pH sensing in living cells using fluorescent microspheres

Bradley, Mark; Alexander, Lois M.; Duncan, Karen A.; Chennaoui, Mourad; Jones, Anita C.; Sánchez‐Martín, Rosario M.

doi:10.1016/j.bmcl.2007.10.075

Cited by 80 publications

(61 citation statements)

References 19 publications

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“…12 While some data support the notion that commercially available microspheres are taken up endocytically, 9,10,13,14 it is hard to reconcile this when their capacity to function as cytoplasmatic pH and calcium sensors has been demonstrated. 15,16 Microspheres have also been used to deliver siRNA intracellularly for the efficient gene silencing of enhanced green fluorescent protein (EGFP) and functional proteins, such as b-galactosidase, which both require cytoplasmic localisation. 17,18 Taken together, these observations are inconsistent with an uptake mechanism that involves traditional endocytosis.…”

Section: Introductionmentioning

confidence: 99%

Investigation of microsphere-mediated cellular delivery by chemical, microscopic and gene expression analysis

et al. 2010

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Section: Introductionmentioning

confidence: 99%

Investigation of microsphere-mediated cellular delivery by chemical, microscopic and gene expression analysis

et al. 2010

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“…The applicability of these systems for fluorimetirc analysis is also well proven [4][5][6][7][8][9][10][11][12][13][14].…”

Section: Introductionmentioning

confidence: 89%

Enzyme Substrates Protective Encapsulation within Polymeric Microspheres

Zajda¹,

Jastrzębska²,

Olszyna³

et al. 2013

AJAC

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Determination of enzymes activity is an important task of analytical and biomedical oriented fluorimetry. Despite of a long track record of application, there is still some room for improvements in this field. In the case of hydrolases, spontaneous decomposition of substrate leads to substantial errors in determination of enzyme activity. An innovative and effective approach is proposed allowing protection of enzyme substrate within the lipophilic moiety of polyacrylate microspheres. It is shown that the introduction of substrate into microspheres is not only an effective method of prevention of unwanted spontaneous process, but also does not disturb the availability of substrate for enzymatic hydrolysis. The effect of presence of proteins in the sample on fluorimetric responses was studied. In contrary to previous studies related to application of lipophilic polymers in biomedical analysis, it is shown that the presence of bovine serum albumins enhances the sensitivity of fluorimetric determination. It is shown that this surprising effect is related to adsorption of proteins on the microspheres surface and change of surface charge of polymer.

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“…Fluorescent nanoparticle sensors possess these properties, but even though many pH-sensitive nanosensors have been developed [8][9][10][11] not many published studies exist wherein thorough measurements of intracellular pH have been performed. 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.…”

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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pH sensing in living cells using fluorescent microspheres

Cited by 80 publications

References 19 publications

Investigation of microsphere-mediated cellular delivery by chemical, microscopic and gene expression analysis

Investigation of microsphere-mediated cellular delivery by chemical, microscopic and gene expression analysis

Enzyme Substrates Protective Encapsulation within Polymeric Microspheres

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

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