Study Of Near-infrared Fluorescent Probe For Zn～(2+) Base On IR-780 As Fluorophore

Zinc ion (Zn²⁺) is an essential component of many enzymes and transcription factors, which exists in cells and body fluid. Zn²⁺ directly participates in various metabolic process, such as cell growth and procreation, tissue repair, genetic transcription, metalloenzyme catalysis and neurotransmission. It has been reported that Zinc deficiency leads to the dysfunction and defect of immune systems. Moreover, Zn²⁺ has been reported to induce selective neuronal cell death that is associated with certain acute conditions, including epilepsy, transient global ischemia and brain injure. Because of the significance of Zn²⁺ in biological system, the research of Zn²⁺ in cell becomes a hot research topic in chemistry, biology and clinical medicine. Fluorescent detection has the advantages of easy operation and high sensitivity, especially, near-infrared fluorescent probe has the predominance of avoiding cell damage and minishing interference by autofluoresence from biological molecules. It has important significance to design and synthesize the near-infrared fluorescent probe of Zn²⁺. Alkaline Phosphatase (ALP) is a kind of metalloenzyme, which extensively exists in various tissues of body and plays an important role in metabolizability of phosphate. ALP is an indicator of hepatobiliary and bone disorder. Therefore, it needs exist to detect ALP sensitively and selectively in many diagnostic and clinical assays. So to design the high sensitivity fluorescent probe of ALP also has important significance. The detailed materials are summarized as following:1. A novel near-infrared fluorescent probe, TAEA-IR-780, was proposed for the measurement of Zn²⁺, which was obtained by directly attaching an acceptor, tris (2-aminoethyl) amine (TAEA), to the near-infrared fluorophore IR-780. The excitation and emission wavelengths of TAEA-IR-780 were 683 nm and 750 nm, respectively, and its fluorescence intensity increased with the addition of Zn²⁺ under physiological condition (pH=7.4) and the detection limit reached 1.00×10-9 mol·L-1. Moreover, the fluorescence intensity of TAEA-IR-780 did not noticeably increase in the presence of other biologically important cations such as Ca²⁺ and Mg²⁺. The results showed that the probe could not only effectively minimize cell damage and autofluorescence by the near-infrared excitation light but also afford sufficient sensitivity and selectivity for the biological detection of Zn²⁺.2. A near-infrared fluorescent probe for Zn²⁺, DPA-IR-780, was designed and synthesized, which based on Di-(2-picoylyl)amine (DPA) as the recognition group. The structure of the probe was charactered by 1HNMR, MS. The excitation and emission wavelengths of the as prepared DPA-IR-780 were 686 nm and 764 nm, respectively, and its fluorescence intensity increased with the addition of Zn²⁺ under physiological condition (pH=7.40) and the detection limit reached 1.00×10-10 mol·L-1. Furthermore, this probe has strong anti-interference ability under the existing other metals. At the same time, because of the long excitation wavelength and emission wavelength, the probe could effectively minimize cell damage and minish interference by autofluoresence from biological molecules.3. A new fluoremic substrate, 3-phoshpase-fluorescein (FMP), detecting alkaline phosphatase (ALP) was developed in this part. The synthetic method was sample, convenient, economical and this new fluoremic substrate was sensitive to ALP. The background fluorescent intensity of FMP was very weak, while the fluorescent intensity was strong when the ALP added after incubation for 40 min at 37℃. And the experiment showed that FMP was relative stable in the Tris·Cl buffer (PH=9.10). The fluorescent intensity increased with the increasing of ALP concentration. About 40 min, the fluorescent intesity could reach a plateau. And the detection limit could reach to 1.83×10-9 mg·mL.