金属纳米诱导的化学发光体系的设计及在免疫分析中的应用
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摘要
论文首先综述了化学发光、化学发光免疫分析、金属纳米材料的制备和性质及其在免疫分析中的应用的研究现状。虽然化学发光的基础理论和免疫分析应用有着多年的研究历史,但一直一来,有关化学发光的研究局限于分子和离子水平。最近,金属纳米粒子直接或者间接参与的化学发光受到了研究者的关注。已经发现金属纳米粒子能够作为化学发光反应的催化剂、还原剂、微尺度反应平台和能量接受体参与化学发光。但是,目前已经开发的纳米化学发光体系仍然十分有限,其中大部分体系的发光量子产率较低,很难应用于分析测定。同时,有关纳米粒子可控的化学发光反应的研究还鲜见报道。另一方面,随着纳米化学发光的研究发展,基于金属纳米标记的化学发光免疫分析开始起步。目前的报道仍局限于“溶出”化学发光免疫分析方法。它需要苛刻的条件溶解金属纳米,方法复杂,难以在实际中获得应用。而直接利用金属纳米催化化学发光反应的非溶出化学发光免疫分析的报道却很少见。基于此,本论文以纳米金和纳米铂为研究对象,从纳米的粒径大小、分散介质两个角度,设计了两个纳米化学发光新体系,探索了这些体系的化学发光行为、规律和机理,详细研究了纳米材料对化学发光体系的调控作用。同时,基于纳米金的化学发光特性,研究了其在非溶出化学发光免疫分析中的应用。主要研究内容如下:
1.发现小粒径纳米金(直径小于5nm)能够抑制鲁米诺—铁氰化钾体系的化学发光;而大粒径纳米金(直径大于10nm)能够增强该体系的化学发光,其中直径为25 nm的纳米金对体系的化学发光具有最大的增强作用。分析了其化学发光光谱,发现其发光体仍然为激发态的3-氨基邻苯二甲酸根离子(AP~(2-))。研究了反应前后的紫外—可见吸收光谱和X-射线光电子能谱(XPS)、鲁米诺和铁氰化钾浓度对化学发光的影响以及纳米金溶胶的荧光猝灭效率,提出了化学发光增强和抑制作用的可能机理。推测小粒径纳米金对化学发光的抑制作用可能源于其竞争消耗铁氰化钾以及对发光体AP~(2-)具有相对较高的猝灭效应。相反地,大粒径纳米金对该体系化学发光的增强作用很可能是由于纳米金对鲁米诺化学发光反应中的电子转移过程具有催化作用,同时大粒径纳米金对发光体AP~(2-)的猝灭作用相对较弱。纳米金对化学发光反应的增强和抑制作用与其粒径大小具有明显依赖性,为开发新型纳米化学发光增强剂和抑制剂提供了一个新的研究思路。
2.发现纳米铂和乙醇同时存在时能够诱导碱性光泽精溶液产生延迟强化学发光现象。化学发光的“诱导期”与注射的纳米铂浓度具有明显的相关性。从注射到出现最大化学发光强度之间的时间(T_p)与纳米铂浓度具有很好的指数关系:而最大化学发光强度与纳米铂浓度之间存在良好的线性关系。利用紫外—可见吸收光谱、荧光光谱和化学发光光谱鉴定了光泽精的反应产物和发光体,发现体系的发光体为激发态的N-甲基吖啶酮(NMA)。实验还对化学发光反应过程中,光泽精及其产物NMA的浓度变化进行了实时检测。结果表明纳米铂和乙醇参与的光泽精化学发光反应是一个自催化反应。采用透射电子显微镜、XPS和核磁共振光谱(NMR)等表征手段对自催化化学发光机理进行了研究。结果表明,纳米铂在体系中作为一个全局催化剂,不仅催化乙醇氧化为乙醛,同时还催化乙醛对光泽精化学发光的增强作用;而乙醇是纳米铂催化反应的一个引发剂。与乙醇类似,甲醇、乙二醇和正丙醇也能与纳米铂共同作用产生光泽精自催化化学发光。这是首次在纳米参与的化学发光反应中观察到自催化现象。该体系的强光发射有望进一步用于生物分析和“冷光源”的构造。
3.基于鲁米诺—硝酸银—纳米金化学发光体系,建立了一种微孔板化学发光免疫分析方法测定人免疫球蛋白G(IgG)。采用聚苯乙烯微孔板同时作为免疫反应的固相载体和化学发光检测容器。一抗(羊抗人IgG)首先包被在微孔板中,然后将抗原(人IgG)和纳米金标记的二抗先后结合到微孔板中,形成夹心式免疫复合物。标记的纳米金能够诱导鲁米诺和硝酸银之间的反应,产生光发射。通过凝絮实验确定形成稳定的纳米金—蛋白质生物复合物所需的最小抗体浓度。实验还对一些化学发光检测条件进行了优化,包括鲁米诺溶液pH、鲁米诺和硝酸银溶液浓度等。在优化的条件下,体系的化学发光强度与人IgG浓度的对数之间存在良好的线性关系,线性范围为25～5000 ng/mL,按三倍信噪比(S/N=3)计算检测限为12.8 ng/mL(～80 pM)。与其它报道的金标化学发光免疫分析方法相比,该方法避免了苛刻的溶出步骤以及难以控制的合成过程,使得方法更简单、省时、易于自动化。该化学发光免疫分析方法可望用于检测临床重要的生物活性物质。
4.建立了一种基于金标银染的非溶出微孔板化学发光免疫分析方法测定人IgG。首先将一抗(羊抗人IgG)固载到聚苯乙烯微孔板中,然后相继结合抗原(人IgG)和金标二抗,形成夹心式免疫复合物。标记的纳米金能够催化对苯二酚还原乙酸银为单质银,并沉积在纳米金表面,之后直接采用鲁米诺—过氧化氢化学发光体系进行检测。实验详细研究了银染时间对化学发光响应的影响。对于不同的银染时间,可以分别获得增强和抑制化学发光响应。在此基础上,初步建立了增强和抑制两种模式的化学发光免疫分析。实验分别对增强模式和抑制模式下的化学发光条件进行了优化,包括鲁米诺的缓冲介质、pH以及鲁米诺和过氧化氢的浓度。在优化的条件下,增强模式的检测限为0.9 ng/mL;抑制模式的检测限为5.6 ng/mL。增强模式的灵敏度高于其它报道的非溶出化学发光免疫分析方法。本项工作首次将金标银染技术用于建立非溶出化学发光免疫分析方法,结果表明银染有利于提高分析的灵敏度。该方法避免了苛刻的溶出过程,以及不规则纳米金的合成。但是,目前这种方法仍有一些不足之处,如线性范围过窄、灵敏度和稳定性仍有待提高。
In this dissertation,the state of arts in the field of chemiluminescence(CL), chemiluminescence immunoassays,the synthesis and properties of metal nano-materials and their applications in immunoassays were reviewed.Although the theory of CL and its application in immunoassay have been investigated for many years,the study of CL was limited to molecular and ion systems.Recently,metal nanoparticle-involved CL has attracted great attentions,in which metal nanoparticles can participate in CL reactions as catalyst,reductant,naonosized platform and energy acceptor.However,most of the reported CL systems involving nanoparticles were of low quantum yield,limiting there applications in analysis.Meanwhile,the study of nanoparticle-controlled CL system has been rarely reported.On the other hand,with the advance of nanoparticle-involved CL,chemiluminescence immunoassays based on metal nanoparticle labels were developed.Most of these reported chemiluminescence immunoassays contained "stripping" process,in which strict conditions were required to dissolve metal nanoparticles,making the method complicated,limiting there practical application.There were few reports about non-stripping chemiluminescence immunoassays based on metal nanoparticle-catalyzed CL reactions.Therefore,in the present dissertation,gold and platinum nanoparticles were utilized to design new CL systems.The nanoparticle-dependent or-controlled CL behaviors,rules and mechanisms of these new CL systems were investigated.Moreover,based on the CL activity of gold nanoparticles,new methods for non-stripping chemiluminescence immunoassays were developed.The main results are as follows:
1.It was found that gold nanoparticles of small size(＜5 nm) could inhibit the chemiluminescence(CL) of the luminol-ferricyanide system,whereas gold nanoparticles of large size(＞10 nm) could enhance this CL,and the most intensive CL signals were obtained with 25-nm-diameter gold nanoparticles.The luminophor was identified as the excited-state 3-aminophthalate anion(Ap~(2-)).The studies of UV-visible spectra,CL spectra,X-ray photoelectron spectra(XPS),effects of concentrations of luminol and ferricyanide solution,and fluorescence quenching efficiency of gold colloids were carried out to explore the CL inhibition and enhancement mechanism.The CL inhibition by gold nanoparticles of small size was supposed to originate from the competitive consumption of ferricyanide by gold nanoparticles and the relatively high quenching efficiency of the luminophor by gold nanoparticles.In contrast,the CL enhancement by gold nanoparticles of large size was ascribed to the catalysis of gold nanoparticles in the electron-transfer process during the luminol CL reaction and the relatively low quenching efficiency of the luminophor by gold nanoparticles.This work demonstrates that gold nanoparticles have the size-dependent inhibition and enhancement in the CL reaction,proposing a perspective for the investigation of new and efficient nanosized inhibitors and enhancers in CL reactions for analytical purposes.
2.An intensive and delayed CL could be observed from the alkaline lucigenin solution in the presence of ethanol and Pt nanopartieles simultaneously.The "induction" period of the CL was largely dependent on the concentration of Pt nanoparticles.The time from injection to the occurrence of the maximum CL intensity, which defined as T_p here,was exponentially related to the concentration of Pt nanoparticles;while the maximum CL intensity was linear with Pt nanoparticles concentration.The products and luminophor of lucigenin CL reaction were characterized by UV-visible spectra,fluorescence spectra and CL spectra.The luminophor of this system was identified as the excited-state N-methylacridone (NMA).The variation of the concentration of lucigenin and its CL product NMA during the process was monitored in situ.The results showed that the reaction of lucigenin and NaOH involving Pt nanoparticles and ethanol was autocatalytic.The studies of transmission electron microscope(TEM),XPS and nuclear magnetic resonance(NMR) were carried out to explore the mechanism of the autocatalytic CL reaction.The results suggested that Pt nanoparticles acted as a "general" catalyst in the CL system that not only catalyzed the oxidation of ethanol to acetaldehyde,but also catalyzed the CL enhancement by acetaldehyde.Ethanol in this system was the initiator for catalysis.Similar to ethanol,other alcohols such as methanol,ethylene glycol and n-propanol could also initiate autocatalytic lucigenin CL in the presence of Pt nanoparticles.This is the first time autocatalytic light emission has been obtained from nanoparticles-involved CL reactions.The strong light emission in this system may find future applications in bioanalysis and fabrication of"cold light sources".
3.A novel microplate-compatible chemiluminescence immunoassay has been developed for the determination of human immunoglobulin G(IgG) based on the luminol-AgNO_3-gold nanoparticles CL system.Polystyrene microtiter plates were used for both immunoreactions and CL measurements.The primary antibody, goat-anti-human IgG,was first immobilized on polystyrene microwells.Then the antigen(human IgG) and the gold-labeled second antibody were connected to the microwells successively to form a sandwich-type immunocomplex.The gold label could trigger the reaction between luminol and AgNO_3,accompanied by light emission.A flocculation test was carried out to determine the minimum amount of goat-anti-human IgG required to form stable nanoparticle-protein bioconjugate.Some experimental parameters influencing the CL reaction,including luminol pH and the concentration of luminol and AgNO_3,were optimized.Under the optimized conditions, the CL intensity of the system was linear with the logarithm of the concentration of human IgG in the range from 25 to 5000 ng/mL,with a detection limit of 12.8 ng/mL (～80 pM) at a signal to noise ratio of three(S/N = 3).Compared with other reported CL immunoassay method based on gold labels,the proposed CL protocol avoids a strict stripping procedure or difficult to control synthesis processes,making the method more simple,time-saving and easily automated.The present CL method is promising for the determination of clinically important bioactive analytes.
4.A new non-stripping and microplate-compatible chemiluminescence immunoassay protocol based on gold label with silver staining has been developed for the determination of human IgG.The primary antibody,goat-anti-human IgG,was first immobilized on polystyrene microwells,and then the antigen(human IgG) and the gold-labeled second antibody were captured onto the microwells successively to form a sandwich-type immunocomplex.The gold label could catalyze the reduction of silver acetate by hydroquinone,leading to the deposition of silver on the gold nanoparticles,followed by direct CL detection using luminol-H_2O_2 system.The effect of staining time on the CL response was carefully studied.With different staining time, both enhanced and inhibited CL detention modes were established for the CL immunoassay.Some CL conditions in both modes,including the buffer media of luminol,luminol pH and the concentration of luminol and H_2O_2,were optimized. Under the optimized conditions,the detection limit of this CL method(S/N = 3) was 0.9 ng/mL for enhanced mode,and 5.6 ng/mL for inhibited mode,respectively.The sensitivity of the enhanced mode was higher than other reported non-stripping CL immunoassay methods.This work revealed that the staining of silver on the gold nanoparticles was useful to improve the sensitivity.This is the first time to imply gold-label-silver-staining technology to develop non-stripping CL immunoassays.The proposed CL protocol avoids strict stripping procedure or the synthesis of irregular gold nanoparticles.However,there are still some disadvantages,such as the narrow linear range,and the further improvement required for the sensitivity and the stability.
1.发现小粒径纳米金(直径小于5nm)能够抑制鲁米诺—铁氰化钾体系的化学发光;而大粒径纳米金(直径大于10nm)能够增强该体系的化学发光,其中直径为25 nm的纳米金对体系的化学发光具有最大的增强作用。分析了其化学发光光谱,发现其发光体仍然为激发态的3-氨基邻苯二甲酸根离子(AP~(2-))。研究了反应前后的紫外—可见吸收光谱和X-射线光电子能谱(XPS)、鲁米诺和铁氰化钾浓度对化学发光的影响以及纳米金溶胶的荧光猝灭效率,提出了化学发光增强和抑制作用的可能机理。推测小粒径纳米金对化学发光的抑制作用可能源于其竞争消耗铁氰化钾以及对发光体AP~(2-)具有相对较高的猝灭效应。相反地,大粒径纳米金对该体系化学发光的增强作用很可能是由于纳米金对鲁米诺化学发光反应中的电子转移过程具有催化作用,同时大粒径纳米金对发光体AP~(2-)的猝灭作用相对较弱。纳米金对化学发光反应的增强和抑制作用与其粒径大小具有明显依赖性,为开发新型纳米化学发光增强剂和抑制剂提供了一个新的研究思路。
2.发现纳米铂和乙醇同时存在时能够诱导碱性光泽精溶液产生延迟强化学发光现象。化学发光的“诱导期”与注射的纳米铂浓度具有明显的相关性。从注射到出现最大化学发光强度之间的时间(T_p)与纳米铂浓度具有很好的指数关系:而最大化学发光强度与纳米铂浓度之间存在良好的线性关系。利用紫外—可见吸收光谱、荧光光谱和化学发光光谱鉴定了光泽精的反应产物和发光体,发现体系的发光体为激发态的N-甲基吖啶酮(NMA)。实验还对化学发光反应过程中,光泽精及其产物NMA的浓度变化进行了实时检测。结果表明纳米铂和乙醇参与的光泽精化学发光反应是一个自催化反应。采用透射电子显微镜、XPS和核磁共振光谱(NMR)等表征手段对自催化化学发光机理进行了研究。结果表明,纳米铂在体系中作为一个全局催化剂,不仅催化乙醇氧化为乙醛,同时还催化乙醛对光泽精化学发光的增强作用;而乙醇是纳米铂催化反应的一个引发剂。与乙醇类似,甲醇、乙二醇和正丙醇也能与纳米铂共同作用产生光泽精自催化化学发光。这是首次在纳米参与的化学发光反应中观察到自催化现象。该体系的强光发射有望进一步用于生物分析和“冷光源”的构造。
3.基于鲁米诺—硝酸银—纳米金化学发光体系,建立了一种微孔板化学发光免疫分析方法测定人免疫球蛋白G(IgG)。采用聚苯乙烯微孔板同时作为免疫反应的固相载体和化学发光检测容器。一抗(羊抗人IgG)首先包被在微孔板中,然后将抗原(人IgG)和纳米金标记的二抗先后结合到微孔板中,形成夹心式免疫复合物。标记的纳米金能够诱导鲁米诺和硝酸银之间的反应,产生光发射。通过凝絮实验确定形成稳定的纳米金—蛋白质生物复合物所需的最小抗体浓度。实验还对一些化学发光检测条件进行了优化,包括鲁米诺溶液pH、鲁米诺和硝酸银溶液浓度等。在优化的条件下,体系的化学发光强度与人IgG浓度的对数之间存在良好的线性关系,线性范围为25～5000 ng/mL,按三倍信噪比(S/N=3)计算检测限为12.8 ng/mL(～80 pM)。与其它报道的金标化学发光免疫分析方法相比,该方法避免了苛刻的溶出步骤以及难以控制的合成过程,使得方法更简单、省时、易于自动化。该化学发光免疫分析方法可望用于检测临床重要的生物活性物质。
4.建立了一种基于金标银染的非溶出微孔板化学发光免疫分析方法测定人IgG。首先将一抗(羊抗人IgG)固载到聚苯乙烯微孔板中,然后相继结合抗原(人IgG)和金标二抗,形成夹心式免疫复合物。标记的纳米金能够催化对苯二酚还原乙酸银为单质银,并沉积在纳米金表面,之后直接采用鲁米诺—过氧化氢化学发光体系进行检测。实验详细研究了银染时间对化学发光响应的影响。对于不同的银染时间,可以分别获得增强和抑制化学发光响应。在此基础上,初步建立了增强和抑制两种模式的化学发光免疫分析。实验分别对增强模式和抑制模式下的化学发光条件进行了优化,包括鲁米诺的缓冲介质、pH以及鲁米诺和过氧化氢的浓度。在优化的条件下,增强模式的检测限为0.9 ng/mL;抑制模式的检测限为5.6 ng/mL。增强模式的灵敏度高于其它报道的非溶出化学发光免疫分析方法。本项工作首次将金标银染技术用于建立非溶出化学发光免疫分析方法,结果表明银染有利于提高分析的灵敏度。该方法避免了苛刻的溶出过程,以及不规则纳米金的合成。但是,目前这种方法仍有一些不足之处,如线性范围过窄、灵敏度和稳定性仍有待提高。
In this dissertation,the state of arts in the field of chemiluminescence(CL), chemiluminescence immunoassays,the synthesis and properties of metal nano-materials and their applications in immunoassays were reviewed.Although the theory of CL and its application in immunoassay have been investigated for many years,the study of CL was limited to molecular and ion systems.Recently,metal nanoparticle-involved CL has attracted great attentions,in which metal nanoparticles can participate in CL reactions as catalyst,reductant,naonosized platform and energy acceptor.However,most of the reported CL systems involving nanoparticles were of low quantum yield,limiting there applications in analysis.Meanwhile,the study of nanoparticle-controlled CL system has been rarely reported.On the other hand,with the advance of nanoparticle-involved CL,chemiluminescence immunoassays based on metal nanoparticle labels were developed.Most of these reported chemiluminescence immunoassays contained "stripping" process,in which strict conditions were required to dissolve metal nanoparticles,making the method complicated,limiting there practical application.There were few reports about non-stripping chemiluminescence immunoassays based on metal nanoparticle-catalyzed CL reactions.Therefore,in the present dissertation,gold and platinum nanoparticles were utilized to design new CL systems.The nanoparticle-dependent or-controlled CL behaviors,rules and mechanisms of these new CL systems were investigated.Moreover,based on the CL activity of gold nanoparticles,new methods for non-stripping chemiluminescence immunoassays were developed.The main results are as follows:
1.It was found that gold nanoparticles of small size(＜5 nm) could inhibit the chemiluminescence(CL) of the luminol-ferricyanide system,whereas gold nanoparticles of large size(＞10 nm) could enhance this CL,and the most intensive CL signals were obtained with 25-nm-diameter gold nanoparticles.The luminophor was identified as the excited-state 3-aminophthalate anion(Ap~(2-)).The studies of UV-visible spectra,CL spectra,X-ray photoelectron spectra(XPS),effects of concentrations of luminol and ferricyanide solution,and fluorescence quenching efficiency of gold colloids were carried out to explore the CL inhibition and enhancement mechanism.The CL inhibition by gold nanoparticles of small size was supposed to originate from the competitive consumption of ferricyanide by gold nanoparticles and the relatively high quenching efficiency of the luminophor by gold nanoparticles.In contrast,the CL enhancement by gold nanoparticles of large size was ascribed to the catalysis of gold nanoparticles in the electron-transfer process during the luminol CL reaction and the relatively low quenching efficiency of the luminophor by gold nanoparticles.This work demonstrates that gold nanoparticles have the size-dependent inhibition and enhancement in the CL reaction,proposing a perspective for the investigation of new and efficient nanosized inhibitors and enhancers in CL reactions for analytical purposes.
2.An intensive and delayed CL could be observed from the alkaline lucigenin solution in the presence of ethanol and Pt nanopartieles simultaneously.The "induction" period of the CL was largely dependent on the concentration of Pt nanoparticles.The time from injection to the occurrence of the maximum CL intensity, which defined as T_p here,was exponentially related to the concentration of Pt nanoparticles;while the maximum CL intensity was linear with Pt nanoparticles concentration.The products and luminophor of lucigenin CL reaction were characterized by UV-visible spectra,fluorescence spectra and CL spectra.The luminophor of this system was identified as the excited-state N-methylacridone (NMA).The variation of the concentration of lucigenin and its CL product NMA during the process was monitored in situ.The results showed that the reaction of lucigenin and NaOH involving Pt nanoparticles and ethanol was autocatalytic.The studies of transmission electron microscope(TEM),XPS and nuclear magnetic resonance(NMR) were carried out to explore the mechanism of the autocatalytic CL reaction.The results suggested that Pt nanoparticles acted as a "general" catalyst in the CL system that not only catalyzed the oxidation of ethanol to acetaldehyde,but also catalyzed the CL enhancement by acetaldehyde.Ethanol in this system was the initiator for catalysis.Similar to ethanol,other alcohols such as methanol,ethylene glycol and n-propanol could also initiate autocatalytic lucigenin CL in the presence of Pt nanoparticles.This is the first time autocatalytic light emission has been obtained from nanoparticles-involved CL reactions.The strong light emission in this system may find future applications in bioanalysis and fabrication of"cold light sources".
3.A novel microplate-compatible chemiluminescence immunoassay has been developed for the determination of human immunoglobulin G(IgG) based on the luminol-AgNO_3-gold nanoparticles CL system.Polystyrene microtiter plates were used for both immunoreactions and CL measurements.The primary antibody, goat-anti-human IgG,was first immobilized on polystyrene microwells.Then the antigen(human IgG) and the gold-labeled second antibody were connected to the microwells successively to form a sandwich-type immunocomplex.The gold label could trigger the reaction between luminol and AgNO_3,accompanied by light emission.A flocculation test was carried out to determine the minimum amount of goat-anti-human IgG required to form stable nanoparticle-protein bioconjugate.Some experimental parameters influencing the CL reaction,including luminol pH and the concentration of luminol and AgNO_3,were optimized.Under the optimized conditions, the CL intensity of the system was linear with the logarithm of the concentration of human IgG in the range from 25 to 5000 ng/mL,with a detection limit of 12.8 ng/mL (～80 pM) at a signal to noise ratio of three(S/N = 3).Compared with other reported CL immunoassay method based on gold labels,the proposed CL protocol avoids a strict stripping procedure or difficult to control synthesis processes,making the method more simple,time-saving and easily automated.The present CL method is promising for the determination of clinically important bioactive analytes.
4.A new non-stripping and microplate-compatible chemiluminescence immunoassay protocol based on gold label with silver staining has been developed for the determination of human IgG.The primary antibody,goat-anti-human IgG,was first immobilized on polystyrene microwells,and then the antigen(human IgG) and the gold-labeled second antibody were captured onto the microwells successively to form a sandwich-type immunocomplex.The gold label could catalyze the reduction of silver acetate by hydroquinone,leading to the deposition of silver on the gold nanoparticles,followed by direct CL detection using luminol-H_2O_2 system.The effect of staining time on the CL response was carefully studied.With different staining time, both enhanced and inhibited CL detention modes were established for the CL immunoassay.Some CL conditions in both modes,including the buffer media of luminol,luminol pH and the concentration of luminol and H_2O_2,were optimized. Under the optimized conditions,the detection limit of this CL method(S/N = 3) was 0.9 ng/mL for enhanced mode,and 5.6 ng/mL for inhibited mode,respectively.The sensitivity of the enhanced mode was higher than other reported non-stripping CL immunoassay methods.This work revealed that the staining of silver on the gold nanoparticles was useful to improve the sensitivity.This is the first time to imply gold-label-silver-staining technology to develop non-stripping CL immunoassays.The proposed CL protocol avoids strict stripping procedure or the synthesis of irregular gold nanoparticles.However,there are still some disadvantages,such as the narrow linear range,and the further improvement required for the sensitivity and the stability.
引文
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