传感器类型
荧光生物传感器
检测对象
蛋白(牛血清白蛋白,BSA);样品基质为蛋白溶液/磷酸盐缓冲液(pH 7)透析体系
检测原理
该检测方法以含双氨基的吡唑双嘧啶荧光染料作为识别与信号元件。BSA表面羧基在EDC/NHS作用下被活化,染料氨基与活化羧基发生碳二亚胺偶联,形成酰胺键共价结合。结合事件改变染料所处微环境和电子结构,导致荧光发射波长发生约30 nm位移并伴随荧光猝灭。游离染料通过磷酸盐缓冲液透析去除,仅保留荧光标记蛋白。荧光分光光度计在固定激发/发射狭缝下读取结合前后或不同染料浓度下的发射强度。随染料浓度增加,染料-BSA结合量逐渐增加,荧光强度升高至饱和;改变蛋白量时结合态发射强度基本不变,从而通过荧光变化反映蛋白结合/蛋白检测。
检测灵敏度
原文未报告LOD、线性范围、灵敏度斜率或相关系数。
效应效果
论文未报告选择性、抗干扰、长期稳定性、重现性(RSD)、实际样品加标回收率,也未与ELISA、HPLC或qPCR等方法进行定量对比。实验显示染料在379 nm吸收、497 nm发射,量子产率为0.375,结合前后发射波长差约30 nm。在染料浓度由0.001增至0.01(原文未明确单位)时,染料与BSA的结合量逐渐增加,高浓度下荧光强度趋于稳定,提示结合位点饱和;改变蛋白量时结合态发射强度基本不变。作者认为该吡唑双嘧啶荧光团与BSA相互作用良好,可在近可见区检测,具有作为蛋白检测荧光探针的应用潜力。
传感器的构成
- 荧光探针/信号标记物:4,4'-(1,1'-(5-(2-甲氧基苯氧基)-[2,2'-双嘧啶]-4,6-二基)双(1H-吡唑-3,1-二基))二苯胺(化合物16),含两个氨基,吸收379 nm、发射497 nm、量子产率0.375,作为荧光信号源。
- 识别/结合对象:牛血清白蛋白(BSA),表面羧基与染料氨基经碳二亚胺偶联形成共价结合,用于蛋白结合检测。
- 偶联/活化试剂:EDC(1-乙基-3-[3-二甲氨基丙基]碳二亚胺盐酸盐)和NHS(N-羟基丁二酰亚胺),活化BSA羧基并促进染料-蛋白酰胺键形成。
- 分离/清洗介质:磷酸盐缓冲液(pH 7)透析,去除未结合游离染料,保留荧光标记蛋白。
- 信号读出:荧光分光光度计(Varian Cary Eclipse),激发/发射狭缝5 nm,检测结合前后荧光发射变化。
中文摘要
背景:具有生物相容性官能团和良好荧光行为的荧光染料可用作生物传感器,用于监测不同生物过程及蛋白检测。已有荧光传感器虽具较高选择性和灵敏度,但仍需开发具有更好荧光性能和生物相容性的新荧光团。结果:以2-苯乙腈、2-氯嘧啶和2-甲氧基苯酚为起始原料,经多步合成得到新型4,4'-(1,1'-(5-(2-甲氧基苯氧基)-[2,2'-双嘧啶]-4,6-二基)双(1H-吡唑-3,1-二基))二苯胺荧光染料。该染料在379 nm处吸收,在497 nm处产生强单发射,量子产率为0.375,并具有一定Stokes位移。中间体与染料经FT-IR、1H NMR、13C NMR和质谱表征。该吡唑双嘧啶荧光染料含有两个适合与蛋白结合的氨基。通过与牛血清白蛋白(BSA)偶联说明其作为生物相容性共轭物的应用。方法基于结合前后对荧光标记蛋白的直接荧光检测,纯化后的荧光共轭物经荧光光度法分析,表明碳二亚胺化学可生成染料-蛋白荧光共轭物。结论:所合成吡唑双嘧啶荧光团与BSA相互作用良好,可作为蛋白检测候选探针。
英文摘要
BACKGROUND: Fluorescent dyes with biocompatible functional group and good fluorescence behavior are used as biosensor for monitoring different biological processes as well as detection of protein assay. All reported fluorophore used as sensors are having high selectivity and sensitivity but till there is more demand to synthesized new fluorophore which have improved fluorescence properties and good biocompatibility.
RESULTS: Novel 4, 4'-(1, 1'-(5-(2-methoxyphenoxy)-[2, 2'-bipyrimidine]-4, 6-diyl)bis(1H-pyrazol-3, 1-diyl)) dianiline fluorescent dye was synthesized by multistep synthesis from 2-phenylacetonitrile, 2-chloropyrimidine and 2-methoxyphenol. This dye has absorption at 379 nm with intense single emission at 497 nm having fairly good quantum yield (0.375) and Stokes shift. The intermediates and dye were characterized by FT-IR, 1H NMR, 13C NMR and Mass spectral analysis. The pyrazole bipyrimidine based fluorescent dye possessing two amino groups suitable for binding with protein is reported. Its utility as a biocompatible conjugate was explained by conjugation with bovine serum albumin. The method is based on direct fluorescence detection of fluorophore-labelled protein before and after conjugation. Purified fluorescent conjugate was subsequently analyzed by fluorimetry. The analysis showed that the tested conjugation reaction yielded fluorescent conjugates of the dye through carbodiimide chemistry.
CONCLUSION: In summery synthesized fluorophore pyrazole-bipyrimidine has very good interaction towards protein bovine serum albumin and it acts as good candidate for protein assay.