Fluorescence based intracellular pH nanoprobes have been developed that overcomes the limitations imposed by shallow penetration depth of ultraviolet excitation, photostability, phototoxicity, and interference from background autofluorescence. In this study, we have constructed a Förster Resonance Energy Transfer (FRET) based pH nanoprobe using upconversion nanoparticle (UCNP) as a donor (excitation/emission @ 980/540 nm, green channel), and mOrange fluorescent protein (excitation/emission @ 548/566 nm, red channel) as acceptor. The UCNP-mOrange nanoprobe could be fluorescently imaged with 980 nm excitation, having deep penetration depth, by a fluorescence microscope on a coverslip, or uptaken in a single HeLa cell. The cellular upatake of these nanoparticles were confirmed by TEM study. The FRET probes, with a FRET efficiency of ∼20% at physiological pH of 7.0, have simultaneous self-ratiometric and ratiometric features varying linearly with local pH. The probe exhibits high accuracy, sensitivity, reversibility, and stability over a wide range of pH (3.0–8.0). The fluorescence intensity ratio from individual green, and red channels in fluorescence microscopic images could be used to estimate the pH of the intracellular compartments of HeLa cell from the pH dependent ratiometric calibration. Nigericin mediated intracellular pH (3.0, 5.0, and 7.0) could be accurately estimated from the CLSM derived FRET ratio. The pH probes demonstrate high stability and reversibility when switched between pH 3, and 8 for at least 5 cycles.

已经开发了基于荧光的细胞内pH纳米探针，该探针克服了紫外线激发的浅穿透深度，光稳定性，光毒性和背景自发荧光的干扰所带来的局限性。在这项研究中，我们使用上转换纳米粒子（UCNP）作为供体（激发/发射@ 980/540 nm，绿色通道）和mOrange荧光蛋白（激发/发射@）构建了基于Förster共振能量转移（FRET）的pH纳米探针。 548/566 nm，红色通道）作为受体。UCNP-mOrange纳米探针可通过盖玻片上的荧光显微镜以980 nm激发光进行荧光成像，具有很深的穿透深度，或被单个HeLa细胞吸收。通过TEM研究证实了这些纳米颗粒的细胞摄取。FRET探针 在7.0的生理pH下具有约20％的FRET效率，同时具有自测度和比例特性，随局部pH线性变化。该探针在很宽的pH值（3.0–8.0）范围内都具有高精度，高灵敏度，可逆性和稳定性。荧光显微镜图像中单个绿色通道和红色通道的荧光强度比可用于根据pH依赖的比例校准来估计HeLa细胞胞内区室的pH。可以从CLSM得出的FRET比值准确估算尼日尔介导的细胞内pH（3.0、5.0和7.0）。当在pH 3和pH 8之间切换至少5个循环时，pH探针显示出高稳定性和可逆性。该探针在广泛的pH值（3.0–8.0）范围内都具有高精度，高灵敏度，可逆性和稳定性。荧光显微镜图像中单个绿色通道和红色通道的荧光强度比可用于根据pH依赖的比例校准来估计HeLa细胞胞内区室的pH。可以从CLSM得出的FRET比值准确估算尼日尔菌介导的细胞内pH（3.0、5.0和7.0）。当在pH 3和pH 8之间切换至少5个循环时，pH探针显示出高稳定性和可逆性。该探针在很宽的pH值（3.0–8.0）范围内都具有高精度，高灵敏度，可逆性和稳定性。荧光显微镜图像中单个绿色通道和红色通道的荧光强度比可用于根据pH依赖的比例校准来估计HeLa细胞胞内区室的pH。可以从CLSM得出的FRET比值准确估算尼日尔菌介导的细胞内pH（3.0、5.0和7.0）。当在pH 3和pH 8之间切换至少5个循环时，pH探针显示出高稳定性和可逆性。荧光显微镜图像中的红色通道和红色通道可用于根据pH依赖的比例校准来估计HeLa细胞的细胞内区室的pH。可以从CLSM得出的FRET比值准确估算尼日尔介导的细胞内pH（3.0、5.0和7.0）。当在pH 3和pH 8之间切换至少5个循环时，pH探针显示出高稳定性和可逆性。荧光显微镜图像中的红色通道和红色通道可用于根据pH依赖的比例校准来估计HeLa细胞的细胞内区室的pH。可以从CLSM得出的FRET比值准确估算尼日尔菌介导的细胞内pH（3.0、5.0和7.0）。当在pH 3和pH 8之间切换至少5个循环时，pH探针显示出高稳定性和可逆性。