration procedure. From time to time, re-calibration of the reader may be necessary.
The individual pH-sensor spots are located at the bottom of the wells for measurement of pH in the range between pH 5.0 to 8.0.

Instrument settings

As HydroPlate^® contains two different dyes, the Berthold Technologies microplate readers have to be measured in dual kinetic mode using two different filter pairs:

Filter pair 1 = Intensity indicator: Excitation 485 nm/Emission 535 nm
Filter pair 2 = Intensity reference: Excitation 485 nm/Emission 620 nm

For a better well to well reproducibility intensity of indicator and intensity of reference are measured and the ratio is calculated.

Filter order numbers
Excitation 485nm: 40271   (Fluorescein Standard Filter in Mithras LB 940)
Emission 535nm: 40273   (Fluorescein Standard Filter in Mithras LB 940)
Emission 620nm: 47731   (TRF Standard Filter in Mithras LB940 TRF models)

The change of fluorescence signal was measured with HydroPlate^® in the Mithras LB 940. Fluorescence bottom reading mode is required as coloured, fluorescent or turbidingredients of the sample will interfere with measurement from the top side. For transparent samples top reading mode can be selected.

Picture 1: Change of fluorescence intensity ratio signal with different pH values (pH 4 – pH 9), HydroPlate® measured with Berthold Mithras LB 940 in top reading mode