Mapping the Pharyngeal and Intestinal pH of Caenorhabditis elegans and Real-Time Luminal pH Oscillations Using Extended Dynamic Range pH-Sensitive Nanosensors
Mapping the Pharyngeal and Intestinal pH of Caenorhabditis elegans and Real-Time Luminal pH Oscillations Using Extended Dynamic Range pH-Sensitive Nanosensors
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DOI：
10.1021/nn401856u
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发表时间：
2013-06-01
期刊：
影响因子：
17.1
通讯作者：
Aylott, Jonathan W.
中科院分区：
文献类型：
--
作者：
Chauhan, Veeren M.;Orsi, Gianni;Aylott, Jonathan W.
扩展动态范围pH敏感比例纳米传感器，能够准确绘制完整的生理pH范围，已被开发并用于实时表征秀丽隐杆线虫咽部和肠腔的pH。将ph敏感荧光团、羧基荧光素（FAM）和俄勒冈绿（OG）以1:1的比例与参考荧光团5-（和6）-羧基四甲基罗丹明（TAMRA）偶联到惰性聚丙烯酰胺基质上，制备了直径为40 nm的纳米传感器。通过测定pH敏感荧光团和参考荧光团之间的荧光比，计算出精确的比例pH值。纳米传感器使用自动图像分析系统进行校准，并验证了pH测量分辨率为+/- 0.17 pH单位。线虫种群活力指标显示，纳米传感器处理过的线虫（浓度为50.00 ~ 3.13 mg/mL）与未处理过的线虫在去除纳米传感器后的4天内（p为24 h）无统计学差异。活线虫管腔的pH值在前咽部为5.96 +/- 031，后肠为339 +/- 0.09。咽部泵送速率决定了摄取的物质从咽部向肠道的转移，这一速率与温度有关。在4℃下对秀丽隐杆线虫进行成像，可将咽泵速降低至7次/min，并可在荧光显微镜下实时重建肠腔内有节奏的pH振荡。
Extended dynamic range pH-sensitive ratiometric nanosensors, capable of accurately mapping the full physiological pH range, have been developed and used to characterize the pH of the pharyngeal and intestinal lumen of Caenorhabditis elegans in real-time. Nanosensors, 40 nm in diameter, were prepared by conjugating pH-sensitive fluorophores, carboxyfluorescein (FAM) and Oregon Green (OG) in a 1:1 ratio, and a reference fluorophore, 5-(and-6)-carboxytetramethylrhodamine (TAMRA) to an inert polyacrylamide matrix. Accurate ratiometric pH measurements were calculated through determination of the fluorescence ratio between the pH-sensitive and reference fluorophores. Nanosensors were calibrated with an automated Image analysis system and validated to demonstrate a pH measurement resolution of +/- 0.17 pH units. The motility of C. elegans populations, as an indicator for viability, showed nematodes treated with nanosensors, for concentrations ranging from 50.00 to 3.13 mg/mL, were not statistically different to nematodes not challenged with nanosensors up to a period of 4 days (p 24 h after nanosensor challenge was removed. The pH of viable C. elegans lumen was found to range from 5.96 +/- 031 in the anterior pharynx to 339 +/- 0.09 in the posterior intestine. The pharyngeal pumping rate, which dictates the transfer of ingested material from the pharynx to the intestine, was found to be temperature dependent. Imaging C. elegans at 4 degrees C reduced the pharyngeal pumping rate to 7 contractions/min and enabled the reconstruction of rhythmic pH oscillations in the intestinal lumen in real-time with fluorescence microscopy.