基于CdTe的II型水溶性近红外量子点的合成及其分析应用
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摘要
量子点(quantum dots, QDs),亦被称为半导体纳米晶体,是一类具有优良光电性能的无机纳米材料,已在化学分析、荧光标记及光电器件等研究领域中展现出巨大的应用潜力。相对于可见光,近红外辐射(650-900nm)在生物组织中穿透性好、光化学损伤小,并且生物组织在该区域的散射、吸收和自发荧光背景都比较低,因此探索和开发新型近红外量子点体系及其相关传感技术已经引起了研究者极大的兴趣。但事实上,由于现阶段近红外量子点合成技术特别是水相直接制备高发光、高稳定性近红外量子点策略大多还处于经验探索阶段、近红外分析技术不够成熟及近红外光检测器的灵敏度低等因素,导致基于量子点近红外荧光、近红外电致化学发光(near-infrared electrogenerated chemiluminescence, NECL)的生物传感体系在生物分析、食品检测等领域中没有被广泛应用。
基于此,本论文以推动近红外分析技术发展为目标,探索水相近红外量子点合成新策略,在制备高发光、高稳定、低毒的水溶性近红外量子点基础上,以研究量子点NECL为重点,发展具有高灵敏、高选择性的新型近红外生物传感器。论文分五个部分,共六章,具体内容如下：
1.以巯基丙酸包裹的CdTe量子点为核的模板,氯化镉为镉源,巯基丙酸为壳层修饰剂,采用硼氢化钠还原亚硒酸钠在线产生Se2-的方法,在150℃水热条件下层层包裹制备了高质量、水溶性、Ⅱ型CdTe/CdSe核壳近红外量子点。与先前CdTe核量子点相比,制备得到的CdTe/CdSe核壳量子点发射波长发生了明显红移,通过调节CdSe壳的厚度,量子点的荧光发射可在620-740nm范围内任意调节,最高荧光量子产率达44.2%。利用高分辨透射电镜、紫外-可见吸收光谱、荧光光谱、X射线粉末衍射、X射线光电子能谱对所制备的量子点进行表征并测定其抗光漂白性能,发现该CdTe/CdSe核壳量子点具有较窄的尺寸分布、清晰的晶体结构及其极高的光稳定性,在生物应用中具有巨大的潜力。
2.以CdTe/CdS近红外量子点为核,以氯化锌为锌源,N-乙酰基-L-半胱氨酸(NAC)为硫源和稳定剂,四乙氧基硅烷为硅前体,借助微波辐射,采用Stober方法直接在水相中合成高发光、高稳定、低毒的NAC稳定的CdTe/CdS@ZnS-SiO2近红外量子点。这个方法不仅缩短了反应时间,增加了荧光强度及其稳定性,而且还减少了材料的细胞毒性。研究发现,Hg2+能通过电子转移过程有效地淬灭CdTe/CdS@ZnS-SiO2量子点的荧光。基于此,我们发展了一个简单的、快速的、特异性检测Hg2+的新方法。在最佳条件下,量子点的荧光强度与Hg2+在浓度为5.0×10-9-1.0x10-6mol/L之间成线性关系,检测限为1.0×10-9mol/L。该方法已经成功地用于奶粉样品中Hg2+的痕量检测。
3.我们系统研究了自行设计的CdTe/CdS/ZnS近红外量子点在裸金电极上的直接阴极NECL行为。实验发现CdTe/CdS/ZnS量子点在电位为-1.25V时(从-0.98V开始)具有强而稳定的NECL信号。这个NECL信号是通过电子注入的CdTe/CdS/ZnS量子点和还原态的过硫酸根离子(S2O82-)之间高效的电子转移反应实现的。相比于CdTe/CdS量子点,ZnS壳层的包裹使其NECL强度提高了9倍。进一步研究发现CdTe/CdS/ZnS量子点的ECL光谱与荧光光谱的最大发射峰位置相吻合,这表明ZnS壳层很好地钝化了量子点的表面缺陷。此外,我们还探讨了pH值、缓冲溶液、电极材料及其K2S2O8浓度对NECL强度的影响,并且阐述了可能的NECL机理。
4.借助信号放大的技术,构建了一个超灵敏、高选择性检测疾病标志物的NECL免疫传感器,在这个检测平台中,以CdTe/CdS小核厚壳近红外量子点为信号分子。通过羊抗人抗体(Ab2)与CdTe/CdS量子点标记的二氧化硅微球(SiO2)共价偶联制备了NECL纳米探针(SiO2-QD-Ab2)。通过超声诱导的自组装过程合成金纳米子/石墨烯(Au-GN)纳米杂合材料并且作为一个生物相容性的微环境用于初始抗体(Ab1)的固定。借助一个“三明治”免疫反应过程,功能化的SiO2-QD-Ab2纳米标签被捕获到电极表面。相对于没有放大的方法,结合Au-GN纳米杂合材料高效电子转移速率和SiO2-QD-Ab2多信号分子的双重放大优势,小核厚壳型的CdTe/CdS近红外量子点的NECL信号强度提高了16.8倍,并且成功实现了人免疫球蛋白G(HIgG)的超灵敏检测,检测限为87fg/mL。此外,构建的NECL免疫传感器已经成功用于血清中HIgG的定量分析。
5.以巯基丙酸修饰的小核厚壳型的CdTe/CdS近红外量子点为NECL能量供体,以金纳米棒(AuNRs)为NECL能量受体,发展了一个高灵敏、高选择检测凝血酶的近红外电致化学发光能量转移(NERET)适配体传感器。通过一个在盐溶液中自然老化的过程,探针DNA修饰的AuNRs (pDNA-AuNRs)被制备,它纵轴吸收峰的位置能够和CdTe/CdS量子点薄膜发出的ECL光谱重合。系统研究了CdTe/CdS量子点与AuNRs两者之间的光谱重叠程度、距离及其杂交时间等因素对NERET猝灭效率的影响。凝血酶的检测通过一个竞争反应实现,凝血酶会取代事先在电极表面发生杂交反应的pDNA-AuNRs,使得量子点的NECL信号得到恢复,从而达到检测凝血酶浓度的目的。在最佳条件下,构建的NERET传感器能够在1.0×10-16-1.0×10-14mol/L浓度范围内对凝血酶进行特异性测定,检测限为2x10-17mol/L。
Quantum dots (QDs), also called semiconductor nanocrystals, are a kind of inorganic nanomaterials with excellent photoelectric properties. In the past few years, QDs have attracted broad attention in the fields of chemical analysis, fluorescence labeling and photoelectric device. Compared to visible light, near-infrared (NIR) emission (650-900nm) offers several unique advantages, including minimal interferential absorption, low biological autofluorescence, and high tissue penetration. Therefore, the exploration and development of new NIR QDs and ralted biosensing methodologies has caused great interest to researchers. But in fact, QD-based NIR fluorescence and NIR electrogenerated chemiluminescence (NECL) biosensors have not been widely used in the fields of biological analysis and food monitoring, due to the relatively primitive synthetic techniques of NIR QDs especially the poor strategies for direct preparation of highly luminescent and stable aqueous NIR QDs, the less mature NIR analytical technique and the low sensitive of detectors towards NIR emission.
Based on this, the main propose of this dissertation is to promote the development of NIR analytical technique. To develop new strategies for the synthesis of aqueous NIR QDs, series of highly luminescent, good stability and low toxic are prepared. The key points are to study the NECL behaviours of NIR QDs and develop highly sensitive and selective NIR biosensors. This dissertation is divided into five parts, a total of six chapters. The specific content is as follow:
1. A simple hydrothermal method is developed for the synthesis of high-quality, water-soluble, and NIR type-II core/shell CdTe/CdSe QDs by employing thiol-capped CdTe QDs as core templates and CdCl2and Na2SeO3as shell precursors. Compared with the original CdTe core QDs, the core/shell CdTe/CdSe QDs exhibit an obvious red-shifted emission, whose color can be tuned between visible and NIR regions (620-740nm) by controlling the thickness of the CdSe shell. The photoluminescence quantum yield (PL QY) of CdTe/CdSe QDs with an optimized thickness of the CdSe shell can reach up to44.2%without any post-preparative treatment. Through a thorough study of the core/shell structure by high-resolution transmission electron microscopy (HRTEM), ultraviolet-visible (UV-vis) absorption spectra, fluorescence spectra, X-ray powder diffraction (XRD) and X-ray photoelectron spectroscopy (XPS), the as-prepared CdTe/CdSe QDs demonstrate good monodispersity, hardened lattice structure and excellent photostability, offering a great potential for biological application.
2. Highly luminescent, good stability and low toxic N-acetyl-L-cysteine (NAC) capped CdTe/CdS@ZnS-SiO2NIR QDs were successfully via a promising microwave strategy, which employed thiol-capped CdTe/CdS QDs as core templates and ZnCl2, NAC and TEOS as shell precursors. This proposed method can greatly shorted reaction time and increase fluorescence intensity and stability, as well as reduce the cytotoxicity. It was found that Hg2+could effectively selective quench the QD emission based on electron transfer process. On the basis of this fact, a simple, rapid and specific method for Hg2+determination was porposed. Under optimal conditions, the fluorescence intensity decreased linearly with the concentration of Hg2+ranging from5.0x10-9to1.0x10"6mol/L. The limit of detection for Hg2+was1.0x10-9mol/L. The developed method was successfully applied to the detection of trace Hg2+in milk power.
3. Cathodic electrochemiluminescence (ECL) from self-designed NIR CdTe/CdS/ZnS QDs on bare Au electrode in aqueous solution was studied. Strong and stable NECL signals at-1.25V with an onset potential of-0.98V produced by the high effectively electron-transfer reaction between electron-injected CdTe/CdS/ZnS QDs and reduced S2O82-are observed. Passivation of the QD surfaces with ZnS shell increased NECL intensity by9times when compared to CdTe/CdS QDs. The good correspondence of the ECL emission peak with the photoluminescence results suggested the surface states of the QDs had been largely passivated by ZnS shell. Furthermore, the effects of pH, buffer solutions, electrode materials and concentrations of K2S2O8on NECL intensity were investigated, and a possible NECL mechanism was also proposed.
4. We reported a NECL immunosensor with amplification techniques for ultrasensitive and selective determination of biomarker. In this sensing platform, CdTe/CdS coresmall/shellthick NIR QDs were first selected as NECL emitters. The NECL nanoprobe (SiO2-QD-Ab2) was designed by covalent assembly of goat anti-human IgG antibody (Ab2) on CdTe/CdS QDs tagged silica nanospheres. Gold nanoparticle-graphene nanosheet (Au-GN) hybrids were prepared by a sonication-induced self-assembly and served as an effective matrix for initial antibodies (Abl) attachment. After a sandwich immunoreaction, the functionalized silica nanosphere labels were captured onto the glass carbon electrode surface. Integrating the dual amplification from the promoting electron transfer rate of Au-GN hybrids and the increasing QD loading of SiO2-QD-Ab2labels, the NECL response from CdTe/CdS QDs enhanced16.8-fold compared to the unamplified protocol and successfully fulfilled the ultrasensitive detection of human IgG (HIgG) with a detection limit of87fg/mL. Moreover, as a practical application, the proposed immunosensor was used to monitor HIgG level in human serum with satisfactory results obtained.
5. We reported a near-infrared electrochemiluminescence resonance energy transfer (NERET) aptasensor for ultrasensitive and selective determination of thrombin, where CdTe/CdS coresmall/shellthick NIR QDs and Au nanorods (AuNRs) were used as a NECL donor and a NECL acceptor, respectively. Probing DNA modified AuNRs (pDNA-AuNRs) was prepared by a salt aging process, whose longitudinal absorption peaks that are easily tuned to match well with the ECL emission spectrum of the CdTe/CdS QDs film. The effect of degree of overlapping spectra, distance, and hybridization time was studied for the quenching efficiency of NERET between the CdTe/CdS QDs film and AuNRs. The protein detection involves a competition binding event, based on thrombin replacing pDNA-AuNRs which is hybridized with capturing aptamer immobilized on a chitosan/CdTe/CdS QDs film modified glass carbon electrode. At the optimized conditions, the NERET system could be used to ultrasensitivly and specifically detect thrombin of the concentration ranging from1.0x10-16to1.0x10-14mol/L. The detection limit was2x10-17mol/L.
基于此,本论文以推动近红外分析技术发展为目标,探索水相近红外量子点合成新策略,在制备高发光、高稳定、低毒的水溶性近红外量子点基础上,以研究量子点NECL为重点,发展具有高灵敏、高选择性的新型近红外生物传感器。论文分五个部分,共六章,具体内容如下：
1.以巯基丙酸包裹的CdTe量子点为核的模板,氯化镉为镉源,巯基丙酸为壳层修饰剂,采用硼氢化钠还原亚硒酸钠在线产生Se2-的方法,在150℃水热条件下层层包裹制备了高质量、水溶性、Ⅱ型CdTe/CdSe核壳近红外量子点。与先前CdTe核量子点相比,制备得到的CdTe/CdSe核壳量子点发射波长发生了明显红移,通过调节CdSe壳的厚度,量子点的荧光发射可在620-740nm范围内任意调节,最高荧光量子产率达44.2%。利用高分辨透射电镜、紫外-可见吸收光谱、荧光光谱、X射线粉末衍射、X射线光电子能谱对所制备的量子点进行表征并测定其抗光漂白性能,发现该CdTe/CdSe核壳量子点具有较窄的尺寸分布、清晰的晶体结构及其极高的光稳定性,在生物应用中具有巨大的潜力。
2.以CdTe/CdS近红外量子点为核,以氯化锌为锌源,N-乙酰基-L-半胱氨酸(NAC)为硫源和稳定剂,四乙氧基硅烷为硅前体,借助微波辐射,采用Stober方法直接在水相中合成高发光、高稳定、低毒的NAC稳定的CdTe/CdS@ZnS-SiO2近红外量子点。这个方法不仅缩短了反应时间,增加了荧光强度及其稳定性,而且还减少了材料的细胞毒性。研究发现,Hg2+能通过电子转移过程有效地淬灭CdTe/CdS@ZnS-SiO2量子点的荧光。基于此,我们发展了一个简单的、快速的、特异性检测Hg2+的新方法。在最佳条件下,量子点的荧光强度与Hg2+在浓度为5.0×10-9-1.0x10-6mol/L之间成线性关系,检测限为1.0×10-9mol/L。该方法已经成功地用于奶粉样品中Hg2+的痕量检测。
3.我们系统研究了自行设计的CdTe/CdS/ZnS近红外量子点在裸金电极上的直接阴极NECL行为。实验发现CdTe/CdS/ZnS量子点在电位为-1.25V时(从-0.98V开始)具有强而稳定的NECL信号。这个NECL信号是通过电子注入的CdTe/CdS/ZnS量子点和还原态的过硫酸根离子(S2O82-)之间高效的电子转移反应实现的。相比于CdTe/CdS量子点,ZnS壳层的包裹使其NECL强度提高了9倍。进一步研究发现CdTe/CdS/ZnS量子点的ECL光谱与荧光光谱的最大发射峰位置相吻合,这表明ZnS壳层很好地钝化了量子点的表面缺陷。此外,我们还探讨了pH值、缓冲溶液、电极材料及其K2S2O8浓度对NECL强度的影响,并且阐述了可能的NECL机理。
4.借助信号放大的技术,构建了一个超灵敏、高选择性检测疾病标志物的NECL免疫传感器,在这个检测平台中,以CdTe/CdS小核厚壳近红外量子点为信号分子。通过羊抗人抗体(Ab2)与CdTe/CdS量子点标记的二氧化硅微球(SiO2)共价偶联制备了NECL纳米探针(SiO2-QD-Ab2)。通过超声诱导的自组装过程合成金纳米子/石墨烯(Au-GN)纳米杂合材料并且作为一个生物相容性的微环境用于初始抗体(Ab1)的固定。借助一个“三明治”免疫反应过程,功能化的SiO2-QD-Ab2纳米标签被捕获到电极表面。相对于没有放大的方法,结合Au-GN纳米杂合材料高效电子转移速率和SiO2-QD-Ab2多信号分子的双重放大优势,小核厚壳型的CdTe/CdS近红外量子点的NECL信号强度提高了16.8倍,并且成功实现了人免疫球蛋白G(HIgG)的超灵敏检测,检测限为87fg/mL。此外,构建的NECL免疫传感器已经成功用于血清中HIgG的定量分析。
5.以巯基丙酸修饰的小核厚壳型的CdTe/CdS近红外量子点为NECL能量供体,以金纳米棒(AuNRs)为NECL能量受体,发展了一个高灵敏、高选择检测凝血酶的近红外电致化学发光能量转移(NERET)适配体传感器。通过一个在盐溶液中自然老化的过程,探针DNA修饰的AuNRs (pDNA-AuNRs)被制备,它纵轴吸收峰的位置能够和CdTe/CdS量子点薄膜发出的ECL光谱重合。系统研究了CdTe/CdS量子点与AuNRs两者之间的光谱重叠程度、距离及其杂交时间等因素对NERET猝灭效率的影响。凝血酶的检测通过一个竞争反应实现,凝血酶会取代事先在电极表面发生杂交反应的pDNA-AuNRs,使得量子点的NECL信号得到恢复,从而达到检测凝血酶浓度的目的。在最佳条件下,构建的NERET传感器能够在1.0×10-16-1.0×10-14mol/L浓度范围内对凝血酶进行特异性测定,检测限为2x10-17mol/L。
Quantum dots (QDs), also called semiconductor nanocrystals, are a kind of inorganic nanomaterials with excellent photoelectric properties. In the past few years, QDs have attracted broad attention in the fields of chemical analysis, fluorescence labeling and photoelectric device. Compared to visible light, near-infrared (NIR) emission (650-900nm) offers several unique advantages, including minimal interferential absorption, low biological autofluorescence, and high tissue penetration. Therefore, the exploration and development of new NIR QDs and ralted biosensing methodologies has caused great interest to researchers. But in fact, QD-based NIR fluorescence and NIR electrogenerated chemiluminescence (NECL) biosensors have not been widely used in the fields of biological analysis and food monitoring, due to the relatively primitive synthetic techniques of NIR QDs especially the poor strategies for direct preparation of highly luminescent and stable aqueous NIR QDs, the less mature NIR analytical technique and the low sensitive of detectors towards NIR emission.
Based on this, the main propose of this dissertation is to promote the development of NIR analytical technique. To develop new strategies for the synthesis of aqueous NIR QDs, series of highly luminescent, good stability and low toxic are prepared. The key points are to study the NECL behaviours of NIR QDs and develop highly sensitive and selective NIR biosensors. This dissertation is divided into five parts, a total of six chapters. The specific content is as follow:
1. A simple hydrothermal method is developed for the synthesis of high-quality, water-soluble, and NIR type-II core/shell CdTe/CdSe QDs by employing thiol-capped CdTe QDs as core templates and CdCl2and Na2SeO3as shell precursors. Compared with the original CdTe core QDs, the core/shell CdTe/CdSe QDs exhibit an obvious red-shifted emission, whose color can be tuned between visible and NIR regions (620-740nm) by controlling the thickness of the CdSe shell. The photoluminescence quantum yield (PL QY) of CdTe/CdSe QDs with an optimized thickness of the CdSe shell can reach up to44.2%without any post-preparative treatment. Through a thorough study of the core/shell structure by high-resolution transmission electron microscopy (HRTEM), ultraviolet-visible (UV-vis) absorption spectra, fluorescence spectra, X-ray powder diffraction (XRD) and X-ray photoelectron spectroscopy (XPS), the as-prepared CdTe/CdSe QDs demonstrate good monodispersity, hardened lattice structure and excellent photostability, offering a great potential for biological application.
2. Highly luminescent, good stability and low toxic N-acetyl-L-cysteine (NAC) capped CdTe/CdS@ZnS-SiO2NIR QDs were successfully via a promising microwave strategy, which employed thiol-capped CdTe/CdS QDs as core templates and ZnCl2, NAC and TEOS as shell precursors. This proposed method can greatly shorted reaction time and increase fluorescence intensity and stability, as well as reduce the cytotoxicity. It was found that Hg2+could effectively selective quench the QD emission based on electron transfer process. On the basis of this fact, a simple, rapid and specific method for Hg2+determination was porposed. Under optimal conditions, the fluorescence intensity decreased linearly with the concentration of Hg2+ranging from5.0x10-9to1.0x10"6mol/L. The limit of detection for Hg2+was1.0x10-9mol/L. The developed method was successfully applied to the detection of trace Hg2+in milk power.
3. Cathodic electrochemiluminescence (ECL) from self-designed NIR CdTe/CdS/ZnS QDs on bare Au electrode in aqueous solution was studied. Strong and stable NECL signals at-1.25V with an onset potential of-0.98V produced by the high effectively electron-transfer reaction between electron-injected CdTe/CdS/ZnS QDs and reduced S2O82-are observed. Passivation of the QD surfaces with ZnS shell increased NECL intensity by9times when compared to CdTe/CdS QDs. The good correspondence of the ECL emission peak with the photoluminescence results suggested the surface states of the QDs had been largely passivated by ZnS shell. Furthermore, the effects of pH, buffer solutions, electrode materials and concentrations of K2S2O8on NECL intensity were investigated, and a possible NECL mechanism was also proposed.
4. We reported a NECL immunosensor with amplification techniques for ultrasensitive and selective determination of biomarker. In this sensing platform, CdTe/CdS coresmall/shellthick NIR QDs were first selected as NECL emitters. The NECL nanoprobe (SiO2-QD-Ab2) was designed by covalent assembly of goat anti-human IgG antibody (Ab2) on CdTe/CdS QDs tagged silica nanospheres. Gold nanoparticle-graphene nanosheet (Au-GN) hybrids were prepared by a sonication-induced self-assembly and served as an effective matrix for initial antibodies (Abl) attachment. After a sandwich immunoreaction, the functionalized silica nanosphere labels were captured onto the glass carbon electrode surface. Integrating the dual amplification from the promoting electron transfer rate of Au-GN hybrids and the increasing QD loading of SiO2-QD-Ab2labels, the NECL response from CdTe/CdS QDs enhanced16.8-fold compared to the unamplified protocol and successfully fulfilled the ultrasensitive detection of human IgG (HIgG) with a detection limit of87fg/mL. Moreover, as a practical application, the proposed immunosensor was used to monitor HIgG level in human serum with satisfactory results obtained.
5. We reported a near-infrared electrochemiluminescence resonance energy transfer (NERET) aptasensor for ultrasensitive and selective determination of thrombin, where CdTe/CdS coresmall/shellthick NIR QDs and Au nanorods (AuNRs) were used as a NECL donor and a NECL acceptor, respectively. Probing DNA modified AuNRs (pDNA-AuNRs) was prepared by a salt aging process, whose longitudinal absorption peaks that are easily tuned to match well with the ECL emission spectrum of the CdTe/CdS QDs film. The effect of degree of overlapping spectra, distance, and hybridization time was studied for the quenching efficiency of NERET between the CdTe/CdS QDs film and AuNRs. The protein detection involves a competition binding event, based on thrombin replacing pDNA-AuNRs which is hybridized with capturing aptamer immobilized on a chitosan/CdTe/CdS QDs film modified glass carbon electrode. At the optimized conditions, the NERET system could be used to ultrasensitivly and specifically detect thrombin of the concentration ranging from1.0x10-16to1.0x10-14mol/L. The detection limit was2x10-17mol/L.
引文
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