基于FRET原理构建金纳米猝灭的荧光探针实现活细胞中葡萄糖的检测
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摘要
半导体纳米粒子或量子点具有独特的光物理学特点,比如量子点的发射波长可通过控制它的大小和组成来调谐,高的荧光量子产率,抗光漂白能力强等。正是由于这些优点,量子点被广泛的应用于生物传感中的荧光标记。将生物材料修饰在纳米颗粒的表面上就可以合成新型的多功能纳米生物复合物。纳米荧光探针作为一种新型的探针,它所特有的量子尺寸效应和小的颗粒尺寸使之呈现出许多与同质单个分子或大块物体不同的光学性质,已经被成功应用于生物样品检测及活体细胞成像中。最近,由蛋白质修饰的纳米颗粒组装的生物纳米传感器已经引起了广泛的关注,例如,半导体量子点已经被作为一种极好的材料用于酶反应的生物分析和应用当中。因此,设计具有良好性能的新型荧光纳米生物传感器用来实现生物活性分子或细胞内活性物质的分析和检测已经成为科学工作者的巨大挑战。
荧光共振能量转移(FRET)作为重要的光物理技术已广泛应用于生物大分子之间距离的定性、定量检测。荧光共振能量转移的效率决定于供体受体之间的距离及其光谱重叠程度。其中,能量给予者为供体(donor),荧光素量子产率高,是最常用的供体分子。能量接受者为受体(acceptor),受体可发射自己的特征荧光(荧光增强),也可作为猝灭剂不发荧光(荧光猝灭),不产生荧光的受体其优势在于能够减少可能由受体本身直接产生的荧光背景的干扰。
作为一种荧光猝灭剂,金纳米颗粒可以有地猝灭荧光团的荧光。纳米金具有比较宽的猝灭范围、对荧光试剂高的猝灭效率以及良好的稳定性,可以提高FRET过程的灵敏度和特异性,使其成为一个研究和应用的热点。与传统的有机猝灭剂相比,金纳米粒子具有特殊的结构特征和光学性质,低的毒性以及好的生物相容性,所以金纳米作为一种极好的猝灭剂已经开拓了高灵敏度检测生物活性分子的前景
葡萄糖是一种重要的生物活性物质,对细胞的健康生长起着非常重要的作用。葡萄糖的缺乏或过量都会对体内的新陈代谢产生不利的影响。目前有很多报道发现相对于正常细胞来说癌变细胞的代谢要消耗更多的葡萄糖。这其中的原因到目前为止还不是很清楚,但是癌细胞的糖代谢旺盛这一点是大家普遍认同的。目前,已经报道许多种有关葡萄糖含量的检测方法。其中,基于右旋糖酐、葡萄糖与葡萄糖氧化酶的竞争性结合的荧光检测方法由于其具有良好的选择性和较低的损伤度已经被广泛地应用于葡萄糖的检测当中。但是这些方法的检测限一般不高,而且毒性问题依然存在,限制了生物样品葡萄糖含量的直接检测,从而限制了细胞信号转换及细胞成像等方面的应用,所以设计一种稳定的高选择性的纳米荧光探针用于活细胞中葡萄糖的成像是非常有意义的。本文主要开展了以下两部分的研究工作:
(一)基于荧光共振能量转移原理(FRET)设计了一种简单的有效的荧光纳米探针用于葡萄糖的检测。探针的思路源于葡萄糖与apo-葡萄糖氧化酶(apo-GOx)是特异性结合的,其结合力大于右旋糖酐(Dextran)与apo-GOx的结合力。探针选择apo-GOx-Dextran作为发生FRET的载体,FITC-Dextran作为能量供体,apo-GOx修饰的金纳米颗粒(AuNPs)作为能量的受体。在体系中不存在葡萄糖的时候,由于apo-GOx与Dextran之间具有结合力,使得Dextran-FITC与AuNPs-apo-GOx相互靠近,满足能量共振转移的条件,在FITC的激发波长下激发时,FITC发射的荧光被金纳米粒子吸收,显示为荧光猝灭。当加入葡萄糖时,葡萄糖会与Dextran竞争与apo-GOx结合,使得FITC-Dextran与AuNPs-apo-GOx远离,能量转移消失,FITC的荧光信号恢复。实验结果显示在优化的实验条件下,荧光光谱分析表明荧光强度随葡萄糖浓度的逐步增大而成线性增强,线性范围为20 nM - 0.2μM。同时,该探针具有高的灵敏度和选择性,检测限为5 nM。细胞中存在的其它糖类和大多数生物物种不会对它的测定产生影响,该纳米探针成功应用于活细胞中葡萄糖的成像。
(二)基于两种不同尺度的金纳米构建的荧光探针实现活细胞中葡萄糖的检测。2 nm的巯基十一酸修饰的金纳米(LAuND)修饰上apo-GOx作为供体,10 nm的Dextran修饰的金纳米颗粒(Dex-Au-NP)作为受体。由于葡萄糖和Dextran与apo-GOx的竞争性结合,当葡萄糖存在时,葡萄糖与apo-GOx特异性结合竞争下来Dextran,导致LAuND荧光的恢复。根据荧光强度的变化来检测葡萄糖,进而实现活细胞中葡萄糖的成像。
Semiconductor nanoparticles (NPs) or quantum dots (QDs) have unique photophysical properties such as size-controlled fluorescence, have high fluorescence quantum yields, and stability against photobleaching, which offer significant advantages as optical labels for biosensing.The integration of nanoparticles with biomaterials yields novel hybrid nanobiomaterials of synergetic properties and functions.The new type of fluorescent nanoprobe with its special quantum size effects and small dimension effects exhibits many different optical characteristics compared to the homogeneous single-molecule or large object and has been successfully applied in the detection of biological samples and cell imaging. Recently, biological assembly of nanosensor with protein-modified nanoparticles applied in biosensing and biodetection has atrracted extensive attention. For example, QDs have been used as a wonderful material in enzyme-based biological analyses and applications. Therefore, the design of elegant new assembled nanobiosensor for realizing the analysis and determination of bioactive molecules in vivo or in vitro has become a great challenge to scientific workers.
Fluorescence resonance energy transfer (FRET) is one of the most powerful and widely used fluorescence technique available for probing structure and dynamics in media. The efficiency of FRET is dependent upon donor-acceptor proximity and spectral overlap. The most used donor is fluorescein due to its high quantum yield. The use of quenching acceptors is becoming increasingly popular in FRET systems, whether the acceptor partner is fluorescent or not.
As a photoluminescent quencher, gold nanoparticles (AuNPs) can ultra-efficiently quench the molecular-excitation energy in chromophore-AuNP composites. AuNPs have been of great interest because of their high extinction coefficient and a broad absorption spectrum in a visible light that is overlapped with the emission wavelength of usual energy donors. In comparison with the organic quencher, AuNPs have unique structural and optical properties, low toxicity, well biocompatibility for new applications in biosensing and molecular engineering.
Glucose, an important bioactive substance, plays a prominent role in the natural growth of cells. Its lack or excess can produce detrimental influence on cellular functions. Currently, there are many references reported cancer cells have increased rates of glucose metabolism relative to normal cells.The reason remains unclear, but that cancer cells metabolize glucose extensively is generally accepted.To date, many methods available for the glucose assays have been reported, among which,fluorescence-assay method based on a competitive binding reaction between glucose oxidase(GOD), Dextran and glucose, which has advantages in terms of good selectivity and nondestructive characteristics, has been extensively used for glucose detection. However, these methods were mostly confined to the toxicity, detection limit and could not determine glucose directly in biological samples, which restricted application in cellular signal transduction and cell imaging. Hence, a design of a selective and stable fluorescent probes for glucose detection in living cells is of special interest for biochemistry. We carried out two aspects of investigation:
First, a simple and effective nanoprobe based on FRET for specific detection of glucose was designed. Dextran-FITC were used as the donor, while AuNPs modified with apo-glucose oxidase (apo-GOx) acting as a quencher. The detection mechanism is based on the switching off FRET through the high specific recognition of apo-GOx to glucose. Evidences available indicated that apo-GOx is highly specific to glucose and a higher affinity of apo-GOx for glucose over Dextran. In the absence of glucose the binding of AuNPs-apo-GOx and FITC-Dextran resulted in a high FRET efficiency. In the presence of glucose, FITC-Dextran of the nanobprobe is displaced by glucose which competes with Dextran on the binding sites of apo-GOx, resulting in the fluorescence recovery of the quenched FITC. The results show that the linear range of this method is 20nM ~ 0.2μM with the detection limit as low as 5 nM, and has excellent selectivity for glucose over other sugars and most biological species present in living cells. The nanoprobe was successfully applied in cellular imaging.
Second, a new-typed nanoprobe with two gold nanoparticles of different sizes was designed for glucose detection.11-MUA modified gold nanodots (LAuND) were emplyed as donors and Dex-Au-NP as quencher. Based on the competitive combination between Dextran, glucose and apo-GOx, in the presence of glucose, Dextran is displaced by glucose which competes with Dextran on the binding sites of apo-GOx, resulting in the fluorescence recovery of quenched LAuND.
荧光共振能量转移(FRET)作为重要的光物理技术已广泛应用于生物大分子之间距离的定性、定量检测。荧光共振能量转移的效率决定于供体受体之间的距离及其光谱重叠程度。其中,能量给予者为供体(donor),荧光素量子产率高,是最常用的供体分子。能量接受者为受体(acceptor),受体可发射自己的特征荧光(荧光增强),也可作为猝灭剂不发荧光(荧光猝灭),不产生荧光的受体其优势在于能够减少可能由受体本身直接产生的荧光背景的干扰。
作为一种荧光猝灭剂,金纳米颗粒可以有地猝灭荧光团的荧光。纳米金具有比较宽的猝灭范围、对荧光试剂高的猝灭效率以及良好的稳定性,可以提高FRET过程的灵敏度和特异性,使其成为一个研究和应用的热点。与传统的有机猝灭剂相比,金纳米粒子具有特殊的结构特征和光学性质,低的毒性以及好的生物相容性,所以金纳米作为一种极好的猝灭剂已经开拓了高灵敏度检测生物活性分子的前景
葡萄糖是一种重要的生物活性物质,对细胞的健康生长起着非常重要的作用。葡萄糖的缺乏或过量都会对体内的新陈代谢产生不利的影响。目前有很多报道发现相对于正常细胞来说癌变细胞的代谢要消耗更多的葡萄糖。这其中的原因到目前为止还不是很清楚,但是癌细胞的糖代谢旺盛这一点是大家普遍认同的。目前,已经报道许多种有关葡萄糖含量的检测方法。其中,基于右旋糖酐、葡萄糖与葡萄糖氧化酶的竞争性结合的荧光检测方法由于其具有良好的选择性和较低的损伤度已经被广泛地应用于葡萄糖的检测当中。但是这些方法的检测限一般不高,而且毒性问题依然存在,限制了生物样品葡萄糖含量的直接检测,从而限制了细胞信号转换及细胞成像等方面的应用,所以设计一种稳定的高选择性的纳米荧光探针用于活细胞中葡萄糖的成像是非常有意义的。本文主要开展了以下两部分的研究工作:
(一)基于荧光共振能量转移原理(FRET)设计了一种简单的有效的荧光纳米探针用于葡萄糖的检测。探针的思路源于葡萄糖与apo-葡萄糖氧化酶(apo-GOx)是特异性结合的,其结合力大于右旋糖酐(Dextran)与apo-GOx的结合力。探针选择apo-GOx-Dextran作为发生FRET的载体,FITC-Dextran作为能量供体,apo-GOx修饰的金纳米颗粒(AuNPs)作为能量的受体。在体系中不存在葡萄糖的时候,由于apo-GOx与Dextran之间具有结合力,使得Dextran-FITC与AuNPs-apo-GOx相互靠近,满足能量共振转移的条件,在FITC的激发波长下激发时,FITC发射的荧光被金纳米粒子吸收,显示为荧光猝灭。当加入葡萄糖时,葡萄糖会与Dextran竞争与apo-GOx结合,使得FITC-Dextran与AuNPs-apo-GOx远离,能量转移消失,FITC的荧光信号恢复。实验结果显示在优化的实验条件下,荧光光谱分析表明荧光强度随葡萄糖浓度的逐步增大而成线性增强,线性范围为20 nM - 0.2μM。同时,该探针具有高的灵敏度和选择性,检测限为5 nM。细胞中存在的其它糖类和大多数生物物种不会对它的测定产生影响,该纳米探针成功应用于活细胞中葡萄糖的成像。
(二)基于两种不同尺度的金纳米构建的荧光探针实现活细胞中葡萄糖的检测。2 nm的巯基十一酸修饰的金纳米(LAuND)修饰上apo-GOx作为供体,10 nm的Dextran修饰的金纳米颗粒(Dex-Au-NP)作为受体。由于葡萄糖和Dextran与apo-GOx的竞争性结合,当葡萄糖存在时,葡萄糖与apo-GOx特异性结合竞争下来Dextran,导致LAuND荧光的恢复。根据荧光强度的变化来检测葡萄糖,进而实现活细胞中葡萄糖的成像。
Semiconductor nanoparticles (NPs) or quantum dots (QDs) have unique photophysical properties such as size-controlled fluorescence, have high fluorescence quantum yields, and stability against photobleaching, which offer significant advantages as optical labels for biosensing.The integration of nanoparticles with biomaterials yields novel hybrid nanobiomaterials of synergetic properties and functions.The new type of fluorescent nanoprobe with its special quantum size effects and small dimension effects exhibits many different optical characteristics compared to the homogeneous single-molecule or large object and has been successfully applied in the detection of biological samples and cell imaging. Recently, biological assembly of nanosensor with protein-modified nanoparticles applied in biosensing and biodetection has atrracted extensive attention. For example, QDs have been used as a wonderful material in enzyme-based biological analyses and applications. Therefore, the design of elegant new assembled nanobiosensor for realizing the analysis and determination of bioactive molecules in vivo or in vitro has become a great challenge to scientific workers.
Fluorescence resonance energy transfer (FRET) is one of the most powerful and widely used fluorescence technique available for probing structure and dynamics in media. The efficiency of FRET is dependent upon donor-acceptor proximity and spectral overlap. The most used donor is fluorescein due to its high quantum yield. The use of quenching acceptors is becoming increasingly popular in FRET systems, whether the acceptor partner is fluorescent or not.
As a photoluminescent quencher, gold nanoparticles (AuNPs) can ultra-efficiently quench the molecular-excitation energy in chromophore-AuNP composites. AuNPs have been of great interest because of their high extinction coefficient and a broad absorption spectrum in a visible light that is overlapped with the emission wavelength of usual energy donors. In comparison with the organic quencher, AuNPs have unique structural and optical properties, low toxicity, well biocompatibility for new applications in biosensing and molecular engineering.
Glucose, an important bioactive substance, plays a prominent role in the natural growth of cells. Its lack or excess can produce detrimental influence on cellular functions. Currently, there are many references reported cancer cells have increased rates of glucose metabolism relative to normal cells.The reason remains unclear, but that cancer cells metabolize glucose extensively is generally accepted.To date, many methods available for the glucose assays have been reported, among which,fluorescence-assay method based on a competitive binding reaction between glucose oxidase(GOD), Dextran and glucose, which has advantages in terms of good selectivity and nondestructive characteristics, has been extensively used for glucose detection. However, these methods were mostly confined to the toxicity, detection limit and could not determine glucose directly in biological samples, which restricted application in cellular signal transduction and cell imaging. Hence, a design of a selective and stable fluorescent probes for glucose detection in living cells is of special interest for biochemistry. We carried out two aspects of investigation:
First, a simple and effective nanoprobe based on FRET for specific detection of glucose was designed. Dextran-FITC were used as the donor, while AuNPs modified with apo-glucose oxidase (apo-GOx) acting as a quencher. The detection mechanism is based on the switching off FRET through the high specific recognition of apo-GOx to glucose. Evidences available indicated that apo-GOx is highly specific to glucose and a higher affinity of apo-GOx for glucose over Dextran. In the absence of glucose the binding of AuNPs-apo-GOx and FITC-Dextran resulted in a high FRET efficiency. In the presence of glucose, FITC-Dextran of the nanobprobe is displaced by glucose which competes with Dextran on the binding sites of apo-GOx, resulting in the fluorescence recovery of the quenched FITC. The results show that the linear range of this method is 20nM ~ 0.2μM with the detection limit as low as 5 nM, and has excellent selectivity for glucose over other sugars and most biological species present in living cells. The nanoprobe was successfully applied in cellular imaging.
Second, a new-typed nanoprobe with two gold nanoparticles of different sizes was designed for glucose detection.11-MUA modified gold nanodots (LAuND) were emplyed as donors and Dex-Au-NP as quencher. Based on the competitive combination between Dextran, glucose and apo-GOx, in the presence of glucose, Dextran is displaced by glucose which competes with Dextran on the binding sites of apo-GOx, resulting in the fluorescence recovery of quenched LAuND.
引文
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