研究目的
To prepare track-etched PET membranes with fluorescent response to pH changes and evaluate their use as biosensors, particularly for sensing pH in biological environments like E. coli cell cultures.
研究成果
The GPIRR process was optimized for functionalizing track-etched PET membranes, enabling the development of a pH biosensor using immobilized GFP. This sensor effectively detected pH changes in buffer solutions and E. coli cultures, demonstrating potential for integration into biological and medical applications. The method offers advantages such as high specificity, protection of fluorophores, and scalability.
研究不足
The grafting yield of GPIRR is very low and not detectable by methods like ATR FT-IR spectroscopy, requiring optimization. Fluorescein-immobilized membranes were not sensitive to pH, limiting their utility. Further studies on sensor stability and time-scale response are needed.
1：Experimental Design and Method Selection:
The study used Grafting Polymerisation Initiated by Remnant Radicals (GPIRR) for selective modification of track-etched membranes (TMs) with poly(glycidyl methacrylate) (poly(GMA)). Fluorescent labeling with Fluorescein isothiocyanate (FITC) and immobilization of Green Fluorescent Protein (GFP) were employed to create pH-sensitive sensors.
2：Sample Selection and Data Sources:
Irradiated PET foils were used, with specific ion types and fluences (e.g., 125Xe ions at
3：95 MeV/amu, fluence 7×10^7 ions/cm2). GFPcys3 protein was expressed and purified from recombinant E. coli. List of Experimental Equipment and Materials:
Materials included glycidyl methacrylate (GMA), tris(2-carboxyethyl)phosphine (TCEP), cysteamine, FITC, and solvents. Equipment included FESEM Carl Zeiss NTS-SUPRA40, AFM Nanoscope IIIA Multimode-AFM, fluorescence microscope Nikon Eclipse TE2000, Nanodrop 3300 fluorospectrometer, and Cytation 5 microplate reader.
4：Experimental Procedures and Operational Workflow:
Etching was done with NaOH 2N at 60°C, followed by GPIRR with GMA in ethanol:water at 60°C. Chemical modifications included cysteamine treatment and FITC labeling or GFP immobilization. Fluorescence measurements were performed at specific wavelengths.
5：Data Analysis Methods:
Fluorescence data were analyzed using calibration curves, ImageJ software for image analysis, and statistical methods for pore diameter and grafting yield calculations.
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