A New Fluorescein Isothiocyanate-Piperidine Self-ordered Ring Phosphorescent Probe for the Determination of Trace Protein

The COOH in fluorescein isothiocyanate (FITC) reacted with NH in piperidine (P) to form FITC‐P on the center of indentation of polyamide membrane (PAM) when drying for 2 min at (92±1)°C. Then, the FITC‐P diffused outward from the indentation center and formed the round SOR‐P‐FITC (containing the FITC‐P self‐ordered rings). Thus, multi‐FITC accumulated on SOR‐P‐FITC, leading to the enhancement of RTP signal on bio‐target, whose Ip increased 2.0 times compared with non‐generated SOR. When bovine serum albumin (BSA) was added to the center of SOR‐P‐FITC, NCS of FITC in SOR‐P‐FITC reacted with NH2 of BSA to form SOR‐P‐FITC‐BSA, which caused the RTP signal of FITC to enhance sharply. The ΔIp of the system was 3.4 times higher than that without β‐CD and 4.0 times higher than that without SOR‐P‐FITC formed. Its ΔIp was linear to the content of BSA. Therefore, a new solid substrate‐room temperature phosphorimetry (SS‐RTP) for the determination of trace protein was established using SOR‐P‐FITC as a phosphorescent probe. Under the optimum condition, the linear range of this method was 0.040–16.0 ag·spot−1 with a detection limit (LD) of 8.5 zg·spot−1 (0.40 µL sample solution per spot, the corresponding concentration was 2.1×10−17 g·mL−1), and the regression equation of working curve was ΔIp=3.848+4.240mBSA (ag·spot‐1), n=6, correlation coefficient (r) was 0.9993. This method with high sensitivity had been applied to determining the content of trace protein in the water samples, and the results coincided well with those obtained with pyrocatechol violet‐Mo(VI) method (P.V.M.M.). At the same time, the mechanism of SS‐RTP using SOR‐P‐FITC as a phosphorescent probe (SOR‐P‐FITC‐SS‐RTP) was discussed. The COOH in fluorescein isothiocyanate (FITC) reacted with NH in piperidine (P) to form FITC‐P on the center of indentation of polyamide membrane (PAM). Then, the FITC‐P formed the round SOR‐P‐FITC (containing the multiple FITC‐P self‐ordered rings), which went through the enrichment process from the spot to the ring. Therefore, multi‐FITC accumulated on detecting facula, causing the RTP signal of bio‐target enhanced. Bovine serum albumin (BSA) caused the RTP signal of FITC enhanced sharply. Thus, a new solid substrate‐room temperature phosphorimetry (SS‐RTP) for the determination of trace protein was established using SOR‐P‐FITC as a phosphorescent probe. The detection limit was 8.5 zg·spot−1 with high sensitivity. It had been successfully used to determine the conte