Expanding the dynamic measurement range for polymeric nanoparticle pH sensors

Sun, Honghao; Almdal, Kristoffer; Andresen, Thomas Lars

doi:10.1039/c1cc10439j

Cited by 64 publications

(69 citation statements)

References 15 publications

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“…This problem has actually been pointed out by Downey et al 32 more than 10 years ago, where they studied the pH in phagocytic cells using fluorescent-labeled zymosan and provided important insight into the challenges associated with pH measurements. We show here that recently reported nanometer-sized polymeric particle sensors, triple-labeled with a dynamic measurement range of almost 4 pH units 33 that covers the entire physiologically relevant range of the endosomeÀlysosome pathway, provide reliable results. We demonstrate the application of this sensor in real-time pH measurements in living cells and show the importance of its design relative to earlier reported sensor types.…”

Section: à24mentioning

confidence: 87%

“…Figure 1b shows ratiometric curves, measured on the microscope, of calibration emission spectra between the sensor and reference fluorophores in the nanoparticle sensors, excited at 488 and 561 nm, respectively, as a function of pH. Two dual-labeled sensors with FS or OG and the recently reported 33 triplelabeled sensor (FS and OG in the same nanoparticle) are shown (all with rhodamine B as the reference fluorophore). The dual-labeled sensors follow a sigmoidal function described by…”

Section: Resultsmentioning

confidence: 99%

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Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

Benjaminsen

Sun²,

Henriksen³

et al. 2011

ACS Nano

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Particle-based nanosensors have over the past decade been designed for optical fluorescent-based ratiometric measurements of pH in living cells. However, quantitative and time-resolved intracellular measurements of pH in endosomes and lysosomes using particle nanosensors are challenging, and there is a need to improve measurement methodology. In the present paper, we have successfully carried out time-resolved pH measurements in endosomes and lyosomes in living cells using nanoparticle sensors and show the importance of sensor choice for successful quantification. We have studied two nanoparticle-based sensor systems that are internalized by endocytosis and elucidated important factors in nanosensor design that should be considered in future development of new sensors. From our experiments it is clear that it is highly important to use sensors that have a broad measurement range, as erroneous quantification of pH is an unfortunate result when measuring pH too close to the limit of the sensitive range of the sensors. Triple-labeled nanosensors with a pH measurement range of 3.2–7.0, which was synthesized by adding two pH-sensitive fluorophores with different pK a to each sensor, seem to be a solution to some of the earlier problems found when measuring pH in the endosome–lysosome pathway.

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Section: à24mentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

Benjaminsen

Sun²,

Henriksen³

et al. 2011

ACS Nano

Self Cite

166

195

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show abstract

“…5. 20 Herein, we have used a nanoparticlebased pH sensor (nano sensor) that we recently developed 21 to measure pH changes of lysosomes after addition of PEI to the cells. This nanosensor is known to reside in the lysosomes and have a dynamic measure ment range from pH 3.2-7.0 covering the whole range of the endosomal system.…”

mentioning

confidence: 99%

The Possible “Proton Sponge ” Effect of Polyethylenimine (PEI) Does Not Include Change in Lysosomal pH

et al. 2013

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Polycations such as polyethylenimine (PEI) are used in many novel nonviral vector designs and there are continuous efforts to increase our mechanistic understanding of their interactions with cells. Even so, the mechanism of polyplex escape from the endosomal/lysosomal pathway after internalization is still elusive. The "proton sponge " hypothesis remains the most generally accepted mechanism, although it is heavily debated. This hypothesis is associated with the large buffering capacity of PEI and other polycations, which has been interpreted to cause an increase in lysosomal pH even though no conclusive proof has been provided. In the present study, we have used a nanoparticle pH sensor that was developed for pH measurements in the endosomal/lysosomal pathway. We have carried out quantitative measurements of lysosomal pH as a function of PEI content and correlate the results to the "proton sponge " hypothesis. Our measurements show that PEI does not induce change in lysosomal pH as previously suggested and quantification of PEI concentrations in lysosomes makes it uncertain that the "proton sponge " effect is the dominant mechanism of polyplex escape.

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“…Stir the mixture for 12 h. (iii) Dialyze against Milli-Q water for 4 d, by changing the water four times. 13. Keep the resulting nanosensors in Milli-Q water at a concentration of 45 mg/ml.…”

Section: Box 1 | Synthesis Of Polyacrylamide Nanosensorsmentioning

confidence: 99%

“…The main issues are the narrow pH ranges the sensors cover 12 and the fact that that proper calibration of the nanosensors is not trivial. In response to these issues, we developed a triple-labeled nanosensor that incorporates two pH-sensitive fluorophores and a reference fluorophore for a dynamic measurement range of almost double the range of conventional dual-labeled nanosensors with one pH-sensitive fluorophore and a reference fluorophore 12,13 .…”

Section: Introductionmentioning

confidence: 99%

Design, calibration and application of broad-range optical nanosensors for determining intracellular pH

2014

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Particle-based nanosensors offer a tool for determining the pH in the endosomal-lysosomal system of living cells. Measurements providing absolute values of pH have so far been restricted by the limited sensitivity range of nanosensors, calibration challenges and the complexity of image analysis. This protocol describes the design and application of a polyacrylamide-based nanosensor (∼60 nm) that covalently incorporates two pH-sensitive fluorophores, fluorescein (FS) and Oregon Green (OG), to broaden the sensitivity range of the sensor (pH 3.1-7.0), and uses the pH-insensitive fluorophore rhodamine as a reference fluorophore. The nanosensors are spontaneously taken up via endocytosis and directed to the lysosomes where dynamic changes in pH can be measured with live-cell confocal microscopy. The most important focus areas of the protocol are the choice of pH-sensitive fluorophores, the design of calibration buffers, the determination of the effective range and especially the description of how to critically evaluate results. The entire procedure typically takes 2-3 weeks.

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Expanding the dynamic measurement range for polymeric nanoparticle pH sensors

Cited by 64 publications

References 15 publications

Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

The Possible “Proton Sponge ” Effect of Polyethylenimine (PEI) Does Not Include Change in Lysosomal pH

Design, calibration and application of broad-range optical nanosensors for determining intracellular pH

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