Nanosensor calibration and pH calculation using Matlab
This protocol is extracted from research article:
Fluorescent nanosensors reveal dynamic pH gradients during biofilm formation
NPJ Biofilms Microbiomes, Jun 17, 2021; DOI: 10.1038/s41522-021-00221-8

Calibration was undertaken in conditions matching each experiment, for each batch and following long term storage. Buffer solutions with a pH ranging from 3.0 to 8.0 in 1.0 steps were prepared using 0.2 M sodium phosphate (Na2HPO4) and 0.1 M citric acid (C6H8O7) as indicated in Supplementary Table 1. The pH was adjusted using 0.2 M sodium phosphate and 0.1 M citric acid.

For the pH calibration of the nanosensors in a BioFlux experiment, 10 mg mL−1 polyacrylamide were prepared in deionised water and mixed 1:1 with each of the pH buffers to get a concentration of 5 mg mL−1. To enable the calculation of a calibration curve, 500 µL of each solution were added to a BioFlux plate and was run for 2 min with a flow rate of 0.25 dyn cm2. Images were taken for each buffer sample using a Zeiss CLSM (OG-FAM = 488 nm, TAMRA = 555 nm). The software MatLab was used to calculate the calibration curve and subsequently to determine the pH of the actual biofilm images taken with the Zeiss CLSM.

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