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A protein – dye hybrid system as a narrow range tunable intracellular pH sensor † † Electronic supplementary information (ESI) available: Figures depicting various photophysical properties, cytotoxicity studies and confocal fluorescence images. See DOI: 10.1039/c6sc02659a Click here for additional data file.

View Article: PubMed Central - PubMed

ABSTRACT

Accurate monitoring of pH variations inside cells is important for the early diagnosis of diseases such as cancer. Even though a variety of different pH sensors are available, construction of a custom-made sensor array for measuring minute variations in a narrow biological pH window, using easily available constituents, is a challenge. Here we report two-component hybrid sensors derived from a protein and organic dye nanoparticles whose sensitivity range can be tuned by choosing different ratios of the components, to monitor the minute pH variations in a given system. The dye interacts noncovalently with the protein at lower pH and covalently at higher pH, triggering two distinguishable fluorescent signals at 700 and 480 nm, respectively. The pH sensitivity region of the probe can be tuned for every unit of the pH window resulting in custom-made pH sensors. These narrow range tunable pH sensors have been used to monitor pH variations in HeLa cells using the fluorescence imaging technique.

No MeSH data available.


Confocal fluorescence images of HeLa cells incubated with BSA–SqNP hybrids in the ratio of 6 : 1, 12 : 1 and 1 : 1 at pH 6.5, 7.0 and 7.5 for 30 min at 37 °C. Green and red images were obtained by exciting at 405 and 640 nm respectively. The corresponding ratio images are also shown.
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fig4: Confocal fluorescence images of HeLa cells incubated with BSA–SqNP hybrids in the ratio of 6 : 1, 12 : 1 and 1 : 1 at pH 6.5, 7.0 and 7.5 for 30 min at 37 °C. Green and red images were obtained by exciting at 405 and 640 nm respectively. The corresponding ratio images are also shown.

Mentions: The pH variations inside cells were imaged using BSA–SqNP hybrid sensors of different ratios. Before the pH monitoring, the cytotoxicity of the probe was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay (Fig. S9†). The result revealed that SqNPs and BSA–SqNPs exhibit minimal toxicity to the cells under the concentrations measured, demonstrating their suitability for biological applications. Furthermore, we investigated the cellular uptake and intracellular distribution of BSA–SqNPs. After incubation of HeLa cells with a 6 : 1 BSA–SqNP combination (in phosphate buffer, pH 7.0) for 30 min, significant red and green fluorescence was detected in the cytoplasm, however not in the nucleus or extracellular environment (Fig. S10†); indicative of the excellent membrane permeability of the probe. Hence, we used a 6 : 1 combination of the BSA–SqNP hybrid to monitor minor pH fluctuations inside live cells. For this purpose, the protein–dye hybrid (6 : 1) was imported into HeLa cells grown in wells of different pH values (6.5–7.5). The intracellular pH was equilibrated to the surrounding medium using the H+/K+ ionophore, nigericin. After the uptake of the BSA–SqNP conjugate for 30 min, confocal fluorescence images of the cells were recorded (Fig. 4). At acidic pH (6.5), the probe gave a strong red emission and a weak green emission due to the noncovalent interaction of the dye with BSA. As the pH of the cells is increased to the basic region (7.5), the probe exhibited a strong green emission and a weak red emission as a result of the covalent interaction of the dye with BSA. The corresponding fluorescence intensity (Fig. S11a and S12b†) obtained from the ratio image exhibits good linearity, which shows the ability of the probe to measure the intracellular pH variation over a small pH range. Cells of pH 6.5–7.5 were incubated with BSA–SqNP hybrids with a 12 : 1 and 1 : 1 ratio (Fig. 4). The cells treated with the 12 : 1 hybrid gave an “always on” signal in the green window and an “always off” signal in the red window irrespective of pH as shown in Fig. 4, S12a and b,† which implies that at the given experimental pH window, this combination is not sensitive. As expected, the 1 : 1 combination gives an “always off” signal in both green and red windows, since it works above the pH range that we chose.


A protein – dye hybrid system as a narrow range tunable intracellular pH sensor † † Electronic supplementary information (ESI) available: Figures depicting various photophysical properties, cytotoxicity studies and confocal fluorescence images. See DOI: 10.1039/c6sc02659a Click here for additional data file.
Confocal fluorescence images of HeLa cells incubated with BSA–SqNP hybrids in the ratio of 6 : 1, 12 : 1 and 1 : 1 at pH 6.5, 7.0 and 7.5 for 30 min at 37 °C. Green and red images were obtained by exciting at 405 and 640 nm respectively. The corresponding ratio images are also shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5134758&req=5

fig4: Confocal fluorescence images of HeLa cells incubated with BSA–SqNP hybrids in the ratio of 6 : 1, 12 : 1 and 1 : 1 at pH 6.5, 7.0 and 7.5 for 30 min at 37 °C. Green and red images were obtained by exciting at 405 and 640 nm respectively. The corresponding ratio images are also shown.
Mentions: The pH variations inside cells were imaged using BSA–SqNP hybrid sensors of different ratios. Before the pH monitoring, the cytotoxicity of the probe was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay (Fig. S9†). The result revealed that SqNPs and BSA–SqNPs exhibit minimal toxicity to the cells under the concentrations measured, demonstrating their suitability for biological applications. Furthermore, we investigated the cellular uptake and intracellular distribution of BSA–SqNPs. After incubation of HeLa cells with a 6 : 1 BSA–SqNP combination (in phosphate buffer, pH 7.0) for 30 min, significant red and green fluorescence was detected in the cytoplasm, however not in the nucleus or extracellular environment (Fig. S10†); indicative of the excellent membrane permeability of the probe. Hence, we used a 6 : 1 combination of the BSA–SqNP hybrid to monitor minor pH fluctuations inside live cells. For this purpose, the protein–dye hybrid (6 : 1) was imported into HeLa cells grown in wells of different pH values (6.5–7.5). The intracellular pH was equilibrated to the surrounding medium using the H+/K+ ionophore, nigericin. After the uptake of the BSA–SqNP conjugate for 30 min, confocal fluorescence images of the cells were recorded (Fig. 4). At acidic pH (6.5), the probe gave a strong red emission and a weak green emission due to the noncovalent interaction of the dye with BSA. As the pH of the cells is increased to the basic region (7.5), the probe exhibited a strong green emission and a weak red emission as a result of the covalent interaction of the dye with BSA. The corresponding fluorescence intensity (Fig. S11a and S12b†) obtained from the ratio image exhibits good linearity, which shows the ability of the probe to measure the intracellular pH variation over a small pH range. Cells of pH 6.5–7.5 were incubated with BSA–SqNP hybrids with a 12 : 1 and 1 : 1 ratio (Fig. 4). The cells treated with the 12 : 1 hybrid gave an “always on” signal in the green window and an “always off” signal in the red window irrespective of pH as shown in Fig. 4, S12a and b,† which implies that at the given experimental pH window, this combination is not sensitive. As expected, the 1 : 1 combination gives an “always off” signal in both green and red windows, since it works above the pH range that we chose.

View Article: PubMed Central - PubMed

ABSTRACT

Accurate monitoring of pH variations inside cells is important for the early diagnosis of diseases such as cancer. Even though a variety of different pH sensors are available, construction of a custom-made sensor array for measuring minute variations in a narrow biological pH window, using easily available constituents, is a challenge. Here we report two-component hybrid sensors derived from a protein and organic dye nanoparticles whose sensitivity range can be tuned by choosing different ratios of the components, to monitor the minute pH variations in a given system. The dye interacts noncovalently with the protein at lower pH and covalently at higher pH, triggering two distinguishable fluorescent signals at 700 and 480 nm, respectively. The pH sensitivity region of the probe can be tuned for every unit of the pH window resulting in custom-made pH sensors. These narrow range tunable pH sensors have been used to monitor pH variations in HeLa cells using the fluorescence imaging technique.

No MeSH data available.