用于蛋白检测的生物条码技术与方法的研究
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摘要
目前癌症是发达国家和大多数发展中国家一个主要的健康问题。每年约有1200万人诊断为肿瘤,其中近800万人死亡。肿瘤治疗的重点在于及早明确诊断,而诊断的关键在于肿瘤在其早期就可被发现与诊断。早期肿瘤的治疗效果要明显优于中晚期肿瘤,同时费用也较中晚期肿瘤明显降低。人们一直在探索、寻求并希望获得肿瘤出现前或是刚刚发生时的标志,借以对肿瘤给予有效、及时的诊断与治疗。相对于肿瘤发生的组织学的改变,肿瘤标志物已成为肿瘤早期诊断和治疗有效的依据。因此目前肿瘤标志物是肿瘤早期诊断有效的客观指标之一已为人所共识。
目前认为肿瘤标志物分为三类,第一种是血清标志物,第二种是细胞标志物,第三种为分子与遗传标志。肿瘤标志物检测方法有酶联免疫法、放射免疫法、免疫荧光测定、放射免疫显像、自动化免疫分析和免疫组化以及目前多用于生物学领域的分子生物学技术等等。但现有的这些检测方法灵敏度尚难以达到在肿瘤早期即发现某些肿瘤标志物含量或水平增高或异常,而多是在肿瘤发展到一定程度或阶段才检测到某些肿瘤标志物水平或含量的异常；或是难以进行高通量的检测；或因检测方法价格昂贵难以进行大规模的普查与重点筛查。
目前以寡核苷酸链为基础的生物条码技术的研究与应用非常广泛。生物条码可以以指数级扩增信号,从而提高检测的灵敏度。目前生物条码结合比色、银染以及电化学发光等技术研究发展迅猛。灵敏度也较上述常用检测方法有明显提高。基因芯片的特点是具备高通量,同时可检测多个指标。将生物条码技术的高灵敏度与基因芯片的高通量相结合针对肿瘤标志物的水平或含量进行检测,在现有基础上对肿瘤的早期诊断具有重要的意义。
本研究应用生物条码与基因芯片对蛋白抗原IgG的水平进行检测。不但可以提高检测的灵敏度,还可以具有检测高通量的特点,可使多个靶标同时得以检测,提高了检测效率。通过本研究可以为针对多个包括蛋白类的指标的检测方法与技术的研究铺垫良好的基础。
本论文具体研究内容如下：
一、分子信标的研究与应用
分子信标是一种特殊的生物条码。是一种设计精妙的新型荧光标记的核酸探针。特殊的发夹结构使其具有很强的特异性识别靶序列的能力。目前分子信标已成为分子生物学和生物技术中一种应用广泛的研究工具。本研究设计了三条不同序列的分子信标。通过应用不同序列的分子信标检测待测蛋白抗原浓度,其测定最小值达到6.67 fM(1 pg/ml)。上述研究证实了应用分子信标技术测定蛋白抗原浓度的可行性、灵敏度以及特异性。
二、量子点的研究与应用
量子点又称为半导体纳米微晶体,是一种由Ⅱ-Ⅵ族或Ⅲ-Ⅴ族元素组成的稳定的、可溶于水、尺寸在2-20 nm之间的纳米晶粒。目前量子点作为荧光探针在各领域的研究中应用非常广泛。与传统的荧光探针相比,量子点优势显著。本研究应用量子点标记生物条码对待测蛋白抗原进行检测,结果稳定可靠。可知测定待测蛋白抗原IgG的浓度最小值为333.5 fM(50 pg／ml)。但尚存在偶联定量的问题。需要进一步研究与探索。
三、寡核苷酸生物条码的研究与应用
自行设计三条不同序列,各由60个碱基组成,用于制备基因芯片用单链DNA生物条码,并由此产生与之部分碱基序列完全互补的三条序列不同的,各由40个碱基组成的单链DNA生物条码。应用待测蛋白抗原IgG的单克隆抗体标记粒径为2-3μm,C3间隔臂胺基修饰的的磁性微球；40个碱基组成的单链DNA生物条码5’端标记荧光素Cy3,3’端标记巯基。然后应用上述双标记的单链DNA生物条码与待测蛋白抗原IgG的多克隆抗体双标记自行制备,粒径为13 nm的纳米金颗粒。通过测定标记荧光素的生物条码的荧光强度,可知测定待测蛋白抗原IgG的最小浓度值为133.4 fM(20 pg／ml)。上述研究证实了应用寡核苷酸生物条码技术测定待测蛋白抗原浓度的可行性、灵敏度以及特异性。为与基因芯片结合生物条码技术的进一步研究与应用奠定基础。
四、基因芯片的研究与应用
随着后基因组时代到来而发展起来的基因芯片技术具有高通量的优势,可同时对同一样本中多种不同的靶标进行分析和测定。本研究应用自行设计,由60个碱基组成的单链DNA生物条码制备基因芯片。通过对待测蛋白抗原进行检测,其灵敏度也可达6.67 fM。结果同时显示应用基因芯片技术具有较好的特异性与高通量的特点。我们分别应用三条不同序列的生物条码制备的基因芯片进行检测,结果具有良好的一致性,进一步证实该方法的稳定与可靠性。
综上所述,本实验分别应用生物条码、量子点、分子信标与基因芯片等技术与方法结合磁性微球与纳米金等生物材料对蛋白抗原水平进行微量检测的可行性、稳定性及检测的灵敏度与特异性进入了比较深入的研究和探索。应用生物条码与基因芯片检测蛋白浓度的方法可行稳定,灵敏度到达6.67 fM,具有进一步研究与开发的潜能。分子信标与量子点在与上述生物材料结合应用方面上有很多问题需要进一步澄清与明确。该研究为进行肿瘤标志物的筛选与检测奠定了良好的基础。
At present, cancer remains a major problem in the developed and most developing countries, and there are approximately 12 million people will be diagnosed with cancer and more 8 million were died every year. The focus of cancer therapy is the diagnosis, and the key question is detected and diagnosed at the early stage of cancer in good time. The early treatment is obviously superior than its advanced stage, and the costs are significantly lower too. People have been doing and exploring the tumor marker indicating the tumorgenesis so as to diagnose and provide effective treatment progressively in time. Tumor marker has been an effective basis for the diagnosis and effective treatment of cancer at its early stage in comparing with the changes of cytohistology at the early stage of tumor. And now, it has been recognized by most people that many tumor markers are one of the effective objective index in diagnosising at early stage of tumor.
There are classified three kinds of tumor markers, the serum tumor marker, the cellular tumor markers, and the molecular and genetic tumor markers. There are many measurement methods to detect the tumor marker, including ELISA, RIA, FIA, RII, IHC, automatic immunoassay system and molecular biology techniques applied in the molecular biology research now. But all the methods sensitivity is limited, and it is difficult to achieve a certain degree to diagnose the cancer at its early stage effectively, or only detect at its advanced stage, or is short of the characteristics of high throughput and difficult to be applied on a large scale general census and screening because of its expensive costs.
The bio-bar-code technology based on the single-strand oligonucleotide is researched applied very comprehensively at present. The bio-bar-code technology can improve the detection sensitivity greatly by its cascade amplification. The research on the the bio-bar-code technology combining with some other methods, such as the scanometric, silver stain and electrochemiluminescence, has made a great progress now, and the detection sensitivity is improved more than before methods mentioned above. The characteristics of genechip are of high throughput, and it can detect many target markers at the same time. It will be of very important significance to detect the concentration or content by the bio-bar-code technology with more high sensitivity combing with the high throughput of genechip.
This research focuses on the detection the concentration of the protein antigen IgG applying with the bio-bar-code and genechip. It not only improves the detection sensitivity, but also be of the characteristics with more high throughput to detect many taget markers at one time, and this method improves the detection efficiency greatly. This research paves the way for the study and research on the detection of many protein target markers at the same time, and including the tumor marker.
The major contents of this paper are shown as follows:
1. The research and application of molecular beacon (MB) Molecular beacon is a special kind of bio-bar-code prober, and is thought to be a new type fluorescein-labeled bio-bar-code probes with the exquisite design. MB is of more strong ability to specifically identify the target sequence for its extraordinary hair-pin structure. And now it has became a powerful research tool in the molecular biology and biotechnology. We designed three different sequence MB for the experiment to detect the target antigen, and the detection limit is 6.67 fM. The research proved the method to be of the availability, sensitivity and speciality in measuring the target antigen.
2. The research and application of the Quantum Dot (QD)
Quantum Dot called semiconductor nanocrystal is a kind of stable, soluble nanocrystal made from the second to sixth group or third to fifth group element, and its diameter is between two to twenty nanometer. QD has been applied in many scopes comprehensively as the fluorescence probes now. There are many advantages of it comparing with the general fluorescence probes. We detected the target antigen applying with the bio-bar-code labeled QD, and the results are stable and reliable. But there are some questions on how to confirm the coupling quantities to be solved.
3. The research and application of the oligonucleotide bio-bar-code assay
Designing three different sequence bio-bar-code, made of 60 bases, are prepared the genechip, hence there are three corresponding different sequence, consist of 40 bases, complementary to the sequence above. The monoclonal-antibody IgG was labeled on the amino-modified magnetic microparticles (MMPs) with C3 space arm, and the diameter of MMPs is two to three micrometer. And the single-strand DNA bio-bar-code was labeled the fluorescein Cy3 and the thiol-group on the terminal of its 5'and the 3'respectively. Then we modified gold nanoparticles (Au-NPs) with above dual-labeled bio-bar-codes and polyclonal-antibody IgG, Au-NPs diameter is about 13 nanometers. We detected the fluorescence intensity of the bio-bar-code to get the low limit of the target protein, and the minimum concentration is 133.4 fM. Ours research proved the availability, sensitivity and specificity of the bio-bar-code assay. It paved the way for the progressive research and application of the genechip combining with the bio-bar-code assay.
4. The research and application of genechip
With the advent of post-genomic era, the developed high-throughput gene chip technology has the characteristics of analyzing and measuring many different target markers in the same sample at one time. We prepared genechip with the single-strand DNA bio-bar-code consisted of 60 bases designed by ours. The sensitivity of the method reached to 6.67 fM too, and the method is of more better specificity and high throughput. We applied with three different sequence bio-bar-code to detect the same target antigen and come to the consistent results, it proved the stability and reliability of the genechip technology.
All in brief, the study proved the availability, stability, sensitivity and specificity of the microconcentration detection method of target protein antigen applying the bio-bar-code assay, quantum dot, molecular beacon and genechip technology with the biomaterials of MMPs and Au-NPs. It showed available and stable of applying the bio-bar-code assay with the genechip technology, the sensitivity could reach to fetomolar level, and about 6.67 fM. The method will be of full potential in the research and application in the future. And there are many questions to be recognized and solved in applying the MB and QD with the biomaterials mentioned above. The research have paved a well way for the next study and applying in screening and measuring the tumor markers in the clinic at future.
目前认为肿瘤标志物分为三类,第一种是血清标志物,第二种是细胞标志物,第三种为分子与遗传标志。肿瘤标志物检测方法有酶联免疫法、放射免疫法、免疫荧光测定、放射免疫显像、自动化免疫分析和免疫组化以及目前多用于生物学领域的分子生物学技术等等。但现有的这些检测方法灵敏度尚难以达到在肿瘤早期即发现某些肿瘤标志物含量或水平增高或异常,而多是在肿瘤发展到一定程度或阶段才检测到某些肿瘤标志物水平或含量的异常；或是难以进行高通量的检测；或因检测方法价格昂贵难以进行大规模的普查与重点筛查。
目前以寡核苷酸链为基础的生物条码技术的研究与应用非常广泛。生物条码可以以指数级扩增信号,从而提高检测的灵敏度。目前生物条码结合比色、银染以及电化学发光等技术研究发展迅猛。灵敏度也较上述常用检测方法有明显提高。基因芯片的特点是具备高通量,同时可检测多个指标。将生物条码技术的高灵敏度与基因芯片的高通量相结合针对肿瘤标志物的水平或含量进行检测,在现有基础上对肿瘤的早期诊断具有重要的意义。
本研究应用生物条码与基因芯片对蛋白抗原IgG的水平进行检测。不但可以提高检测的灵敏度,还可以具有检测高通量的特点,可使多个靶标同时得以检测,提高了检测效率。通过本研究可以为针对多个包括蛋白类的指标的检测方法与技术的研究铺垫良好的基础。
本论文具体研究内容如下：
一、分子信标的研究与应用
分子信标是一种特殊的生物条码。是一种设计精妙的新型荧光标记的核酸探针。特殊的发夹结构使其具有很强的特异性识别靶序列的能力。目前分子信标已成为分子生物学和生物技术中一种应用广泛的研究工具。本研究设计了三条不同序列的分子信标。通过应用不同序列的分子信标检测待测蛋白抗原浓度,其测定最小值达到6.67 fM(1 pg/ml)。上述研究证实了应用分子信标技术测定蛋白抗原浓度的可行性、灵敏度以及特异性。
二、量子点的研究与应用
量子点又称为半导体纳米微晶体,是一种由Ⅱ-Ⅵ族或Ⅲ-Ⅴ族元素组成的稳定的、可溶于水、尺寸在2-20 nm之间的纳米晶粒。目前量子点作为荧光探针在各领域的研究中应用非常广泛。与传统的荧光探针相比,量子点优势显著。本研究应用量子点标记生物条码对待测蛋白抗原进行检测,结果稳定可靠。可知测定待测蛋白抗原IgG的浓度最小值为333.5 fM(50 pg／ml)。但尚存在偶联定量的问题。需要进一步研究与探索。
三、寡核苷酸生物条码的研究与应用
自行设计三条不同序列,各由60个碱基组成,用于制备基因芯片用单链DNA生物条码,并由此产生与之部分碱基序列完全互补的三条序列不同的,各由40个碱基组成的单链DNA生物条码。应用待测蛋白抗原IgG的单克隆抗体标记粒径为2-3μm,C3间隔臂胺基修饰的的磁性微球；40个碱基组成的单链DNA生物条码5’端标记荧光素Cy3,3’端标记巯基。然后应用上述双标记的单链DNA生物条码与待测蛋白抗原IgG的多克隆抗体双标记自行制备,粒径为13 nm的纳米金颗粒。通过测定标记荧光素的生物条码的荧光强度,可知测定待测蛋白抗原IgG的最小浓度值为133.4 fM(20 pg／ml)。上述研究证实了应用寡核苷酸生物条码技术测定待测蛋白抗原浓度的可行性、灵敏度以及特异性。为与基因芯片结合生物条码技术的进一步研究与应用奠定基础。
四、基因芯片的研究与应用
随着后基因组时代到来而发展起来的基因芯片技术具有高通量的优势,可同时对同一样本中多种不同的靶标进行分析和测定。本研究应用自行设计,由60个碱基组成的单链DNA生物条码制备基因芯片。通过对待测蛋白抗原进行检测,其灵敏度也可达6.67 fM。结果同时显示应用基因芯片技术具有较好的特异性与高通量的特点。我们分别应用三条不同序列的生物条码制备的基因芯片进行检测,结果具有良好的一致性,进一步证实该方法的稳定与可靠性。
综上所述,本实验分别应用生物条码、量子点、分子信标与基因芯片等技术与方法结合磁性微球与纳米金等生物材料对蛋白抗原水平进行微量检测的可行性、稳定性及检测的灵敏度与特异性进入了比较深入的研究和探索。应用生物条码与基因芯片检测蛋白浓度的方法可行稳定,灵敏度到达6.67 fM,具有进一步研究与开发的潜能。分子信标与量子点在与上述生物材料结合应用方面上有很多问题需要进一步澄清与明确。该研究为进行肿瘤标志物的筛选与检测奠定了良好的基础。
At present, cancer remains a major problem in the developed and most developing countries, and there are approximately 12 million people will be diagnosed with cancer and more 8 million were died every year. The focus of cancer therapy is the diagnosis, and the key question is detected and diagnosed at the early stage of cancer in good time. The early treatment is obviously superior than its advanced stage, and the costs are significantly lower too. People have been doing and exploring the tumor marker indicating the tumorgenesis so as to diagnose and provide effective treatment progressively in time. Tumor marker has been an effective basis for the diagnosis and effective treatment of cancer at its early stage in comparing with the changes of cytohistology at the early stage of tumor. And now, it has been recognized by most people that many tumor markers are one of the effective objective index in diagnosising at early stage of tumor.
There are classified three kinds of tumor markers, the serum tumor marker, the cellular tumor markers, and the molecular and genetic tumor markers. There are many measurement methods to detect the tumor marker, including ELISA, RIA, FIA, RII, IHC, automatic immunoassay system and molecular biology techniques applied in the molecular biology research now. But all the methods sensitivity is limited, and it is difficult to achieve a certain degree to diagnose the cancer at its early stage effectively, or only detect at its advanced stage, or is short of the characteristics of high throughput and difficult to be applied on a large scale general census and screening because of its expensive costs.
The bio-bar-code technology based on the single-strand oligonucleotide is researched applied very comprehensively at present. The bio-bar-code technology can improve the detection sensitivity greatly by its cascade amplification. The research on the the bio-bar-code technology combining with some other methods, such as the scanometric, silver stain and electrochemiluminescence, has made a great progress now, and the detection sensitivity is improved more than before methods mentioned above. The characteristics of genechip are of high throughput, and it can detect many target markers at the same time. It will be of very important significance to detect the concentration or content by the bio-bar-code technology with more high sensitivity combing with the high throughput of genechip.
This research focuses on the detection the concentration of the protein antigen IgG applying with the bio-bar-code and genechip. It not only improves the detection sensitivity, but also be of the characteristics with more high throughput to detect many taget markers at one time, and this method improves the detection efficiency greatly. This research paves the way for the study and research on the detection of many protein target markers at the same time, and including the tumor marker.
The major contents of this paper are shown as follows:
1. The research and application of molecular beacon (MB) Molecular beacon is a special kind of bio-bar-code prober, and is thought to be a new type fluorescein-labeled bio-bar-code probes with the exquisite design. MB is of more strong ability to specifically identify the target sequence for its extraordinary hair-pin structure. And now it has became a powerful research tool in the molecular biology and biotechnology. We designed three different sequence MB for the experiment to detect the target antigen, and the detection limit is 6.67 fM. The research proved the method to be of the availability, sensitivity and speciality in measuring the target antigen.
2. The research and application of the Quantum Dot (QD)
Quantum Dot called semiconductor nanocrystal is a kind of stable, soluble nanocrystal made from the second to sixth group or third to fifth group element, and its diameter is between two to twenty nanometer. QD has been applied in many scopes comprehensively as the fluorescence probes now. There are many advantages of it comparing with the general fluorescence probes. We detected the target antigen applying with the bio-bar-code labeled QD, and the results are stable and reliable. But there are some questions on how to confirm the coupling quantities to be solved.
3. The research and application of the oligonucleotide bio-bar-code assay
Designing three different sequence bio-bar-code, made of 60 bases, are prepared the genechip, hence there are three corresponding different sequence, consist of 40 bases, complementary to the sequence above. The monoclonal-antibody IgG was labeled on the amino-modified magnetic microparticles (MMPs) with C3 space arm, and the diameter of MMPs is two to three micrometer. And the single-strand DNA bio-bar-code was labeled the fluorescein Cy3 and the thiol-group on the terminal of its 5'and the 3'respectively. Then we modified gold nanoparticles (Au-NPs) with above dual-labeled bio-bar-codes and polyclonal-antibody IgG, Au-NPs diameter is about 13 nanometers. We detected the fluorescence intensity of the bio-bar-code to get the low limit of the target protein, and the minimum concentration is 133.4 fM. Ours research proved the availability, sensitivity and specificity of the bio-bar-code assay. It paved the way for the progressive research and application of the genechip combining with the bio-bar-code assay.
4. The research and application of genechip
With the advent of post-genomic era, the developed high-throughput gene chip technology has the characteristics of analyzing and measuring many different target markers in the same sample at one time. We prepared genechip with the single-strand DNA bio-bar-code consisted of 60 bases designed by ours. The sensitivity of the method reached to 6.67 fM too, and the method is of more better specificity and high throughput. We applied with three different sequence bio-bar-code to detect the same target antigen and come to the consistent results, it proved the stability and reliability of the genechip technology.
All in brief, the study proved the availability, stability, sensitivity and specificity of the microconcentration detection method of target protein antigen applying the bio-bar-code assay, quantum dot, molecular beacon and genechip technology with the biomaterials of MMPs and Au-NPs. It showed available and stable of applying the bio-bar-code assay with the genechip technology, the sensitivity could reach to fetomolar level, and about 6.67 fM. The method will be of full potential in the research and application in the future. And there are many questions to be recognized and solved in applying the MB and QD with the biomaterials mentioned above. The research have paved a well way for the next study and applying in screening and measuring the tumor markers in the clinic at future.
引文
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