Superoxide anion (O 2 .- ), as the first generated reactive oxygen species (ROS), has been considered to be highly deleterious to cell functions. The measurement of intracellular O 2 .- level is of great importance to uncover its roles in a variety of oxidative damage diseases. Hydroethidium (HE) fluorescence-based method is dominating intracellular O 2 .- assay by monitoring the unique product 2-OH-E + of HE/O 2 .- reaction. However, the avoid-less cross-interference of red fluorescence limited its ability to provide trustworthy information on intracellular O 2 .- formation. By the detection of 2-OH-E + , we herein developed and validated an improved LC/MS-based method for the measurement of intracellular O 2 .- . Firstly, we demonstrated the proportionality of HE/O 2 .- reaction. Secondly, ungerimine was used as internal standard to eliminate daily basis and matrix effect in the LC/MS-based detection of 2-OH-E + . Afterward, the total protein concentration was utilized for cell number normalization. Accordingly, an equation was further proposed to calculate the relative abundance (RA) of intracellular O 2 .- . Finally, the developed method has been successfully utilized to evaluate the inhibitory effects of natural compounds on O 2 .- generation, the result of which was validated by the HE-based fluorescent measurement. Compared with the fluorescent measurement, the LC/MS-based intracellular O 2 .- assay method is more sensitive, selective and accurate.
Ying Zhou,Jie Ding,Tingxizi Liang,E. S. Abdel-Halim,Liping Jiang,Jun-Jie Zhu
Fluorescent nanosensors have been widely applied in recognition and imaging of bioactive small molecules; however, the complicated surface modification process and background interference limit their applications in practical biological samples. Here, a simple, universal method was developed for ratiometric fluorescent determination of general small molecules. Taking superoxide anion (O2•–) as an example, the designed sensor was composed of three main moieties: probe carrier, rattle-type silica colloidal particles (mSiO2@hmSiO2 NPs); reference fluorophore doped into the core of NPs, fluorescein isothiocyanate (FITC); fluorescent probe for superoxide anion, hydroethidine (HE). In the absence of O2•–, the sensor just emitted green fluorescence of FITC at 518 nm. When released HE was oxidized by O2•–, the oxidation product exhibited red fluorescence at 570 nm and the intensity was linearly associated with the concentration of O2•–, while that of reference element remained constant. Accordingly, ratiometric determination of O2•– was sensitively and selectively achieved with a linear range of 0.2–20 μM, and the detection limit was calculated as low as 80 nM. Besides, the technique was also successfully applied for dual-emission imaging of O2•– in live cells and realized visual recognition with obvious fluorescence color change in normal conditions or under oxidative stress. As long as appropriate reference dyes and sensing probes are selected, ratiometric biosensing and imaging of bioactive small molecules would be achieved. Therefore, the design could provide a simple, accurate, universal platform for biological applications.
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