蛋白质和DNA多组分化学发光检测新技术的研究
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摘要
蛋白质和DNA的多组分同时检测是目前临床诊断、环境监测、微生物检验等领域的一个研究热点,具有分析通量高、所需时间短、样品消耗少、分析成本低等突出优点。目前多组分检测通常采用标记物识别模式或空间位置识别模式,但由于受到标记物数目以及所用仪器设备较为昂贵等因素的影响,在一定程度上限制了多组分检测技术的广泛应用。
化学发光(CL)分析法不需光源,仪器设备简单、操作简便,具有极高的灵敏度,已成为近年来一个非常活跃的研究领域,目前已成功地应用在药学、生物学、分子生物学、临床医学和环境学等诸多领域。为此,本论文采用化学发光分析法,利用不同载体物化性质差异分离识别不同待测物,发展了三种具有创新意义的多组分同时检测技术,实现了同一份样品中多组分的同时检测,整个论文由以下四部分构成:
第一章:绪论
本绪论由三节构成,其中第一节简单介绍了基于标记物识别模式的多组分检测技术的研究进展及其意义,主要内容包括:荧光标记、金属标记、酶标记等,并列举了近年来它们在该分析领域的部分典型示例;第二节中介绍了基于空间位置识别模式的多组分检测技术的研究进展及其意义,也列举了近年来它们在该分析领域的部分典型示例;第三节阐述了本博士论文的目的、意义、主要研究内容及创新之处。
第二章:基于不同载体的无标记多组分DNA化学发光检测新技术
无标记简单快速检测特定序列DNA在临床诊断、环境监测等领域具有非常重要的意义,尤其是无标记多组分同时检测技术,本章基于特异性化学发光试剂3,4,5-三甲氧基苯甲酰甲醛(TMPG)与鸟嘌呤(G)碱基之间的瞬时衍生反应,能够产生CL的原理,实现了单份样品中无标记多组分特定序列DNA的同时检测。具体采用磁珠、聚苯乙烯微球和温度敏感高分子(PNIP)三种载体,以乙型肝炎病毒(HBV)的三条DNA合成片段为例,优化构建了新型的基于不同载体的无标记多组分DNA化学发光检测新技术。整个分析过程由以下两步构成:(1)分别制备三种载体修饰探针、混合后与以上三条DNA合成片段在同一试管中杂交;(2)依据三种载体的物化性质差异分离洗涤,利用TMPG与目标DNA中的G碱基之间的瞬时衍生反应,产生特异性CL来定量三条DNA合成片段。结果表明:该方法具有操作简便、分析速度快和灵敏度高等特点,目标序列在0.1-6 pmol范围内具有良好的线性相关性,最低检测限可达100 fmol。为了进一步提高检测灵敏度,随后又采用G30放大检测技术,在载体修饰探针与DNA合成片段杂交后,再与一段富含G的放大序列(含有两个功能序列段:一是能够与目标序列的15个核苷酸单位杂交的序列段,另一个是富含G的放大信号序列段---(T2G15)2)进行第二次杂交,最低检测限可达15 fmol。方法选择性研究表明:本法对单碱基错配和完全不互补序列有较好的识别能力。综合而言:这种新型的载体识别无标记多组分DNA化学发光检测技术,不同于目前的多组分编码策略,首次采用不同载体物理化学差异分辨、瞬时衍生化学发光反应来同时检测三种目标序列DNA:其次采用仪器设备简单、操作简便的CL分析法来替代目前常用于多组分检测的流动注射或成像系统等昂贵设备,有望为临床、环境、生物防护等领域的DNA多组分定量分析提供有价值的手段。
第三章:基于不同载体的无标记多组分PCR扩增产物的化学发光检测新技术
第二章测定HBV的PCR真实样品的研究过程中发现:双链PCR扩增产物(PCR amplicon)只有一条链与探针互补,所以在PCR扩增产物检测时,探针会与扩增产物另一条链存在竞争杂交,这将显著降低检测灵敏度;不对称PCR扩增技术的使用虽可改善检测灵敏度,但PCR扩增效率也会因引物量的减少而下降。为此,本章旨在设计能够直接进行双链PCR产物同时检测的载体识别无标记多组分检测新技术,进一步提高和发展第二章所构建的无标记多组分检测技术,具体分析过程由以下三步构成:(1)通过改造上游引物的方式使三条PCR扩增产物上分别标记地高辛(digoxin),生物素(biotin)和荧光素(FITC);(2)三条标记物修饰的PCR扩增产物的混合物分别与相应的标记有抗地高辛(anti-digoxin),链霉亲和素(SA),抗荧光素(anti-FITC)的载体混合物发生免疫反应;(3)依据三种载体的物化性质差异分离洗涤,利用TMPG与PCR产物中的G碱基之间的瞬时衍生反应,产生特异性CL来定量三条PCR产物。相比于目前的PCR检测方法,本法具有以下特点,第一,传统的PCR检测技术多采用PCR产物95℃加热解链实现竞争杂交反应,我们的检测可直接检测双链PCR产物,无需将双链DNA加热变性,避免了竞争杂交对检测灵敏度的影响;第二,多组分实时PCR技术必须采用发射波长不相互交叉的荧光标记物来实现多组分同时检测;本法采用瞬时衍生载体分辨无标记检测技术,直接进行双链PCR产物的多组分同时检测;第三,实时PCR仪器设备相对昂贵,而化学发光仪仪器设备简单、操作简便。综合而言,本章发展的载体分辨无标记多组分PCR扩增产物化学发光检测新技术,具有简单,快速,灵敏度高等特点,有望在临床诊断、环境监测、微生物检验等领域发挥作用。
第四章:基于温度敏感的四种蛋白质同时检测化学发光新技术
鉴于以上两章中多组分DNA检测技术取得的成果,本章尝试将这种新型的载体分辨化学发光技术拓宽至多种蛋白质的同时检测。为此,本章以免疫球蛋白IgG、IgM、IgA和蛋白多肽药物GH作为四个模型蛋白质分析物,采用磁珠和温敏高分子两种载体以及碱性膦酸酯酶(AP)和辣根过氧化物酶(HRP)两种标记物,构建了一个均相的非竞争的ELISA反应,实现了四种蛋白质的化学发光同时检测。整个分析过程由以下三步构成:(1)分别制备两对第一抗体偶联物:抗IgG-磁珠、抗IgM-磁珠、抗IgA-PNIP和抗GH-PNIP,混合并与四种抗原混合物IgG、IgM、IgA和GH在同一试管中进行免疫反应:(2)再与四种标记了HRP、AP的第二抗体混合物进行夹心反应:(3)依据两种载体的性质差异进行分离洗涤,将两种载体各均分两份,用相应的试剂盒进行检测。研究结果表明:该法IgG、IgM、IgA和GH的检测限分别为0.5,0.5,1.0和0.5 ng/mL。此外,还可通过引入不同的载体,如:对不同温度敏感的温敏高分子或对不同PH值敏感的PH敏感高分子;以及采用多种标记物,如采用酶(HRP和ALP)或胶体金等,进一步增加分析物的数目,提高检测的灵敏度。综合而言,该技术有望在多组分蛋白质的分析、测定方面发挥重要的作用。
Simultaneous detection of multiplex has gained considerable interest in clinical, environmental,and biodefense applications,etc.Numerous multianalyte assays have been developed due to its high sample throughput,short assay time,low sample consumption and reduced overall cost,as compared to parallel single-analyte assays. In recent years,considerable effort is mainly focused on developing multi-label-based multianalyte assay methods.Most of the current developed approaches for multianalyte are based on spatially resolution or multiple-labels mode.However,there are some restrictions in the number of labels and,therefore,of analytes that could be determined simultaneously.
Chemiluminescence(CL) has been exploited within a wide range of applications in various fields,due to their extremely high sensitivity along with their extra advantages such as simple instrumentation,wide calibration ranges,and suitability for miniaturization in analytical chemistry.In contrast to current multi-label-based detection techniques,we took good advantage of three different kinds of carriers to separate targets by thermo-triggered precipitation,and one label rather than multiple was employed.According to their character in common,all of them could be seperated by centrifugation at 35℃.Meanwhile,each of them has its unique attributes;namely,they can be seperated from each by magnetic force,centrifugation under room temperature and 35℃,respectively.Description of research in my thesis is presented as follows:
Chapter 1:This thesis first addresses multi-label-based detection techniques,and then presents current state of knowledge,including fluorescent-labe,enzyme-labe,etc. The second phase of this chapter reviews current main spatially resolution mode and its applications in various fields,and discusses the development in this field. Following that,objectives and significance of this research are summarized.
Chapter 2:Instantaneous Derivatization Technology for Simultaneous and Homogeneous Determination of Multiple DNA Targets
There are potential advantages,in terms of simplicity and speed,for detecting DNA hybridization steps directly without using any external labels,especially for the multiplexed assays.In the current paper,we describe the use of a carrier-resolved label-free multiplexed assay for the simultaneous detection of multiple DNA targets. Herein we demonstrate that this protocol,using three homogeneous carriers' thermosensitive poly(N-isopropylacrylamide),polystyrene beads,and magnetic beads, respectively,for simultaneous determination of three short DNA fragments specific to hepatitis B virus.Briefly,one hybridization occurs between a mixture of three different capture probe DNAs immobilized onto three carriers and three targets in a single vessel,and then chemiluminescence(CL) detection proceeds via an instantaneous derivatization reaction between the specific CL reagent 3,4,5-trimethoxylphenylglyoxal(TMPG) and the guanine nucleotide-rich regions within the target DNA.An excellent linearity is found within the range between 0.1 and 6.0 pmol with the lowest detection limit of 100 fmol.In contrast to current encoding strategies,every hybridization signal for the corresponding DNA target in our protocol is uniquely immobilized onto one carrier vehicle with a unique and intrinsic physical-chemical signature.Moreover,an instantaneous derivatization reaction is employed for the label-free determination of three targets in a single vessel. In addition,a simple CL setup is employed to read the carrier code instead of an expensive and complicated flow cytometer or imaging system commonly used for multiplexed assays.Further signal amplification is achieved by employing three amplified DNAs for second hybridization,which include a guanine nucleobase-rich sequence domain for the generation of light and an additional tethered nucleic acid domain complementary with one of the target DNA as an amplification platform. Such simple amplified CL transduction allows detection of DNA targets down to the 15-fmol level.This new protocol also provided a good capability in discriminating perfectly complementary DNA from single-base mismatches and noncomplementary sequences.Overall,the protocol described here may have value in a variety of clinical, environmental,and biodefense applications for which the accurate quantitative analysis of multiple DNA targets is desired.
Chapter 3:Carrier-resolved Technology for Homogeneous and Simultaneous Detection of Multiple PCR Amplicons
A general purpose label-free chemiluminescence(CL) platform is developed wherein polymerase chain reaction(PCR) amplicons labeled with one hapten are detected via an instantaneous derivatization reaction between the specific CL reagent 3,4,5-trimethoxyl-phenylglyoxal(TMPG) and the guanine nucleotides within the PCR target DNA.As a proof of principle,the response selectivity of this platform is evauated for simultaneous determination of three PCR amplicons specific to Hepatitis B Virus(HBV) by using three homogeneous carriers magnetic beads,polystyrene beads,and thermo-sensitive poly-N-isopropylacrylamide(PNIP),respectively. Briefly,PCR amplicons are labeled with digoxin,biotin or FITC via the modified up-stream primers.After PCR amplification,the immunoreactions occur between a mixture of three target PCR amplicons and three modified carriers with either anti-digoxin,streptavidin or anti-FITC in a single vessel and then each carrier is separated from the others under different conditions based on their attributes for direct CL detection.Different from conventional endpoint PCR detection techniques where dsDNA PCR amplicons have to be denatured(e.g.,heated to 95℃for a few minutes) before the detection hybridization,firstly this new protocol offers the enhanced sensitivity due to the absence of competitive hybridization.Secondly, multiplex real-time PCR required the use of multiple fluorophores with emission peaks that can be unequivocally separated,whereas herein an instantaneous derivatization reaction is employed for the label-free determination of three targets in a single vessel,and every signal for the corresponding PCR amplicon in our protocol is uniquely immobilized onto one carrier with a unique and intrinsic physical-chemical signature.Thirdly,specialized expensive equipment is required for the detection of real-time PCR amplicons whereas herein a simple CL setup was employed to perform our novel multiplexed PCR assays.Overall,the assay is sentive,safe,and cost-effetive when compared to conventional agarose gel electrophoresis,real-time PCR for the detection of PCR amplicons.Furthermore, the protocol described here may have value in a variety of clinical,environmental, and biodefense applications for which the accurate quantitative analysis of multiple PCR amplicons is desired.
Chapter 4:Sequential Determination of Four Proteins by Temperature-Triggered Homogeneous Chemiluminescent Immunoassay
A novel protocol for performing a sequential multiplex immunoassay,based on a temperature-triggered separation/mixing process and alkaline phosphatase(AP) and horseradish peroxidase(HRP) catalyzed chemiluminescence(CL) detection,is described.Herein we introduce poly(N-isopropylacrylamide)(PNIP) and magnetic beads as bimolecular immobilizing carriers to separate different antigens by taking advantage of thermal response.PNIP is known to aggregate and precipitate out of water when the temperature is raised above the lower critical solution temperature (LCST) of 31℃;thus,it can be separated from supernatant by centrifugation.Besides, magnetic beads can be separated from PNIP by magnetic force as the temperature is lower than LCST.A homogeneous noncompetitive ELISA was employed,formed by using IgG,IgM,IgA,and growth hormone(GH) as two couples of carrier analytes, two couples of capture antibodies were immobilized onto the surface of magnetic beads and PNIP,respectively and AP and HRP labeled second antibodies.With a sandwich format,highly sensitive CL detection of HRP was applied,and the detection limits ofIgG and IgA were as low as 2.0 and 1.5 ng/mL,respectively.
化学发光(CL)分析法不需光源,仪器设备简单、操作简便,具有极高的灵敏度,已成为近年来一个非常活跃的研究领域,目前已成功地应用在药学、生物学、分子生物学、临床医学和环境学等诸多领域。为此,本论文采用化学发光分析法,利用不同载体物化性质差异分离识别不同待测物,发展了三种具有创新意义的多组分同时检测技术,实现了同一份样品中多组分的同时检测,整个论文由以下四部分构成:
第一章:绪论
本绪论由三节构成,其中第一节简单介绍了基于标记物识别模式的多组分检测技术的研究进展及其意义,主要内容包括:荧光标记、金属标记、酶标记等,并列举了近年来它们在该分析领域的部分典型示例;第二节中介绍了基于空间位置识别模式的多组分检测技术的研究进展及其意义,也列举了近年来它们在该分析领域的部分典型示例;第三节阐述了本博士论文的目的、意义、主要研究内容及创新之处。
第二章:基于不同载体的无标记多组分DNA化学发光检测新技术
无标记简单快速检测特定序列DNA在临床诊断、环境监测等领域具有非常重要的意义,尤其是无标记多组分同时检测技术,本章基于特异性化学发光试剂3,4,5-三甲氧基苯甲酰甲醛(TMPG)与鸟嘌呤(G)碱基之间的瞬时衍生反应,能够产生CL的原理,实现了单份样品中无标记多组分特定序列DNA的同时检测。具体采用磁珠、聚苯乙烯微球和温度敏感高分子(PNIP)三种载体,以乙型肝炎病毒(HBV)的三条DNA合成片段为例,优化构建了新型的基于不同载体的无标记多组分DNA化学发光检测新技术。整个分析过程由以下两步构成:(1)分别制备三种载体修饰探针、混合后与以上三条DNA合成片段在同一试管中杂交;(2)依据三种载体的物化性质差异分离洗涤,利用TMPG与目标DNA中的G碱基之间的瞬时衍生反应,产生特异性CL来定量三条DNA合成片段。结果表明:该方法具有操作简便、分析速度快和灵敏度高等特点,目标序列在0.1-6 pmol范围内具有良好的线性相关性,最低检测限可达100 fmol。为了进一步提高检测灵敏度,随后又采用G30放大检测技术,在载体修饰探针与DNA合成片段杂交后,再与一段富含G的放大序列(含有两个功能序列段:一是能够与目标序列的15个核苷酸单位杂交的序列段,另一个是富含G的放大信号序列段---(T2G15)2)进行第二次杂交,最低检测限可达15 fmol。方法选择性研究表明:本法对单碱基错配和完全不互补序列有较好的识别能力。综合而言:这种新型的载体识别无标记多组分DNA化学发光检测技术,不同于目前的多组分编码策略,首次采用不同载体物理化学差异分辨、瞬时衍生化学发光反应来同时检测三种目标序列DNA:其次采用仪器设备简单、操作简便的CL分析法来替代目前常用于多组分检测的流动注射或成像系统等昂贵设备,有望为临床、环境、生物防护等领域的DNA多组分定量分析提供有价值的手段。
第三章:基于不同载体的无标记多组分PCR扩增产物的化学发光检测新技术
第二章测定HBV的PCR真实样品的研究过程中发现:双链PCR扩增产物(PCR amplicon)只有一条链与探针互补,所以在PCR扩增产物检测时,探针会与扩增产物另一条链存在竞争杂交,这将显著降低检测灵敏度;不对称PCR扩增技术的使用虽可改善检测灵敏度,但PCR扩增效率也会因引物量的减少而下降。为此,本章旨在设计能够直接进行双链PCR产物同时检测的载体识别无标记多组分检测新技术,进一步提高和发展第二章所构建的无标记多组分检测技术,具体分析过程由以下三步构成:(1)通过改造上游引物的方式使三条PCR扩增产物上分别标记地高辛(digoxin),生物素(biotin)和荧光素(FITC);(2)三条标记物修饰的PCR扩增产物的混合物分别与相应的标记有抗地高辛(anti-digoxin),链霉亲和素(SA),抗荧光素(anti-FITC)的载体混合物发生免疫反应;(3)依据三种载体的物化性质差异分离洗涤,利用TMPG与PCR产物中的G碱基之间的瞬时衍生反应,产生特异性CL来定量三条PCR产物。相比于目前的PCR检测方法,本法具有以下特点,第一,传统的PCR检测技术多采用PCR产物95℃加热解链实现竞争杂交反应,我们的检测可直接检测双链PCR产物,无需将双链DNA加热变性,避免了竞争杂交对检测灵敏度的影响;第二,多组分实时PCR技术必须采用发射波长不相互交叉的荧光标记物来实现多组分同时检测;本法采用瞬时衍生载体分辨无标记检测技术,直接进行双链PCR产物的多组分同时检测;第三,实时PCR仪器设备相对昂贵,而化学发光仪仪器设备简单、操作简便。综合而言,本章发展的载体分辨无标记多组分PCR扩增产物化学发光检测新技术,具有简单,快速,灵敏度高等特点,有望在临床诊断、环境监测、微生物检验等领域发挥作用。
第四章:基于温度敏感的四种蛋白质同时检测化学发光新技术
鉴于以上两章中多组分DNA检测技术取得的成果,本章尝试将这种新型的载体分辨化学发光技术拓宽至多种蛋白质的同时检测。为此,本章以免疫球蛋白IgG、IgM、IgA和蛋白多肽药物GH作为四个模型蛋白质分析物,采用磁珠和温敏高分子两种载体以及碱性膦酸酯酶(AP)和辣根过氧化物酶(HRP)两种标记物,构建了一个均相的非竞争的ELISA反应,实现了四种蛋白质的化学发光同时检测。整个分析过程由以下三步构成:(1)分别制备两对第一抗体偶联物:抗IgG-磁珠、抗IgM-磁珠、抗IgA-PNIP和抗GH-PNIP,混合并与四种抗原混合物IgG、IgM、IgA和GH在同一试管中进行免疫反应:(2)再与四种标记了HRP、AP的第二抗体混合物进行夹心反应:(3)依据两种载体的性质差异进行分离洗涤,将两种载体各均分两份,用相应的试剂盒进行检测。研究结果表明:该法IgG、IgM、IgA和GH的检测限分别为0.5,0.5,1.0和0.5 ng/mL。此外,还可通过引入不同的载体,如:对不同温度敏感的温敏高分子或对不同PH值敏感的PH敏感高分子;以及采用多种标记物,如采用酶(HRP和ALP)或胶体金等,进一步增加分析物的数目,提高检测的灵敏度。综合而言,该技术有望在多组分蛋白质的分析、测定方面发挥重要的作用。
Simultaneous detection of multiplex has gained considerable interest in clinical, environmental,and biodefense applications,etc.Numerous multianalyte assays have been developed due to its high sample throughput,short assay time,low sample consumption and reduced overall cost,as compared to parallel single-analyte assays. In recent years,considerable effort is mainly focused on developing multi-label-based multianalyte assay methods.Most of the current developed approaches for multianalyte are based on spatially resolution or multiple-labels mode.However,there are some restrictions in the number of labels and,therefore,of analytes that could be determined simultaneously.
Chemiluminescence(CL) has been exploited within a wide range of applications in various fields,due to their extremely high sensitivity along with their extra advantages such as simple instrumentation,wide calibration ranges,and suitability for miniaturization in analytical chemistry.In contrast to current multi-label-based detection techniques,we took good advantage of three different kinds of carriers to separate targets by thermo-triggered precipitation,and one label rather than multiple was employed.According to their character in common,all of them could be seperated by centrifugation at 35℃.Meanwhile,each of them has its unique attributes;namely,they can be seperated from each by magnetic force,centrifugation under room temperature and 35℃,respectively.Description of research in my thesis is presented as follows:
Chapter 1:This thesis first addresses multi-label-based detection techniques,and then presents current state of knowledge,including fluorescent-labe,enzyme-labe,etc. The second phase of this chapter reviews current main spatially resolution mode and its applications in various fields,and discusses the development in this field. Following that,objectives and significance of this research are summarized.
Chapter 2:Instantaneous Derivatization Technology for Simultaneous and Homogeneous Determination of Multiple DNA Targets
There are potential advantages,in terms of simplicity and speed,for detecting DNA hybridization steps directly without using any external labels,especially for the multiplexed assays.In the current paper,we describe the use of a carrier-resolved label-free multiplexed assay for the simultaneous detection of multiple DNA targets. Herein we demonstrate that this protocol,using three homogeneous carriers' thermosensitive poly(N-isopropylacrylamide),polystyrene beads,and magnetic beads, respectively,for simultaneous determination of three short DNA fragments specific to hepatitis B virus.Briefly,one hybridization occurs between a mixture of three different capture probe DNAs immobilized onto three carriers and three targets in a single vessel,and then chemiluminescence(CL) detection proceeds via an instantaneous derivatization reaction between the specific CL reagent 3,4,5-trimethoxylphenylglyoxal(TMPG) and the guanine nucleotide-rich regions within the target DNA.An excellent linearity is found within the range between 0.1 and 6.0 pmol with the lowest detection limit of 100 fmol.In contrast to current encoding strategies,every hybridization signal for the corresponding DNA target in our protocol is uniquely immobilized onto one carrier vehicle with a unique and intrinsic physical-chemical signature.Moreover,an instantaneous derivatization reaction is employed for the label-free determination of three targets in a single vessel. In addition,a simple CL setup is employed to read the carrier code instead of an expensive and complicated flow cytometer or imaging system commonly used for multiplexed assays.Further signal amplification is achieved by employing three amplified DNAs for second hybridization,which include a guanine nucleobase-rich sequence domain for the generation of light and an additional tethered nucleic acid domain complementary with one of the target DNA as an amplification platform. Such simple amplified CL transduction allows detection of DNA targets down to the 15-fmol level.This new protocol also provided a good capability in discriminating perfectly complementary DNA from single-base mismatches and noncomplementary sequences.Overall,the protocol described here may have value in a variety of clinical, environmental,and biodefense applications for which the accurate quantitative analysis of multiple DNA targets is desired.
Chapter 3:Carrier-resolved Technology for Homogeneous and Simultaneous Detection of Multiple PCR Amplicons
A general purpose label-free chemiluminescence(CL) platform is developed wherein polymerase chain reaction(PCR) amplicons labeled with one hapten are detected via an instantaneous derivatization reaction between the specific CL reagent 3,4,5-trimethoxyl-phenylglyoxal(TMPG) and the guanine nucleotides within the PCR target DNA.As a proof of principle,the response selectivity of this platform is evauated for simultaneous determination of three PCR amplicons specific to Hepatitis B Virus(HBV) by using three homogeneous carriers magnetic beads,polystyrene beads,and thermo-sensitive poly-N-isopropylacrylamide(PNIP),respectively. Briefly,PCR amplicons are labeled with digoxin,biotin or FITC via the modified up-stream primers.After PCR amplification,the immunoreactions occur between a mixture of three target PCR amplicons and three modified carriers with either anti-digoxin,streptavidin or anti-FITC in a single vessel and then each carrier is separated from the others under different conditions based on their attributes for direct CL detection.Different from conventional endpoint PCR detection techniques where dsDNA PCR amplicons have to be denatured(e.g.,heated to 95℃for a few minutes) before the detection hybridization,firstly this new protocol offers the enhanced sensitivity due to the absence of competitive hybridization.Secondly, multiplex real-time PCR required the use of multiple fluorophores with emission peaks that can be unequivocally separated,whereas herein an instantaneous derivatization reaction is employed for the label-free determination of three targets in a single vessel,and every signal for the corresponding PCR amplicon in our protocol is uniquely immobilized onto one carrier with a unique and intrinsic physical-chemical signature.Thirdly,specialized expensive equipment is required for the detection of real-time PCR amplicons whereas herein a simple CL setup was employed to perform our novel multiplexed PCR assays.Overall,the assay is sentive,safe,and cost-effetive when compared to conventional agarose gel electrophoresis,real-time PCR for the detection of PCR amplicons.Furthermore, the protocol described here may have value in a variety of clinical,environmental, and biodefense applications for which the accurate quantitative analysis of multiple PCR amplicons is desired.
Chapter 4:Sequential Determination of Four Proteins by Temperature-Triggered Homogeneous Chemiluminescent Immunoassay
A novel protocol for performing a sequential multiplex immunoassay,based on a temperature-triggered separation/mixing process and alkaline phosphatase(AP) and horseradish peroxidase(HRP) catalyzed chemiluminescence(CL) detection,is described.Herein we introduce poly(N-isopropylacrylamide)(PNIP) and magnetic beads as bimolecular immobilizing carriers to separate different antigens by taking advantage of thermal response.PNIP is known to aggregate and precipitate out of water when the temperature is raised above the lower critical solution temperature (LCST) of 31℃;thus,it can be separated from supernatant by centrifugation.Besides, magnetic beads can be separated from PNIP by magnetic force as the temperature is lower than LCST.A homogeneous noncompetitive ELISA was employed,formed by using IgG,IgM,IgA,and growth hormone(GH) as two couples of carrier analytes, two couples of capture antibodies were immobilized onto the surface of magnetic beads and PNIP,respectively and AP and HRP labeled second antibodies.With a sandwich format,highly sensitive CL detection of HRP was applied,and the detection limits ofIgG and IgA were as low as 2.0 and 1.5 ng/mL,respectively.
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