Polymeric gel nanoparticle pH sensors for intracellular measurements
Andresen, Thomas Lars
Benjaminsen, Rikke Vicki
Christensen, Nynne Meyn
Henriksen, Jonas Rosager
Precise measurements of pH in cells and intracellular compartments are of importance to both the fundamental understanding of metabolism and to the development of drugs that are released from the endosomes-lysome pathway. We have developed polymer gel nanoparticles as carriers of covalently bound fluorophores for ratiometric measurements of pH. One pH insensitive fluorophore serves as a reference while one or more pH sensitive fluorophores serve to give the desired pH dependence of the output. With one pH sensitive fluorophore a dynamic range of ~2 pH units is achieved. The physiologically interesting pH range is approximately 4 pH units and thus a nanoparticle sensor with two pH sensitive fluorophores is appropriate. With one pH sensitive fluorophore the output from the sensor follows R=R0+R1/10(pKa-pH), where R is the ratio of fluorescence for the two fluorophores, R0 is the minimum value of R, and R0+R1 is the maximum value of R. Suteable choice of fluorophores, here Rhodamin B as reference and Oregon green (OG) and Flurescin (FA) as pH sensitive fluorophores gives a nanoparticle sensor with the calibration curve shown in the Figure. The fluorophores are derivatized to allow for covalent bonding to the gel nanoparticle. The synthesis also allows for introduction of charged groups. Particle charge influence the cell uptake heavily. The synthesis scheme is outlined below. The sensors are internalized in cells through endocytosis and the sensor allows for the accurate measurement of pH as a function of position in the cell and time.