Department of Chemical Engineering, Bio-IN𝙫ENT Lab, Siddaganga Institute of Technology, Tumakuru, India.
J Liposome Res. 2023 Sep;33(3):300-313. doi: 10.1080/08982104.2023.2172582. Epub 2023 Feb 6.
Measurement of osmolarity is critical for optimizing bioprocesses including antibody production and detecting pathologies. Thus, rapid, sensitive, and sensing of osmolarity is desirable. This study aims to develop and assess the suitability of calcein- and sulforhodamine-loaded nanoliposomes for ratiometric sensing of osmolarity by fluorescence spectroscopy and evaluate the range of detection. The detection is based on concentration-dependent self-quenching of calcein fluorescence (sensor dye at 6-15 mM) and concentration-independent fluorescence of sulforhodamine (reference dye) due to osmotic shrinkage of the nanoliposomes when exposed to hyperosmotic solutions. Using mathematical modeling, 6 mM calcein loading was found to be optimal to sense osmolarity between 300 and 3000 mOsM. Calcein (6 mM)- and sulforhodamine (2 mM)-loaded nanoliposomes were produced by thin-film hydration and serial extrusion. The nanoliposomes were unilamellar, spherical (108 ± 9 nm), and uniform in size (polydispersity index [PDI] 0.12 ± 0.04). Their shrinkage induced by exposure to hyperosmotic solutions led to rapid self-quenching of calcein fluorescence (F), but no effect on sulforhodamine fluorescence (F) was observed. F/F decreased linearly with increasing osmolarity, obeying Boyle van't Hoff's relationship, thus proving that the nanoliposomes are osmosensitive. A calibration curve was generated to compute osmolarity based on F/F measurements. As a proof-of-concept, dynamic changes in osmolarity in a yeast-based fermentation process was demonstrated. Thus, the nanoliposomes have great potential as sensors to rapidly and sensitively measure wide-ranging osmolarities.
渗透压的测量对于优化生物工艺过程包括抗体生产和检测病理学至关重要。因此，理想的情况是快速、灵敏和感测渗透压。本研究旨在开发并评估钙黄绿素和磺基罗丹明负载的纳米脂质体用于通过荧光光谱学进行渗透压比色感应的适用性，并评估检测范围。该检测基于当暴露于高渗溶液时纳米脂质体由于渗透压收缩而导致的钙黄绿素荧光（传感器染料在 6-15mM 时）浓度依赖性自猝灭和磺基罗丹明（参考染料）的浓度独立荧光。使用数学建模，发现 6mM 钙黄绿素负载量最适合感应 300 至 3000mOsM 之间的渗透压。通过薄膜水合和连续挤压制备负载有钙黄绿素（6mM）和磺基罗丹明（2mM）的纳米脂质体。纳米脂质体为单分子层、球形（108±9nm）且大小均匀（多分散指数 [PDI]0.12±0.04）。它们在暴露于高渗溶液时的收缩导致钙黄绿素荧光（F）的快速自猝灭，但对磺基罗丹明荧光（F）没有影响。F/F 随渗透压的增加呈线性下降，遵循 Boyle van't Hoff 关系，从而证明纳米脂质体是渗透压敏感的。生成了校准曲线，以基于 F/F 测量值计算渗透压。作为概念验证，演示了酵母发酵过程中渗透压的动态变化。因此，纳米脂质体作为快速灵敏地测量广泛渗透压的传感器具有很大的潜力。
