联吡啶钌结合纳米材料构建信号增强型及多组分检测型电致化学发光免疫传感器研究
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摘要
电致化学发光免疫传感器是一种将电致化学发光技术与免疫学分析方法相结合而发展起来的具有高灵敏度、高选择性、低背景等特点的生物传感器。免疫生物传感器敏感界面上生物识别系统的构建是将生物识别事件转化为可检测物理化学信号的关键。利用化学、材料及生物等多种技术放大并特异性转化与免疫反应有关的检测信号,是实现传感器超灵敏检测的有效方法。本文针对电致化学发光信号探针联吡啶钌因水溶性强、分子结构无官能团可共价交联而难于固载标记的缺点,以复合纳米材料为载体,用掺杂、吸附或包埋联吡啶钌的方法增加联吡啶钌固载量,并标记抗体；采用不同的信号放大策略及巧妙的检测策略构建信号增强型及多组分检测型电致化学发光药物及免疫传感器。研究工作分为以下几个部分：
1.多壁碳纳米管和联吡啶钉/纳米金复合纳米球为有效基质自组装构建新型固相电致化学发光药物传感器研究
利用纳米金及多壁碳纳米管等纳米材料比表面积大、导电性好,可增加联吡啶钉固载量并有效增加单位时间内光量子激发态数量,放大Ru(bpy)32+ -TPrA体系电致化学发光强度的特点,我们新制备了联吡啶钌掺杂纳米金的纳米球颗粒用于固相电致化学发光传感器构建,并将该传感器用于对苦参碱药物的定量测定。该部分研究工作,成功通过定量计算对比不同纳米颗粒修饰电极的电活性比表面积,探讨不同纳米颗粒对电致化学发光传感器性能的贡献,实现了电致化学发光性能的定量分析。实验结果表明多壁碳纳米管与纳米金相比,多壁碳纳米管能为修饰电极提供更大的电活性比表面积,更有利于联吡啶钌固相电致化学发光。将多壁碳纳米管与纳米金结合构建的固相电致化学发光传感器具有更高的灵敏度,其对苦参碱线性响应范围为2.0 X 10-6-6.0X 10-3 mol·L-1,最低检测限为0.7μmol·L-1。
2.联吡啶钌掺杂二氧化钛纳米颗粒标记抗体构建超灵敏电致化学发光免疫传感器研究
该部分研究工作,以TiO2纳米颗粒作为免疫标记载体,利用TiO2半导体纳米材料比表面积大,能降低光量子激发态能垒,有助于增强电致化学发光的优点,制备了Nafion包裹的TiO2复合纳米颗粒,将联吡啶钉分子通过阳离子交换作用固载在纳米颗粒表面,并利用Nafion嵌套抗体,将信号探针联吡啶钌标记到抗体上。该方法巧妙的运用联吡啶钌掺杂TiO2复合纳米颗粒既固载探针又标记抗体,解决了联吡啶钉无官能团可交联标记的缺陷,同时利用纳米颗粒大比表面积增加了联吡啶钉的标记量,从而放大检测信号,提高免疫传感器灵敏度。实验结果表明目标ECL免疫传感器对HCG抗原有较灵敏的分析表现,线性范围是0.02-25 mIU·mL-1,最低检测下限是0.007 mlU·mL-1 (S/N=3).
3.基于电位控制小麦状银颗粒高效催化联吡啶钌发光构建信号增强型电致化学发光免疫传感器的研究
我们用循环伏安扫描法制得一种新的小麦状银颗粒,并发现该颗粒在-0.4-1.25 V电位下,当发生Ag+/Ag氧化还原时,对联吡啶钌的电致化学发光有明显增强作用,我们将此作为信号放大步骤之一用于构建ECL免疫传感器。同时,利用Pt纳米颗粒掺杂及亲和素/生物素多位点耦联技术增加联吡啶钌分子在二抗上的标记量作为又一信号放大步骤。二者联用以多级放大免疫检测信号,提高传感器灵敏度。实验结果表明该免疫传感器在0.01-20 ng-mL-1范围内对IgG抗原有较好的线性响应,检测限为3 pg-mL-1 (S/N=3)。在这一没有共反应试剂的ECL体系中,Ru(bpy)32+发光信号通过DpAg纳米颗粒催化,Pt纳米材料掺杂及亲和素/生物素多位点耦联得以充分放大。
4.脂质体包裹增强信号小分子做为传感和放大平台结合适配体传感器用于超灵敏电致化学发光免疫测定
我们成功地合成了包裹可卡因分子的脂质体纳米颗粒,联合免疫脂质体可放大水溶性信号物标记量,适体链可特异结合识别小分子的特点,将这两种高灵敏高特异的生物分析技术相结合通过多级放大用于电致化学发光免疫分析。实验选择对联吡啶钉发光有增强作用的小分子-可卡因作为信号报告物,利用包裹可卡因分子的免疫脂质体建立与可卡因浓度相关的免疫检测平台,然后利用可卡因对联吡啶钉发光信号的增强作用,通过新设计的特异识别可卡因的适配体传感器检测从免疫脂质体释放的可卡因浓度,从而间接实现对免疫抗原的定量检测。该检测策略避免了水溶性联吡啶钌的固载,通过脂质体放大信号报告分子可卡因的量,再通过可卡因对联吡啶钉的共反应试剂作用增强联吡啶钌发光,实现免疫检测信号的特异性多重放大。该多种放大技术联用的超灵敏免疫检测策略用于心衰标志物NT-proBNP检测,检测限可达0.77pg-mL-1。
5.基于多组分肿瘤标志物同时检测的电致化学发光免疫传感器研究
我们最新的研究发现过硫酸根S2O82-除了文献报道的可以增强联吡啶钌阴极发光外,其本身在电位-1.4 V也有电致化学发光。我们成功地将S2O82-的阴极发光与联吡啶钌的阳极发光相结合,以脂质体为免疫标记载体,将联吡啶钌和过硫酸钾分别用脂质体包裹并分别标记到两种不同肿瘤标志物上,按夹心反应模式构建多种肿瘤标志物同时检测的电致化学发光免疫传感器,初步探索用电致化学发光技术在同一敏感界面实现多组分的同时组装同时检测。实验结果表明S2O82-标记抗体对应的AFP抗原检测线性响应范围为20-250 ng-mL-1,检测限为6.7ng·mL-1 (S/N=3), Ru(bpy)32+标记抗体对应的CEA抗原检测线性响应范围为5～150 ng·mL-1,检测限为1.6 ng-mL-1 (S/N=3)。由于溶解氧及联吡啶钌对S2O82-阴极发光的影响,S2O82-标记抗体对应的检测信号更大,故得到的对应AFP抗原检测限更低。
Electrochemiluminescence (ECL) immunosensors combined the merits of ECL technology and immunoassay, are valuable tools for monitoring the antibody or antigen due to the advantages of high sensitivity, high selectivity and low background. The biological recognition system on an immunosensor is pivotal for the trancduction between the biorecognition event and detected signal. In the system, using the material and biological amplificatory technology to specifically transduce and amplify the signal is an effective approach for improving the sensitivity. This research focuses on the preparation of the multi-functionalized nanomaterials, the construction of the immunoreaction interface, the strategy of sensitivity enhancement and the multiplexed assay by Ru(bpy)32+ -based ECL immunosensors. The detail contents are as follows:
1. Multi-walled carbon nanotubes and Ru(bpy)32+/nano-Au nano-sphere as efficient matrixes for a novel solid-state electrochemiluminescence sensor.
As the large surface area and conductivity of gold nanoparticles, it was employed to prepare the Ru(bpy)32+/nano-Au nano-sphere (abbreviate as Ru-AuNPs) via the electrostatic interactions. And an effective method for immobilization of Ru(bpy)32+ is developed by using the Ru-AuNPs and MWCNTs-Nafion composite nanoparticles for a novel ECL sensor. The experiment confirms that the enhancement of the ECL intensity on the sensor is attributed to following reasons. One hand, the employment of MWCNTs in the Nafion film enlarged the electro-active surface areas to benefit the contact between the signal probe on the composite film and co-reactant used as reinforcing agent. On the other hand, the nano-materials of MWCNTs and nano-Au also improve the conductivity of the self-assembled film so as to increase the quantity of excited state of Ru(bpy)32+ in the unit time and finally cause better properties in luminescence. The ECL sensor was used to detect a kind of alkaloid medicine, Matrine. It showed the good response to the concentration of the Matrine from 2.0×10-6 mol·L-1 to 6.0×10-3 mol·L-1. Furthermore, this work innovatively employs the quantificational count of electro-active surface area to explain the contribution of nanomaterial for ECL response, which brings the new approach into the ECL research region of nanomaterial.
2. A new electrochemiluminescence immunosensor based on Ru(bpy)32+ -doped TiO2 nanoparticles labeling for ultrasensitive detection of human chorionic gonadotrophin.
As the large surface area and low protons excited energy of TiO2 nanoparticles, a sensitive ECL immunosensor was constructed on the basis of sandwich type immunoreaction with Nafion functionalized TiO2 composite nanoparticles as label. The composite nanoparticles labeled antibody were synthesized by successively loading the antibody and large amounts of Ru(bpy)32+ molecules onto the Nafion coated TiO2 nanoparticles, which is named as Ru-Nafion@TiO2 labeled secondary antibody. They together with the electro-deposited gold nanoparticles (DpAu) were employed to assemble the ECL immunosensor. The capability of the immunosensor was evaluated by detecting the ECL responses of immunosensor on different concentrations of human chorionic gonadotrophin (HCG). Comparing with other immunoassays, the sandwich-type ECL immunosensor assembled with DpAu and Ru-Nafion@TiO2 nanoparticles can perform the ultrasensitive detection to HCG with a low detection limit 0.007 mIU·mL-1.
3. Potential controlling highly-efficient catalysis of wheat-like silver particles for electrochemiluminescence immunosensor labeled by nano-Pt@Ru and multi-sites biotin/streptavidin affinity.
The potential controlling silver catalysis for Ru(bpy)32+ ECL signal at a special potential-0.4-1.25 V was newly developed as the ECL signal amplification strategy for ultrasensitive protein detection. In the proposed Ru(bpy)32+ ECL immune system without any co-reactant, the detected ECL signal was amplified due to the following multiple amplification strategies:(1) the ECL catalysis for Ru(bpy)32+ were performed by electro-inducing the DpAg particles to generate Ag+ ion and controlled by the special potential. The catalyzer Ag+ produced near the electrode surface and reproduced by cyclic potential scan, which improved the catalytical efficiency. (2) The amount of the ECL signal probes linked to secondary antibodies were amplified by the adsorption of Pt nanoparticles and the multiple sites bridge linkage of biotin/SA. These new multiple signal amplification strategies made the proposed ECL immunosensor achieve ultrasensitive detection for model protein human IgG with the detection limit down to 3 pg·mL-1.
4. Signal-enhancer molecules encapsulated liposome as a valuable sensing and amplification platform combining the aptasensor for ultrasensitive ECL immunoassay.
Based on the associated signal amplificatory technology, an innovatory ECL immunoassay strategy was proposed to detect the newly-developing heart failure biomarker N-terminal pro-brain natriuretic peptide (NT-proBNP). Firstly, this strategy used small molecules encapsulated liposome as immune label to construct a sandwich immune sensing platform for NT-proBNP. Then the ECL aptasensor was prepared to collect and detect the small molecules released from the liposome. Finally, based on the ECL signal changes caused by the small molecules, the ECL signal indirectly reflected the level of NT-proBNP antigen. In this experiment, the cocaine was chosen as the proper small molecule that can act as signal-enhancer to enhance the ECL of Ru(bpy)32+ The quantificational calculation proved the -5.3x103 cocaine molecules per liposome enough to perform the assignment of signal amplification. The cocaine-binding ECL aptasensor further promoted the work aimed at amplifying signal. The performance of NT-proBNP assay by the proposed strategy exhibited high sensitivity and high specificities with a linear relationship over 0.01-500 ng-mL-1 range, and a detection limit down to 0.77 pg·mL-1.
5. The study on the electrochemiluminescence immunosensor based on the simultaneous multiplexed immunoassay.
A new discovery that the cathodal ECL of S2O82- anion appears at -1.4 V promotes the multiplexed assay by ECL immunosensor. The K2S2O8 and Ru(bpy)32+ were chosen as the ECL signal probe and respectively encapsulated in liposome as immune label. Relative to the Ru(bpy)32+ -encapsulated liposome as the label of anti-CEA, the K2S2O8-encapsulated liposome was used to label the anti-AFP. Then they were orderly subjected the sandwich immunoassay on one electrode surface. Finally, these ECL probes were released by the lysing and adsorbed on the electrode suface for the detection. The cathodal and anodic ECL scans were executed synchronously at the potential scope of -1.6 V-1.25 V. And the ECL reponses appeared at -1.4 V for the AFP antigen and at 1.2 V for the CEA. Due to the enhancement of the dissolved oxygen, the ECL response with the S2O82- anion label is larger than that with the Ru(bpy)32+ label. The immunosensor showed linear ranges of 20-250 ng·mL-1 for AFP and 5-150 ng·mL-1 for CEA. This result testifies that the multiplexed assay can be realized in the ECL.
1.多壁碳纳米管和联吡啶钉/纳米金复合纳米球为有效基质自组装构建新型固相电致化学发光药物传感器研究
利用纳米金及多壁碳纳米管等纳米材料比表面积大、导电性好,可增加联吡啶钉固载量并有效增加单位时间内光量子激发态数量,放大Ru(bpy)32+ -TPrA体系电致化学发光强度的特点,我们新制备了联吡啶钌掺杂纳米金的纳米球颗粒用于固相电致化学发光传感器构建,并将该传感器用于对苦参碱药物的定量测定。该部分研究工作,成功通过定量计算对比不同纳米颗粒修饰电极的电活性比表面积,探讨不同纳米颗粒对电致化学发光传感器性能的贡献,实现了电致化学发光性能的定量分析。实验结果表明多壁碳纳米管与纳米金相比,多壁碳纳米管能为修饰电极提供更大的电活性比表面积,更有利于联吡啶钌固相电致化学发光。将多壁碳纳米管与纳米金结合构建的固相电致化学发光传感器具有更高的灵敏度,其对苦参碱线性响应范围为2.0 X 10-6-6.0X 10-3 mol·L-1,最低检测限为0.7μmol·L-1。
2.联吡啶钌掺杂二氧化钛纳米颗粒标记抗体构建超灵敏电致化学发光免疫传感器研究
该部分研究工作,以TiO2纳米颗粒作为免疫标记载体,利用TiO2半导体纳米材料比表面积大,能降低光量子激发态能垒,有助于增强电致化学发光的优点,制备了Nafion包裹的TiO2复合纳米颗粒,将联吡啶钉分子通过阳离子交换作用固载在纳米颗粒表面,并利用Nafion嵌套抗体,将信号探针联吡啶钌标记到抗体上。该方法巧妙的运用联吡啶钌掺杂TiO2复合纳米颗粒既固载探针又标记抗体,解决了联吡啶钉无官能团可交联标记的缺陷,同时利用纳米颗粒大比表面积增加了联吡啶钉的标记量,从而放大检测信号,提高免疫传感器灵敏度。实验结果表明目标ECL免疫传感器对HCG抗原有较灵敏的分析表现,线性范围是0.02-25 mIU·mL-1,最低检测下限是0.007 mlU·mL-1 (S/N=3).
3.基于电位控制小麦状银颗粒高效催化联吡啶钌发光构建信号增强型电致化学发光免疫传感器的研究
我们用循环伏安扫描法制得一种新的小麦状银颗粒,并发现该颗粒在-0.4-1.25 V电位下,当发生Ag+/Ag氧化还原时,对联吡啶钌的电致化学发光有明显增强作用,我们将此作为信号放大步骤之一用于构建ECL免疫传感器。同时,利用Pt纳米颗粒掺杂及亲和素/生物素多位点耦联技术增加联吡啶钌分子在二抗上的标记量作为又一信号放大步骤。二者联用以多级放大免疫检测信号,提高传感器灵敏度。实验结果表明该免疫传感器在0.01-20 ng-mL-1范围内对IgG抗原有较好的线性响应,检测限为3 pg-mL-1 (S/N=3)。在这一没有共反应试剂的ECL体系中,Ru(bpy)32+发光信号通过DpAg纳米颗粒催化,Pt纳米材料掺杂及亲和素/生物素多位点耦联得以充分放大。
4.脂质体包裹增强信号小分子做为传感和放大平台结合适配体传感器用于超灵敏电致化学发光免疫测定
我们成功地合成了包裹可卡因分子的脂质体纳米颗粒,联合免疫脂质体可放大水溶性信号物标记量,适体链可特异结合识别小分子的特点,将这两种高灵敏高特异的生物分析技术相结合通过多级放大用于电致化学发光免疫分析。实验选择对联吡啶钉发光有增强作用的小分子-可卡因作为信号报告物,利用包裹可卡因分子的免疫脂质体建立与可卡因浓度相关的免疫检测平台,然后利用可卡因对联吡啶钉发光信号的增强作用,通过新设计的特异识别可卡因的适配体传感器检测从免疫脂质体释放的可卡因浓度,从而间接实现对免疫抗原的定量检测。该检测策略避免了水溶性联吡啶钌的固载,通过脂质体放大信号报告分子可卡因的量,再通过可卡因对联吡啶钉的共反应试剂作用增强联吡啶钌发光,实现免疫检测信号的特异性多重放大。该多种放大技术联用的超灵敏免疫检测策略用于心衰标志物NT-proBNP检测,检测限可达0.77pg-mL-1。
5.基于多组分肿瘤标志物同时检测的电致化学发光免疫传感器研究
我们最新的研究发现过硫酸根S2O82-除了文献报道的可以增强联吡啶钌阴极发光外,其本身在电位-1.4 V也有电致化学发光。我们成功地将S2O82-的阴极发光与联吡啶钌的阳极发光相结合,以脂质体为免疫标记载体,将联吡啶钌和过硫酸钾分别用脂质体包裹并分别标记到两种不同肿瘤标志物上,按夹心反应模式构建多种肿瘤标志物同时检测的电致化学发光免疫传感器,初步探索用电致化学发光技术在同一敏感界面实现多组分的同时组装同时检测。实验结果表明S2O82-标记抗体对应的AFP抗原检测线性响应范围为20-250 ng-mL-1,检测限为6.7ng·mL-1 (S/N=3), Ru(bpy)32+标记抗体对应的CEA抗原检测线性响应范围为5～150 ng·mL-1,检测限为1.6 ng-mL-1 (S/N=3)。由于溶解氧及联吡啶钌对S2O82-阴极发光的影响,S2O82-标记抗体对应的检测信号更大,故得到的对应AFP抗原检测限更低。
Electrochemiluminescence (ECL) immunosensors combined the merits of ECL technology and immunoassay, are valuable tools for monitoring the antibody or antigen due to the advantages of high sensitivity, high selectivity and low background. The biological recognition system on an immunosensor is pivotal for the trancduction between the biorecognition event and detected signal. In the system, using the material and biological amplificatory technology to specifically transduce and amplify the signal is an effective approach for improving the sensitivity. This research focuses on the preparation of the multi-functionalized nanomaterials, the construction of the immunoreaction interface, the strategy of sensitivity enhancement and the multiplexed assay by Ru(bpy)32+ -based ECL immunosensors. The detail contents are as follows:
1. Multi-walled carbon nanotubes and Ru(bpy)32+/nano-Au nano-sphere as efficient matrixes for a novel solid-state electrochemiluminescence sensor.
As the large surface area and conductivity of gold nanoparticles, it was employed to prepare the Ru(bpy)32+/nano-Au nano-sphere (abbreviate as Ru-AuNPs) via the electrostatic interactions. And an effective method for immobilization of Ru(bpy)32+ is developed by using the Ru-AuNPs and MWCNTs-Nafion composite nanoparticles for a novel ECL sensor. The experiment confirms that the enhancement of the ECL intensity on the sensor is attributed to following reasons. One hand, the employment of MWCNTs in the Nafion film enlarged the electro-active surface areas to benefit the contact between the signal probe on the composite film and co-reactant used as reinforcing agent. On the other hand, the nano-materials of MWCNTs and nano-Au also improve the conductivity of the self-assembled film so as to increase the quantity of excited state of Ru(bpy)32+ in the unit time and finally cause better properties in luminescence. The ECL sensor was used to detect a kind of alkaloid medicine, Matrine. It showed the good response to the concentration of the Matrine from 2.0×10-6 mol·L-1 to 6.0×10-3 mol·L-1. Furthermore, this work innovatively employs the quantificational count of electro-active surface area to explain the contribution of nanomaterial for ECL response, which brings the new approach into the ECL research region of nanomaterial.
2. A new electrochemiluminescence immunosensor based on Ru(bpy)32+ -doped TiO2 nanoparticles labeling for ultrasensitive detection of human chorionic gonadotrophin.
As the large surface area and low protons excited energy of TiO2 nanoparticles, a sensitive ECL immunosensor was constructed on the basis of sandwich type immunoreaction with Nafion functionalized TiO2 composite nanoparticles as label. The composite nanoparticles labeled antibody were synthesized by successively loading the antibody and large amounts of Ru(bpy)32+ molecules onto the Nafion coated TiO2 nanoparticles, which is named as Ru-Nafion@TiO2 labeled secondary antibody. They together with the electro-deposited gold nanoparticles (DpAu) were employed to assemble the ECL immunosensor. The capability of the immunosensor was evaluated by detecting the ECL responses of immunosensor on different concentrations of human chorionic gonadotrophin (HCG). Comparing with other immunoassays, the sandwich-type ECL immunosensor assembled with DpAu and Ru-Nafion@TiO2 nanoparticles can perform the ultrasensitive detection to HCG with a low detection limit 0.007 mIU·mL-1.
3. Potential controlling highly-efficient catalysis of wheat-like silver particles for electrochemiluminescence immunosensor labeled by nano-Pt@Ru and multi-sites biotin/streptavidin affinity.
The potential controlling silver catalysis for Ru(bpy)32+ ECL signal at a special potential-0.4-1.25 V was newly developed as the ECL signal amplification strategy for ultrasensitive protein detection. In the proposed Ru(bpy)32+ ECL immune system without any co-reactant, the detected ECL signal was amplified due to the following multiple amplification strategies:(1) the ECL catalysis for Ru(bpy)32+ were performed by electro-inducing the DpAg particles to generate Ag+ ion and controlled by the special potential. The catalyzer Ag+ produced near the electrode surface and reproduced by cyclic potential scan, which improved the catalytical efficiency. (2) The amount of the ECL signal probes linked to secondary antibodies were amplified by the adsorption of Pt nanoparticles and the multiple sites bridge linkage of biotin/SA. These new multiple signal amplification strategies made the proposed ECL immunosensor achieve ultrasensitive detection for model protein human IgG with the detection limit down to 3 pg·mL-1.
4. Signal-enhancer molecules encapsulated liposome as a valuable sensing and amplification platform combining the aptasensor for ultrasensitive ECL immunoassay.
Based on the associated signal amplificatory technology, an innovatory ECL immunoassay strategy was proposed to detect the newly-developing heart failure biomarker N-terminal pro-brain natriuretic peptide (NT-proBNP). Firstly, this strategy used small molecules encapsulated liposome as immune label to construct a sandwich immune sensing platform for NT-proBNP. Then the ECL aptasensor was prepared to collect and detect the small molecules released from the liposome. Finally, based on the ECL signal changes caused by the small molecules, the ECL signal indirectly reflected the level of NT-proBNP antigen. In this experiment, the cocaine was chosen as the proper small molecule that can act as signal-enhancer to enhance the ECL of Ru(bpy)32+ The quantificational calculation proved the -5.3x103 cocaine molecules per liposome enough to perform the assignment of signal amplification. The cocaine-binding ECL aptasensor further promoted the work aimed at amplifying signal. The performance of NT-proBNP assay by the proposed strategy exhibited high sensitivity and high specificities with a linear relationship over 0.01-500 ng-mL-1 range, and a detection limit down to 0.77 pg·mL-1.
5. The study on the electrochemiluminescence immunosensor based on the simultaneous multiplexed immunoassay.
A new discovery that the cathodal ECL of S2O82- anion appears at -1.4 V promotes the multiplexed assay by ECL immunosensor. The K2S2O8 and Ru(bpy)32+ were chosen as the ECL signal probe and respectively encapsulated in liposome as immune label. Relative to the Ru(bpy)32+ -encapsulated liposome as the label of anti-CEA, the K2S2O8-encapsulated liposome was used to label the anti-AFP. Then they were orderly subjected the sandwich immunoassay on one electrode surface. Finally, these ECL probes were released by the lysing and adsorbed on the electrode suface for the detection. The cathodal and anodic ECL scans were executed synchronously at the potential scope of -1.6 V-1.25 V. And the ECL reponses appeared at -1.4 V for the AFP antigen and at 1.2 V for the CEA. Due to the enhancement of the dissolved oxygen, the ECL response with the S2O82- anion label is larger than that with the Ru(bpy)32+ label. The immunosensor showed linear ranges of 20-250 ng·mL-1 for AFP and 5-150 ng·mL-1 for CEA. This result testifies that the multiplexed assay can be realized in the ECL.
引文
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