用于生物微环境检测的极性比例荧光探针制备及性能分析

Preparation and properties of ratio fluorescent probe for polarity detecting of biological microenvironment

为了构建准确检测生物微环境的极性比例荧光探针,首先通过在苊酮上引入丙二腈和N,N-二甲基基团合成以萘环荧光团为基础的探针IA,再通过氨基缩合成环反应合成极性响应型的比例荧光探针IAN,并利用~1H NMR、C NMR和HRMS等方法对探针IA、IAN进行结构及质量表征后,用紫外-可见分光光度计和荧光光谱仪对探针IA和IAN在不同极性溶剂中的吸收、荧光发射光谱进行检测。结果表明:探针IA吸收峰和荧光发射峰为单峰,其荧光发射峰强度随着溶剂极性升高而增强;探针IAN则具有双吸收和双荧光发射峰(λ_(em)=474、552 nm),且荧光发射峰比值I_(474nm)/I_(552nm)对溶剂极性变化产生灵敏的Boltzmann函数响应;与探针IA相比,探针IAN能够作为比例荧光探针准确响应环境极性变化;运用探针IAN对患乳腺癌的小鼠和患肝胆综合症的珍珠龙胆石斑鱼组织检测显示,病变组织的荧光强度明显低于健康组织。研究表明,氨基发生缩合反应生成含氮原子杂环,可提高氮原子孤对电子与π^(*)体系的共轭程度,从而使探针分子同时产生n→π^(*)与π^(*)→π^(*)分子跃迁,基于此机理,可构建灵敏响应极性变化的比例荧光探针。

To construct a polar ratio fluorescent probe for accurate detection of biological microenvironment,the probe IA based on naphthalene ring fluorophore was firstly synthesized by introducing malononitrile and N,N-dimethyl group on acenaphthene ketone,and then the polarity responsive ratio fluorescent probe IAN was synthesized by condensation reaction of IA amino group into heterocycle.The molecular structure and mass of probes IA and IAN were characterized by~1H NMR,C NMR and HRMS,and then the absorption and fluorescence spectra of probes IA and IAN in different polarity solvents were examined and compared by UV-Vis and fluorescence spectrophotometer.The results showed that IA exhibited single absorption and fluorescence emission peaks and fluorescence intensity of IA was gradually enhanced with solvent polarity increasing.Differently,double absorption and fluorescence emission peaks of IAN were simultaneously detected in solvents(λ_(em)=474 nm,and 552 nm),and the fluorescence intensity ratio I_(474nm)/I_(552nm)produced a sensitive Boltzmann function response to solvent polarity changes.Compared with IA,IAN was able to respond accurately to environmental polarity changes as a ratio fluorescent probe.The fluorescence intensity of the lesions was shown to be significantly lower than those of the healthy tissues by using IAN on mice with breast cancer and grouper hybrid Epinephelus fuscoguttatus♀×E.lanceolatus♂with hepatobiliary syndrome.These findings indicated that amino group undergoing a condensation reaction to generate a heterocycle containing nitrogen atom increased the conjugation degree of lone pair electrons of the nitrogen atom to theπ^(*)system,so that the probe IAN produced n→π^(*)andπ^(*)→π^(*)molecular transition at the same time,and then enabled the construction of a ratio fluorescent probe responding sensitively to polarity changes.