基于信号放大技术的压电生物传感器制备及应用
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摘要
随着分析科学的不断发展,生命科学领域中的各种分析和检测过程,越来越多地要借助于生物传感技术获取所需的信息。经过40多年的不断发展,随着现代测量技术、分子生物学、生物电子学和仿生学的迅猛发展及相互结合,生物传感技术在基础研究、应用研究、新产品开发和商品化等方面都取得了长足进展。各种新型生物传感器不断涌现,仪器性能不断提高,目前报道的生物传感器已有几百种,在医疗保健、食品卫生、环境监测、国防与安全等方面已逐步推广应用。本研究论文针对当前传感技术发展中的一些瓶颈问题,重点关注如何提高传感器信号等问题。结合压电免疫传感分析技术简便、快速、可实时输出数据的特点及电化学传感技术的灵敏度高的优点,利用纳米材料的优越性能及酶催化信号放大技术发展了几种新型的生物传感器,对黄曲霉毒素B1、人IgG、α-地贫突变基因、模拟DNA序列进行了定量测定,所得结果对比传统方法,在检测的灵敏度、线性范围方面都有所提高,并验证了技术的实用性。
压电免疫传感器是结合了压电效应的高灵敏性和免疫反应的高特异性的一种生物传感器,具有简便、快速、灵敏、成本低、响应谱广、可实时数据输出等优点,在生物技术、临床诊断、环境监测、食品工业、医药和军事等领域具有广泛的应用前景。我们利用酶和纳米材料在生物传感器中的应用,结合有效的生物活性组分的固定方法,采用信号放大技术提高分析信号、降低检测下限,提出了三种新型的压电免疫传感器。同时,尝试用石英晶体微天平作为敏感元件,在其表面固定具有发夹结构的DNA探针,用于识别并结合目标DNA序列产生相应的信号构建一种简单有效的DNA检测方法。还提出了一种新的电化学检测DNA的方法,以二茂铁标记的寡核苷酸构建了一种简单通用的信号打开型的分子开关,实现了对DNA序列的无试剂检测。主要内容如下:
(1)研制了一种简单、快速、灵敏的压电免疫传感器,用于黄曲霉毒素B1的检测。黄曲霉毒素B1是一种小分子物质,很难用压电法直接检测,本文采用间接竞争免疫法。纳米金标记羊抗鼠IgG抗体用于放大响应信号。在0.10～100 ng mL?1的范围内,该传感器的响应信号与AFB1浓度的对数呈现良好的线性关系。该传感器可以检测到AFB1的最低浓度为0.01 ng mL?1,定量能力与经典酶联免疫吸附法(ELISA)相接近。该传感器界面可用甘氨酸缓冲液可顺利再生,并可重复使用至少9次而其响应信号没有明显降低(第二章)。
(2)发展了一种基于间接竞争免疫反应和酶催化沉积放大的黄曲霉毒素B1压电检测方法。先在石英晶振表面包被一层3-巯基丙酸自组装膜,再共价结合BSA-AFB1偶联抗原,接着待测物AFB1与BSA-AFB1偶联抗原竞争结合鼠抗AFB1抗体,然后结合辣根过氧化物酶(HRP)标记的羊抗鼠IgG抗体。在H2O2存在时,HRP酶催化氧化4-氯-1-萘酚生成不溶物4-氯-1-萘醌沉积在电极表面,引起显著的质量变化,从而导致明显的频率响应。该传感器可获得黄曲霉毒素B1的浓度检测范围为0.01～10 ng mL?1。通过对模拟样品的分析结果表明,该传感器能够有效地检测牛奶样品中AFB1的含量,可望用于实际样品检测(第三章)。
(3)提出了一种简便的基于SiO2纳米颗粒免疫凝集反应的压电传感器对人IgG的直接检测方法。待测物人IgG会同时被晶振表面固定的羊抗人IgG抗体和检测体系中SiO2标记的羊抗人IgG抗体识别,引起检测体系中的SiO2在传感界面上特异性凝集,从而引起晶振表面的巨大质量变化和检测介质密度和粘度变化。实验结果表明,该方法修饰的探针不仅能充分地减少背景干扰,而且能显著地放大响应信号。实验中,采用扫描电镜(SEM)考察了探针表面在发生免疫凝集反应前后的形貌变化。此外,还考察了免疫凝集促进剂PEG及离子强度控制剂NaCl的使用对实验的影响。该传感器的频率变化与人IgG浓度为正相关关系,其检测下限为0.084μg mL-1(第四章)。
(4)尝试用石英晶体微天平作为敏感元件,在其表面固定具有发夹结构的DNA探针,用于识别并结合目标DNA序列产生相应的信号构建一种简单有效的DNA检测方法。其特点在于结合使用限制性核酸内切酶ECoR I及纳米金标记的检测探针,能够很好的降低背景值并有效增强信号。方案如下:先在石英晶振表面通过巯基自组装固定一段具有发夹结构的DNA探针,其发夹部分能够被内切酶ECoR I特异性识别并切割。再用巯基己醇封闭,然后与目标DNA作用。如果待测体系中含有目标DNA,则DNA探针的发夹结构打开且随后的内切酶对其没有作用,并可以结合纳米金标记的检测探针而使信号放大。否则,在没有目标DNA时,发夹结构的DNA探针可被酶切,与纳米金标记的检测探针作用也不能产生任何信号。实验结果表明,此方法是一种简单实用、灵敏度高的适应于DNA分析检测的技术(第五章)。
(5)在本工作中,我们提出了一种新的电化学检测DNA的方法,以二茂铁标记的寡核苷酸构建了一种简单通用的信号打开型的分子开关,实现了对DNA序列的无试剂检测。这一方法实际应用于基因突变导致α-地贫的分析检测中,用于检测染色体末端142编码子(Hb Constant Spring codon 142)单碱基突变(TAA→CAA)的存在,取得了较好的实验结果。目标DNA浓度在0.01到100 pM之间与电流信号具有较好的线性关系(R2=0.9777),该方法检测限为0.01 pM。该DNA传感器在1 M NaOH溶液中可顺利实现再生。所有这些表明,此方法是一种简单快速而且比较灵敏的适用于单碱基突变检测的技术,具有较强的通用性(第六章)。
With the continuous progress of the analytical science, a variety of analysis and testing process in life sciences, more and more with the aid of bio-sensing technology to obtain the required information. After 40 years the continuous rapid development and mutual integrationin modern measurement techniques, molecular biology, bio-electronics and bionics, biosensing technology has made remarkable progress in basic research, applied research, new product development as well as in commercialization. The current biosensors have been reported in hundreds of species and new types of biosensors are emerging, equipment performance continuously improving, have made a wide range of applications in health care, food hygiene, environmental monitoring, defense and security. This research paper concerns on a number of bottleneck problems for the current development of sensor technology, focusing on how to improve the sensor signals and so on. Combination of the advantages of simple, rapid, real-time output data with piezoelectric immunosensor sensing analysis and high sensitivity, characteristics of electrochemical sensing technology, using superior performance of nano-materials, and the enzyme-catalyzed signal amplification technology, the several new biological sensors for aflatoxin B1, human IgG,α-thalassemia mutations, the quantitative simulation of DNA sequence determination were developed and the results were compared with conventional methods, the detection sensitivity, linear range has improved and verified the practicality of the techniques.
Piezoelectric immunosensor combines the high sensitivity of the piezoelectric effect and high specificity of immune response as a biological sensor, has the characteristics of simple, rapid, sensitive, low cost, to respond in a broad spectrum, real-time data output, etc., with wide application prospects in the biological technology, clinical diagnostics, environmental monitoring, food industry, medicine and military fields. We use enzymes and nano-materials in the biological sensor applications, combined with the effective fixed method of bio-active component in analysis using signal amplification technology to improve the signal, lower detection limit, has developed three new types of piezoelectric immunosensor. At the same time, try to use quartz crystal microbalance as a sensor on its surface fixed with a hairpin DNA probes used to identify and target DNA to produce a signal to build a simple and efficient DNA detection method. Also proposed a new method of electrochemical detection of DNA in order to ferrocene labeled oligonucleotide construct a simple general-purpose the type of signal opening molecular switch, realized its reagent-free detection of DNA sequences. The main contents are as follows:
(1) A simple, rapid and highly sensitive piezoelectric immunosensor has been proposed and applied to detect aflatoxin B1 (AFB1). It is unlikely that direct binding of small molecules such like AFB1 to the piezoelectric sensor surface could result in a satisfactory detection limit and sensitivity. Thus, indirect competitive immunoassay technique had been used for the detection of the target and gold nanoparticles (GNP) been employed as a‘weight label’to the secondary antibody for amplifying the response. This method is proven in its ability to detect AFB1 down to a level of 0.01 ng mL-1 in artificially contaminated milk, which is comparable to or even exceeding the sensitivity of microtitre plate ELISA. Furthermore, the frequency responses of the immunoassay are linearly correlated to the logarithm of AFB1 concentration in the range of 0.10 ~ 100 ng mL-1. The sensor could be regenerated under very mild conditions simply by immersing the sensor into glycine buffer solution to desorb the combined antibody. It is found that the as-renewed sensor could be reused at least 9 runs without obvious loss of sensing sensitivity (Chapter 2).
(2) An ultrasensitive piezoelectric method for the detection of the aflatoxin B1 based on the indirect competitive immunoassay and the biocatalyzed deposition amplification has been developed. In this method, the quartz crystal surface was coated with a self-assembled monolayer of 3-mercaptopropionic acid (MPA) for covalently immobilization of the BSA-AFB1 conjugate, which could compete with the free AFB1 for binding to the anti-AFB1 antibody (MsIgG). After the competitive immunoreaction, the horseradish peroxidase (HRP) labeled goat anti-mouse IgG (G-Anti-MsIgG) was introduced into the detection cell to combine with the anti-AFB1 antibody on the crystal surface. The enzyme labeled G-Anti-MsIgG as a biocatalyst could accelerate the oxidation of 4-chloro-1-naphthol by H2O2 to yield the insoluble product benzo-4-chlorohexadienone on the surface of quartz crystal microbalance (QCM), resulting in a mass increase that was reflected by a decrease in the resonance frequency of the QCM. The proposed approach could allow for the determination of AFB1 in the concentration range of 0.01 ~ 10.0 ng mL-1. Furthermore, several artificially contaminated milk samples were analyzed with good recoveries obtained, which demonstrated the suitability of the proposed method for detecting AFB1 (Chapter 3).
(3) A simple piezoelectric immunoagglutination assay technique with antibody-modified nanoparticles has been developed for direct quantitative detection of protein. The proposed technique is based on the specific agglutination of goat anti-hIgG-coated silica nanoparticles in the presence of human immunoglobulin G (hIgG),which causes a frequency change and is monitored by a piezoelectric device. The antibody modified on the probe surface would combine with antibody-coated nanoparticles in the presence of antigen (hIgG) when the surface agglutination reaction took place, which couples both the mass effect and viscoelastic effect acting on the probe. The results indicate that the background interference can be substantially minimized and the probe signal can be observably multiplied. In addition, the surfaces of the modified probe and that after combining the complex of immunoagglutination were imaged by scanning electronic microscopy (SEM). Moreover, an optimization of assay medium composition with the addition of poly(ethylene glycol) (PEG) serving as immunoagglutination enhancer and sodium chloride to control the ion-strength was investigated. The frequency responses of the immunoagglutination assay were found to correlate well with the hIgG concentration with a detection limit of 0.084μg mL-1 (Chapter 4).
(4) Try to use quartz crystal microbalance as a sensor on its surface fixed with a hairpin DNA probes used to identify and target DNA to produce a signal to build a simple and efficient DNA detection method. It features combined with restriction endonuclease ECoR I and nano-gold-labeled detection probe that can reduce the background of a very good value and effective way to boost the signal. The program are as follows: first, the quartz crystal surface through the thiol-based self-assembly of a fixed period with a hairpin DNA probes can be part of its hairpin endonuclease ECoR I specifically recognize and cut. Mercapto-hexanol then closed, then the role of the target DNA. If the system under test contains a target DNA, the DNA probe of the hairpin turn and the subsequent enzyme is not their role, and can be combined with nano-gold-labeled detection probe making signal amplification. Otherwise, in the absence of target DNA, the hairpin DNA probe can be digested, and nano-Au labeled probes role can not produce any signal. The experimental results show that this method is a simple and practical, high sensitivity analytics adapting the detection of DNA (Chapter 5).
(5) In the present approach, we revealed a new scheme for electrochemical detection the target DNA sequence using Fc-labeled oligonucleotide as a molecular switch, which is a signal-on sensor featuring both generalizability and simplicity in design toward reagentless detection of DNA. The present approach has been demonstrated with the identification of a single-base mutation in Hb Constant Spring codon 142 (terminating codon TAA→CAA) that is one of the major types ofα-thalassemia point mutations for clinical diagnosis. After reaction with various concentration of target DNA under the optimum experimental conditions, the calibration curve was plotted. The results showed that the current intensity was linear to the logarithm of the target concentration in the range from 0.01 to 100 pM with a detection limit 0.01 pM. The experiment found that the DNA sensor could be reused via immersed into NaOH solution to be easily and successfully regenerated. All these features revealed that the system is a promising candidate for single-base mutation discrimination, owing the advantages of both generalizability and simplicity toward reagentless detection of DNA with sensitivity and selectivity (Chapter 6).
压电免疫传感器是结合了压电效应的高灵敏性和免疫反应的高特异性的一种生物传感器,具有简便、快速、灵敏、成本低、响应谱广、可实时数据输出等优点,在生物技术、临床诊断、环境监测、食品工业、医药和军事等领域具有广泛的应用前景。我们利用酶和纳米材料在生物传感器中的应用,结合有效的生物活性组分的固定方法,采用信号放大技术提高分析信号、降低检测下限,提出了三种新型的压电免疫传感器。同时,尝试用石英晶体微天平作为敏感元件,在其表面固定具有发夹结构的DNA探针,用于识别并结合目标DNA序列产生相应的信号构建一种简单有效的DNA检测方法。还提出了一种新的电化学检测DNA的方法,以二茂铁标记的寡核苷酸构建了一种简单通用的信号打开型的分子开关,实现了对DNA序列的无试剂检测。主要内容如下:
(1)研制了一种简单、快速、灵敏的压电免疫传感器,用于黄曲霉毒素B1的检测。黄曲霉毒素B1是一种小分子物质,很难用压电法直接检测,本文采用间接竞争免疫法。纳米金标记羊抗鼠IgG抗体用于放大响应信号。在0.10～100 ng mL?1的范围内,该传感器的响应信号与AFB1浓度的对数呈现良好的线性关系。该传感器可以检测到AFB1的最低浓度为0.01 ng mL?1,定量能力与经典酶联免疫吸附法(ELISA)相接近。该传感器界面可用甘氨酸缓冲液可顺利再生,并可重复使用至少9次而其响应信号没有明显降低(第二章)。
(2)发展了一种基于间接竞争免疫反应和酶催化沉积放大的黄曲霉毒素B1压电检测方法。先在石英晶振表面包被一层3-巯基丙酸自组装膜,再共价结合BSA-AFB1偶联抗原,接着待测物AFB1与BSA-AFB1偶联抗原竞争结合鼠抗AFB1抗体,然后结合辣根过氧化物酶(HRP)标记的羊抗鼠IgG抗体。在H2O2存在时,HRP酶催化氧化4-氯-1-萘酚生成不溶物4-氯-1-萘醌沉积在电极表面,引起显著的质量变化,从而导致明显的频率响应。该传感器可获得黄曲霉毒素B1的浓度检测范围为0.01～10 ng mL?1。通过对模拟样品的分析结果表明,该传感器能够有效地检测牛奶样品中AFB1的含量,可望用于实际样品检测(第三章)。
(3)提出了一种简便的基于SiO2纳米颗粒免疫凝集反应的压电传感器对人IgG的直接检测方法。待测物人IgG会同时被晶振表面固定的羊抗人IgG抗体和检测体系中SiO2标记的羊抗人IgG抗体识别,引起检测体系中的SiO2在传感界面上特异性凝集,从而引起晶振表面的巨大质量变化和检测介质密度和粘度变化。实验结果表明,该方法修饰的探针不仅能充分地减少背景干扰,而且能显著地放大响应信号。实验中,采用扫描电镜(SEM)考察了探针表面在发生免疫凝集反应前后的形貌变化。此外,还考察了免疫凝集促进剂PEG及离子强度控制剂NaCl的使用对实验的影响。该传感器的频率变化与人IgG浓度为正相关关系,其检测下限为0.084μg mL-1(第四章)。
(4)尝试用石英晶体微天平作为敏感元件,在其表面固定具有发夹结构的DNA探针,用于识别并结合目标DNA序列产生相应的信号构建一种简单有效的DNA检测方法。其特点在于结合使用限制性核酸内切酶ECoR I及纳米金标记的检测探针,能够很好的降低背景值并有效增强信号。方案如下:先在石英晶振表面通过巯基自组装固定一段具有发夹结构的DNA探针,其发夹部分能够被内切酶ECoR I特异性识别并切割。再用巯基己醇封闭,然后与目标DNA作用。如果待测体系中含有目标DNA,则DNA探针的发夹结构打开且随后的内切酶对其没有作用,并可以结合纳米金标记的检测探针而使信号放大。否则,在没有目标DNA时,发夹结构的DNA探针可被酶切,与纳米金标记的检测探针作用也不能产生任何信号。实验结果表明,此方法是一种简单实用、灵敏度高的适应于DNA分析检测的技术(第五章)。
(5)在本工作中,我们提出了一种新的电化学检测DNA的方法,以二茂铁标记的寡核苷酸构建了一种简单通用的信号打开型的分子开关,实现了对DNA序列的无试剂检测。这一方法实际应用于基因突变导致α-地贫的分析检测中,用于检测染色体末端142编码子(Hb Constant Spring codon 142)单碱基突变(TAA→CAA)的存在,取得了较好的实验结果。目标DNA浓度在0.01到100 pM之间与电流信号具有较好的线性关系(R2=0.9777),该方法检测限为0.01 pM。该DNA传感器在1 M NaOH溶液中可顺利实现再生。所有这些表明,此方法是一种简单快速而且比较灵敏的适用于单碱基突变检测的技术,具有较强的通用性(第六章)。
With the continuous progress of the analytical science, a variety of analysis and testing process in life sciences, more and more with the aid of bio-sensing technology to obtain the required information. After 40 years the continuous rapid development and mutual integrationin modern measurement techniques, molecular biology, bio-electronics and bionics, biosensing technology has made remarkable progress in basic research, applied research, new product development as well as in commercialization. The current biosensors have been reported in hundreds of species and new types of biosensors are emerging, equipment performance continuously improving, have made a wide range of applications in health care, food hygiene, environmental monitoring, defense and security. This research paper concerns on a number of bottleneck problems for the current development of sensor technology, focusing on how to improve the sensor signals and so on. Combination of the advantages of simple, rapid, real-time output data with piezoelectric immunosensor sensing analysis and high sensitivity, characteristics of electrochemical sensing technology, using superior performance of nano-materials, and the enzyme-catalyzed signal amplification technology, the several new biological sensors for aflatoxin B1, human IgG,α-thalassemia mutations, the quantitative simulation of DNA sequence determination were developed and the results were compared with conventional methods, the detection sensitivity, linear range has improved and verified the practicality of the techniques.
Piezoelectric immunosensor combines the high sensitivity of the piezoelectric effect and high specificity of immune response as a biological sensor, has the characteristics of simple, rapid, sensitive, low cost, to respond in a broad spectrum, real-time data output, etc., with wide application prospects in the biological technology, clinical diagnostics, environmental monitoring, food industry, medicine and military fields. We use enzymes and nano-materials in the biological sensor applications, combined with the effective fixed method of bio-active component in analysis using signal amplification technology to improve the signal, lower detection limit, has developed three new types of piezoelectric immunosensor. At the same time, try to use quartz crystal microbalance as a sensor on its surface fixed with a hairpin DNA probes used to identify and target DNA to produce a signal to build a simple and efficient DNA detection method. Also proposed a new method of electrochemical detection of DNA in order to ferrocene labeled oligonucleotide construct a simple general-purpose the type of signal opening molecular switch, realized its reagent-free detection of DNA sequences. The main contents are as follows:
(1) A simple, rapid and highly sensitive piezoelectric immunosensor has been proposed and applied to detect aflatoxin B1 (AFB1). It is unlikely that direct binding of small molecules such like AFB1 to the piezoelectric sensor surface could result in a satisfactory detection limit and sensitivity. Thus, indirect competitive immunoassay technique had been used for the detection of the target and gold nanoparticles (GNP) been employed as a‘weight label’to the secondary antibody for amplifying the response. This method is proven in its ability to detect AFB1 down to a level of 0.01 ng mL-1 in artificially contaminated milk, which is comparable to or even exceeding the sensitivity of microtitre plate ELISA. Furthermore, the frequency responses of the immunoassay are linearly correlated to the logarithm of AFB1 concentration in the range of 0.10 ~ 100 ng mL-1. The sensor could be regenerated under very mild conditions simply by immersing the sensor into glycine buffer solution to desorb the combined antibody. It is found that the as-renewed sensor could be reused at least 9 runs without obvious loss of sensing sensitivity (Chapter 2).
(2) An ultrasensitive piezoelectric method for the detection of the aflatoxin B1 based on the indirect competitive immunoassay and the biocatalyzed deposition amplification has been developed. In this method, the quartz crystal surface was coated with a self-assembled monolayer of 3-mercaptopropionic acid (MPA) for covalently immobilization of the BSA-AFB1 conjugate, which could compete with the free AFB1 for binding to the anti-AFB1 antibody (MsIgG). After the competitive immunoreaction, the horseradish peroxidase (HRP) labeled goat anti-mouse IgG (G-Anti-MsIgG) was introduced into the detection cell to combine with the anti-AFB1 antibody on the crystal surface. The enzyme labeled G-Anti-MsIgG as a biocatalyst could accelerate the oxidation of 4-chloro-1-naphthol by H2O2 to yield the insoluble product benzo-4-chlorohexadienone on the surface of quartz crystal microbalance (QCM), resulting in a mass increase that was reflected by a decrease in the resonance frequency of the QCM. The proposed approach could allow for the determination of AFB1 in the concentration range of 0.01 ~ 10.0 ng mL-1. Furthermore, several artificially contaminated milk samples were analyzed with good recoveries obtained, which demonstrated the suitability of the proposed method for detecting AFB1 (Chapter 3).
(3) A simple piezoelectric immunoagglutination assay technique with antibody-modified nanoparticles has been developed for direct quantitative detection of protein. The proposed technique is based on the specific agglutination of goat anti-hIgG-coated silica nanoparticles in the presence of human immunoglobulin G (hIgG),which causes a frequency change and is monitored by a piezoelectric device. The antibody modified on the probe surface would combine with antibody-coated nanoparticles in the presence of antigen (hIgG) when the surface agglutination reaction took place, which couples both the mass effect and viscoelastic effect acting on the probe. The results indicate that the background interference can be substantially minimized and the probe signal can be observably multiplied. In addition, the surfaces of the modified probe and that after combining the complex of immunoagglutination were imaged by scanning electronic microscopy (SEM). Moreover, an optimization of assay medium composition with the addition of poly(ethylene glycol) (PEG) serving as immunoagglutination enhancer and sodium chloride to control the ion-strength was investigated. The frequency responses of the immunoagglutination assay were found to correlate well with the hIgG concentration with a detection limit of 0.084μg mL-1 (Chapter 4).
(4) Try to use quartz crystal microbalance as a sensor on its surface fixed with a hairpin DNA probes used to identify and target DNA to produce a signal to build a simple and efficient DNA detection method. It features combined with restriction endonuclease ECoR I and nano-gold-labeled detection probe that can reduce the background of a very good value and effective way to boost the signal. The program are as follows: first, the quartz crystal surface through the thiol-based self-assembly of a fixed period with a hairpin DNA probes can be part of its hairpin endonuclease ECoR I specifically recognize and cut. Mercapto-hexanol then closed, then the role of the target DNA. If the system under test contains a target DNA, the DNA probe of the hairpin turn and the subsequent enzyme is not their role, and can be combined with nano-gold-labeled detection probe making signal amplification. Otherwise, in the absence of target DNA, the hairpin DNA probe can be digested, and nano-Au labeled probes role can not produce any signal. The experimental results show that this method is a simple and practical, high sensitivity analytics adapting the detection of DNA (Chapter 5).
(5) In the present approach, we revealed a new scheme for electrochemical detection the target DNA sequence using Fc-labeled oligonucleotide as a molecular switch, which is a signal-on sensor featuring both generalizability and simplicity in design toward reagentless detection of DNA. The present approach has been demonstrated with the identification of a single-base mutation in Hb Constant Spring codon 142 (terminating codon TAA→CAA) that is one of the major types ofα-thalassemia point mutations for clinical diagnosis. After reaction with various concentration of target DNA under the optimum experimental conditions, the calibration curve was plotted. The results showed that the current intensity was linear to the logarithm of the target concentration in the range from 0.01 to 100 pM with a detection limit 0.01 pM. The experiment found that the DNA sensor could be reused via immersed into NaOH solution to be easily and successfully regenerated. All these features revealed that the system is a promising candidate for single-base mutation discrimination, owing the advantages of both generalizability and simplicity toward reagentless detection of DNA with sensitivity and selectivity (Chapter 6).
引文
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