多肿瘤标志物诊断模式的建立和新型电化学免疫传感器的研制
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摘要
世界卫生组织指出,如能早期诊断并及时治疗,95%的肿瘤可以治愈。肿瘤标志物在肿瘤普查、诊断、判断预后、评价疗效和高危人群随访观察等方面都具有较大的实用价值。随着现代生物技术(包括蛋白质组、ELISA、RT-PCR、FISH和SELDI-TOF等)的飞速发展,发现了许多有应用前景的肿瘤标志物。目前临床应用的标志物达到20左右,但因单项肿瘤标志物敏感度和特异度较低,无法准确诊断肿瘤和预测预后。国内外研究表明,多肿瘤标志物联合动态检测是提高肿瘤诊断可行性策略。目前临床上常用的多肿瘤标志物蛋白芯片检测(C-12蛋白芯片),含有12种(糖类抗原19-9(carbohydrate antigen19-9,CA19-9),神经元特异性烯醇化酶(neuron specific enolase,NSE),癌胚抗原(carcinoembryonic antigen,CEA),糖类抗原242(carbohydrate antigen 242,CA242),糖类抗原125(carbohydrate antigen125,CA125),糖类抗原15-3(carbohydrate antigen153,CA15-3),甲胎蛋白(alpha-fetoprotein,AFP),铁蛋白(Ferritin, Fer),游离型前列腺特异性抗原(free prostate specific antigen,F-PSA),前列腺特异性抗原(prostate specific antigen,PSA),生长激素(human growth hormone,HGH)及绒毛膜促性腺激素β(β-HCG)等12种肿瘤标志物,涵盖了大约10种常见的恶性肿瘤,适用于健康人群的体检、筛查及进行肿瘤的辅助诊断。但在具体应用过程中发现C-12蛋白芯片具有诊断灵敏度低、特异度低、分析困难、无法准确定位等缺点,限制其临床的进一步应用。
发展高灵敏的免疫分析技术对于肿瘤标志物的检测有着至关重要的意义。电化学免疫传感器利用抗原和抗体间的高度特异性结合,将免疫分析法和电化学传感器相结合,该传感器具有灵敏度高,分析速度快,操作简便、价格低廉及选择性好等优点,被广泛应用于临床诊断领域。纳米生物技术是纳米技术与生物技术交叉渗透形成的新技术,是生物医学领域的一个重要发展方向。纳米粒子具有优良的电学、磁学、光学性质以及优越的生物相容性,可用于固定抗体,在免疫分析中已得到广泛的应用。现今,免疫分析法和免疫传感器的研制成为检测肿瘤标志物的研究新兴技术。大量研究用于研发操作简便易行的传感器。尽管这个领域的技术具有不少优点,但是寻求一种更加简便,灵敏度更高,选择性更好的免疫方法仍然是现代医学检验和生物医学研究领域的研究热点。
本研究用C-12蛋白芯片法检测2177例恶性肿瘤患者(肺癌836例,肝癌318例,胰腺癌84例,胃癌123例,肠癌338例,乳腺癌206例,卵巢癌47例,子宫内膜癌28例,食管癌197例)和2111例正常及良性疾病的非肿瘤患者的12项常见肿瘤标志物,并结合临床资料进行回顾性研究。进而应用Bayes法建立肿瘤判别诊断函数,提高C-12蛋白芯片对肿瘤诊断的准确率并探讨多肿瘤标志物蛋白芯片检测系统对恶性肿瘤诊断的临床意义。采用HTA、结合VBscript及Javascript语言编程,研制《多蛋白芯片肿瘤判别》软件。同时将纳米技术、生物传感技术和电化学分析技术结合用于免疫分析。应用纳米二氧化硅颗粒、纳米金包裹石墨烯纳米复合物、PB- SiO2纳米复合物和包裹了抗坏血酸(AA)和尿酸(UA)的脂质体等纳米材料,采用不同方法构建功能化生物分子固定界面,制备了一系列高灵敏的电化学免疫传感器。研究目的
1.探讨多肿瘤标志物在10种常见肿瘤中的表达及其临床意义。
2.通过Bayes法建立多肿瘤标志物判别函数,以明确肿瘤类型,提高肿瘤诊断准确率,比较多肿瘤标志物判别函数与C-12多肿瘤标志物蛋白芯片检测系统的差异和对肿瘤诊断的临床意义。进而研制基于多肿瘤蛋白芯片检测结果的肿瘤判别工具软件系统。
3.将IgG抗体标记到辣根过氧化物酶(HRP)掺杂二氧化硅纳米颗粒表面(anti-IgG-SiO2-HRP),建立一种简单又灵敏的电化学免疫分析方法检测人血清中IgG的水平。
4.制备纳米金包裹的石墨烯纳米复合物(NGGN)标记二抗,基于夹心免疫分析模式,建立一种新型高灵敏电化学免疫免疫传感器,用于检测血清中癌胚抗原(CEA)含量。
5.使用PB-SiO2复合纳米粒子,壳聚糖-金纳米粒子复合物(CS-nanoAu)制备检测小细胞肺癌的性能优良的新型电化学免疫传感器。
6.采用分别包裹了抗坏血酸(AA)和尿酸(UA)的脂质体作为信号放大的载体,构建了一种可同时检测小细胞肺癌血清标志物ProGRP和NSE的生物传感技术。
研究内容和方法
1.多肿瘤标志物在恶性肿瘤中的表达及其临床意义用蛋白芯片法检测2177例恶性肿瘤患者(肺癌836例,肝癌318例,胰腺癌84例,
胃癌123例,肠癌338例,乳腺癌206例,卵巢癌47例,子宫内膜癌28例,食管癌197例)和2111例正常及患良性疾病的非肿瘤患者的12项常见肿瘤标志物,并结合临床资料进行回顾性研究。建立肿瘤蛋白芯片数据库和临床数据库。进而分析各肿瘤标志物在常见十种肿瘤中的表达及其临床意义。
2.多肿瘤标志物判别函数的建立及诊断软件的研制
应用Bayes法建立肿瘤判别诊断函数,比较12种肿瘤标志物和判别诊断函数对肿瘤诊断的准确率差异。并采用HTA、结合VBscript及Javascript语言编程。
3.双重标记纳米二氧化硅颗粒电化学免疫传感器检测血清中IgG
合成HRP掺杂纳米二氧化硅,然后将IgG抗体键合到纳米二氧化硅的表面,制得anti-IgG-SiO2-HRP并作为检测抗体,使用夹心免疫分析方法,在酶底物过氧化氢存在的条件下,免疫反应前后电流的变化与抗原的浓度成正比。
4.新型纳米金包裹石墨烯纳米复合物检测癌胚抗原高灵敏电化学免疫传感器的研究。
电沉积普鲁士蓝修饰玻碳电极,并在其表面电沉积一层纳米金,固载癌胚抗体蛋白。制备辣根过氧化物酶(HRP)交联的二抗标记的NGGN颗粒,利用夹心免疫分析方法实现对癌胚抗原的高灵敏检测。
5.基于PB- SiO2纳米复合物的信号增强的免标记电化学免疫传感器用于检测神经化烯醇酶(NSE)
利用层层自组装技术将PB-SiO2复合纳米粒子,壳聚糖-金纳米粒子复合物(CS-nanoAu)依次组装到玻碳电极表面形成具有高表面能和良好氧化还原活性的夹心结构纳米复合膜。
6.基于脂质体信号增强的电化学免疫传感器同时检测血清中ProGRP和NSE
采用分别包裹了抗坏血酸(AA)和尿酸(UA)的脂质体作为信号放大的载体,利用免疫反应的高特异性,结合电化学的高灵敏性,构建了一种可同时检测ProGRP和NSE的生物传感技术。采用“三明治”夹心形式,当固载的捕获抗体与待测物发生特异性的结合,再用表面固定了检测抗体并同时内部包埋了UA或者AA的脂质体夹心检测,最后用表面活性剂破乳,释放出电活性分子,通过检测UA和AA的线性扫描伏安(LSV)峰电流信号强度来检测样品中对应的ProGRP和NSE的浓度。
研究结果
1.多肿瘤标志物在恶性肿瘤中的表达及其临床意义
1) 1209例体检者中,128例结果异常,阳性率为10.59%,显著低于肿瘤组(P<0.01)。12项肿瘤标志物检测结果显示其在体检组的诊断准确率为89.66%,肿瘤患者的诊断准确率为62.77%,灵敏度为66.11%,特异度为48.68%。
2)单项指标中CEA、CA125和CA242阳性率分别为40.93%、29.78%和13.48%,与肺部良性疾病以及正常人群阳性率有统计学意义(P<0.05),其中CEA对肺癌诊断有统计学差异。肺腺癌的临床疗效病情分级与CA19-9、CEA、CA242、AFP以及CA125有相关性,鳞癌只与CA125具有相关性,小细胞肺癌与CA19-9、CA125有相关性。
3)结直肠癌组蛋白芯片的阳性率为71.97%,显著高于结直肠良性病变和正常体检组。C-12芯片检测结直肠癌的灵敏度是71.97%,特异度是55.7%,阳性预测值是83.09%,阴性预测值是39.64%;联合检测的阳性率显著高于单项肿瘤标志物的检测(P<0.05)。CA19-9、CEA、CA242、AFP、β-HCG,HGH以及CA125阳性率明显高于良性病变组和正常体检组(P<0.05),同时CA19-9、CEA、CA242和CA125联合检测的灵敏度和准确率明显高于单项检测指标。CA19-9、CEA、CA242以及CA125表达强度和肿瘤预后相关。
4)胰腺癌患者的阳性率(81.82%)显著高于胰腺良性病变患者(48.72%)和健康体检者(10.59%)(P<0.05)。CA19-9,CEA和CA242,其指标两两联合或三项联合检测较单项指标检测的敏感度和准确率都有所升高,但特异度有所下降。联合检测可提高敏感度和准确率,对诊断更有价值。
5)用C-12芯片检测胃癌的灵敏度是50% ,显著高于胃良性病变患者(34.82%)和健康体检者(10.59%) ( P<0.05) ,有诊断意义的指标是CA19-9、CEA、CA125、CA242和HGH ( P<0.05) ,前面3项指标联合检测的敏感度为44.12 % ,准确率为59.22 %。
6)用C-12芯片检测泌尿系肿瘤的阳性预测值是93.33%,阴性预测值是23.68%,特异度是81.81%,灵敏度是49.12%;男性生殖系肿瘤的阳性预测值是64.86%,阴性预测值是46.15%,特异度是48%,灵敏度是62.16%。在前列腺癌中,PSA和f-PSA联合检测时在其灵敏度不变的情况下,特异度可达到90%,并与前列腺良性病变之间有显著性差异(P<0.01)。对于泌尿系肿瘤和男性生殖系肿瘤,C-12芯片的联合检测的阳性率与单项肿瘤标志物检测有明显差异(P<0.05)。
2.多肿瘤标志物判别函数的建立及诊断软件的研制
1)成功建立了多肿瘤标志物的三级诊断判别函数。
2)一级判别函数诊断的特异度为82.11%,灵敏度为71.28%,准确率为83.97%。而C-12蛋白芯片法检测12项肿瘤标志物的特异度为70.11%,灵敏度为66.10%,准确率为68.07%。
3)二级判别函数能显著提高10种常规肿瘤诊断的灵敏度、特异度和准确率。
4)三级诊断判别函数对确定部分肿瘤的类型有意义。
5)成功编制出《多蛋白芯片肿瘤判别》软件,该软件功能主要包括:肿瘤蛋白芯片数据的录入,十种常见肿瘤判别结果提示,数据查询及报告打印等功能。
3.双重标记纳米二氧化硅颗粒电化学免疫传感器检测血清中IgG
1)该免疫传感器的线性范围为0.01-15 nmol/L IgG,检测限为5.0 pmol/L。而使用HRP标记的IgG抗体(HRP-anti-IgG)作为检测抗体,线性范围为1.0-10 nmol/L,检测限为0.1 nmol/L IgG。
2)该免疫传感器具有良好的重现性、稳定性和特异性。
3)使用该免疫传感器对人血清中IgG的检测,其结果和酶联免疫分析方法检测的结果一致。
4.新型纳米金包裹石墨烯纳米复合物检测癌胚抗原高灵敏电化学免疫传感器的研究
1)与传统的HRP-anti-CEA和纳米金标记的HRP-anti-CEA相比,其灵敏度能提高100倍,检测线可达到0.01 ng/mL CEA。
2)该免疫传感器具有良好的重现性、稳定性和特异性。
3)与前期工作相比,本研究中石墨烯能改变该传感器的导电性,有利于电子的传输,可用于临床诊断。
5.基于PB- SiO2纳米复合物的信号增强的免标记电化学免疫传感器用于检测神经化烯醇酶(NSE)
1)利用PB-SiO2纳米复合物对H2O2明显的电催化作用,构建信号增强的电流型免疫传感器,提高传感器的灵敏度。
2)在含有0.75 mM H2O2的测试底液中,NSE的浓度与还原峰电流的变化值成正比。该免疫传感器对NSE抗原的标准血清样品的线性响应范围为0.25-5.0 ng/mL和5.0-75 ng/mL,检测限为0.08ng/mL。
3)免疫传感器与ELISA法的测定NSE结果一致,表明该免疫传感器可较好的应用于人血清中NSE的直接检测。
6.基于脂质体信号增强的电化学免疫传感器同时检测血清中ProGRP和NSE
1)通过包裹了不同电活性分子的脂质体作为信号放大的载体,可有效增强免疫反应电流响应信号,提高传感器的灵敏度。
2)成功研制同时检测NSE和ProGRP的免疫传感器,ProGRP检测限是100-1000 pg/mL,NSE检测限是5-50 ng/mL。
3)将该免疫传感器用于人血清中NSE和ProGRP的同时检测,并与ELISA法的测定结果比较,结果表明该免疫传感器与经典的ELISA法的测定结果具有良好相关性。
结论
1.多肿瘤标志物在恶性肿瘤中的表达及其临床意义
C-12蛋白芯片在肿瘤普查、诊断、判断预后、评价疗效和高危人群随访观察等方面都具有较大的实用价值。但其具有诊断灵敏度低、特异度低,分析困难,无法准确定位等缺点,限制其临床的进一步应用。
2.多肿瘤标志物判别函数的建立及诊断软件的研制
Bayes法建立多肿瘤标志物判别函数具有较高灵敏度、特异度和准确率,对肿瘤的诊断具有重要临床价值。研制的软件自动完成肿瘤诊断判别方程计算,为肿瘤临床医师提供了一种科学、便捷、高效的工具。
3.双重标记纳米二氧化硅颗粒电化学免疫传感器检测血清中IgG
成功研制一种灵敏度高、制备简单的电化学免疫传感器,通过使用HRP掺杂的二氧化硅作为标记,使用夹心免疫分析模式,实现对人体血清中IgG的检测。该方法的优点就在于使用了双重标记纳米颗粒作为检测抗体,改善了夹心免疫分析方法,提高了该免疫分析的灵敏度。
4.新型纳米金包裹石墨烯纳米复合物检测癌胚抗原高灵敏电化学免疫传感器的研究。
成功研制了一种新型的高灵敏的导电的纳米标记物用于构建电化学免疫传感器,该免疫传感器可用于对痕量血清中CEA的检测,为临床肿瘤的诊断提供了一个新的方法。
5.基于PB- SiO2纳米复合物的信号增强的免标记电化学免疫传感器用于检测神经化烯醇酶(NSE)
成功构建了检测NSE电化学传感器免疫传感器,具有灵敏度高、响应时间快、线性范围宽等特点,实现了微量NSE的定量检测,表明该传感器在NSE的诊断方面具有一定的应用前景。
6.基于脂质体信号增强的电化学免疫传感器同时检测血清中ProGRP和NSE成功构建了同时检测NSE和ProGRP的电化学传感器,具有敏感性高、特异性强、检测快速、成本低廉的特点,建立了血清学诊断小细胞肺癌的临床快速检测方法。
The World Health Organization pointed out, 95 percent cancer can be cured with early diagnosis and timely treatment. Up to now, tumor marker detective technology is almost the only non-invasive effective way to find out malignant tumor. Tumor markers have been used in the tumor screen, diagnosis, prognosis, evaluation of efficacy and the follow-up of high-risk groups. With recent advances in biotechnology such as proteomics, FISH, ELISA, RT-PCR, and SELDI-TOF, many promising biomarkers have been identified and are currently under investigation and validation. More than twenty tumor marker were used in clinic, no one can exactly predict tumor with low specificity and low sensitivity. Domestic and foreign research indicated that recombination of multiple tumor marker would be a realistic way to improve diagnostic accurate. At present, the C-12 protein chip diognostic system including carbohydrate antigen19-9, neuron specific enolase, carcinoembryonic antigen, carbohydrate antigen242, carbohydrate antigen125,carbohydrate antigen153,alpha-fetoprotein,Ferritin, free prostate specific antigen,prostate specific antigen,human growth hormone andβ-HCG tumor marker were used to diagnosis in normally 10 tumor . It can be been used in the tumor screen, diagnosis, evaluation of efficacy and the follow-up of high-risk groups. It can’t be wildly used with low specificity, sensitivity, accuracy and difficulty analysis in clinical practice.
Developing of sensitive immunoassay technology is crucial for realizing the detection of tumor markers. The electrochemical immunosensor uses specificity of the reaction of antigen and antibody to combine electroanalysis with immunoassay, has been broadly applied in clinical diagnosis owing to the advantages of their high sensitivity, fast analytical time, simple measurement, low cost and high selectivity. Nanometer biotechnology developed from nanometer technique and biology is an novel technique of interdisciplinary studies and is the most advanced research program in the field of biomedicine. Nanoparticles have excellent properties in electricity, magnetism, optics and predominant biocompatibility, it can be used as a platform to immobilize antibodies by adsorption which exert crucial functions in the fields of biochemical immunoanalysis. In recent years, many immunoassay methods and immunosensors as currently popular techniques are being developed for the detection of tumor-related biomarkers. And great efforts have been made worldwide to develop and improve immunoassays for the detection of biomarkers with the aim of making portable and affordable devices. Despite of many advances in this field, it is still a challenge to exploit new approaches that can improve the simplicity, selectivity, and sensitivity of clinical immunoassay, to meet the requirements of modern medical diagnostics and biomedical research applications.
A retrospective study was done on data of 2177 patients with malignant tumors (836 with lung cancer, 318 with liver cancer, 84 with pancreatic cancer, 123 with gastric cancer, 338 with colorectal cancer, 206 with breast cancer, 47 with ovarian cancer, 28 with endometrial cancer and 197 with esophageal cancer) and 2111 normal and benign lesion (control group), in whom 12 tumor markers were detected by protein chip technology. Furthermore, the diagnostic function was established to evaluate the accuracy rate in cancer by bayesian methods and evaluate the clinical values of C-12 multiple tumor marker protein chip detective system in diagnosis of cancer. The < multiple tumor marker analysis system V1.0> was programmed by HTA based on VBscript and Javascript language. Ten tumor scorings can be calculated and printed by this software. We also focuse on designing and fabricating novel biomimetic interface combining some kinds of nanoparticles with double-codified nanosilica particles, nanogold-enwrapped graphene nanocomposites, Prussian blue-SiO2 nanocomposite and the liposomes containing ascorbic acid (AA) or Uric acid (UA) for the immobilization of biomolecules.
Objective
1. To research the expression of 12 tumor markers in 10 common tumors, also evaluate its role in tumor diagnosis, treatment and patient’s prognosis.
2. To establish the diagnostic function by bayesian methods to identify the type of tumor and evaluate the accuracy rate. The difference of the diagnostic function and the C-12 multiple tumor marker protein chip detective system was compared to evaluate their clinical value in diagnosis of tumor. Furthermore, to develop a simple, convenient tumor analysis software with detective results of multiple tumor marker.
3. A simple and sensitive method for in situ amplified electrochemical immunoassay of human serum IgG has been developed by using double-codified nanosilica particles as labels based on horseradish peroxidase-doped nanosilica particles (HRP-SiO2) with the conjugation of anti-IgG antibodies (anti-IgG-SiO2-HRP).
4. A new, highly sensitive electrochemical immunosensor with a sandwich-type immunoassay format was designed to quantify carcinoembryonic antigen (CEA) in serum, as a model tumor marker, using nanogold-enwrapped graphene nanocomposites (NGGNs) as trace labels in clinical immunoassays.
5. A signal-enhanced label-free electrochemical immunosensor was constructed to quantify neuron-specific enolase (NSE), a putative serum marker of small-cell lung carcinoma (SCLC), by the employment of Prussian blue doped silica dioxide (PB-SiO2) nanocomposite and chitosan stabled gold nanoparticle (CS-nanoAu).
6. A multiplex immunoassay with high diagnostic sensitivity and specificity was constructed to quantify neuron-specific enolase (NSE) and pro-gastrin-releasing peptide (ProGRP), with the amplification strategy of the liposomes containing ascorbic acid (AA) or Uric acid (UA) as entrapped marker.
Materials and Methods
1. The clinical significance and expression of C-12 multiple tumor marker protein chip detective system in tumors
A retrospective study was done on data of 2177 patients with malignant tumors (836 with lung cancer, 318 with liver cancer, 84 with pancreatic cancer, 123 with gastric cancer, 338 with colorectal cancer, 206 with breast cancer, 47 with ovarian cancer, 28 with endometrial cancer and 197 with esophageal cancer) and 2111 normal and benign lesion (control group), in whom 12 tumor markers were detected by protein chip technology. And established tumor markers database and clinical database. To analysis 12 tumor marker expressed in ten common malignant tumor and it’s Clinical Significance.
2. Establishment of diagnostic function by Bayesian methods and Development of < Multiple Tumor Marker Analysis System V1.0> software
The diagnostic function was established by Bayesian method to compare the accuracy difference in diagnosis of tumors with 12 tumor markers. And the < multiple tumor marker analysis system V1.0> was programmed by HTA based on VBscript and Javascript language.
3. Signal amplification of electrochemical immunosensor for the detection of human serum IgG using double-codified nanosilica particles as labels
Electrochemical immunoassay method for the detection of human serum IgG by using double-codified nanosilica particles as labels based on HRP-doped nanosilica particles modified with anti-IgG antibodies. The detection is based on the catalytic reduction of the carried HRP in the nanosilica particles relative to the H2O2 system with a sandwich-type immunoassay format.
4. Nanogold-enwrapped graphene nanocomposites as trace labels for sensitivity enhancement of electrochemical immunosensors in clinical immunoassays: Carcinoembryonic antigen as a model
Prussian Blue (PB) was first modified on a glassy carbon electrode, then gold nanoparticles monolayer were obtained by electrochemically deposition for the anti-CEA antibodies loading. The immunoassay was performed using horseradish peroxidase (HRP)-conjugated anti-CEA as secondary antibodies attached on the NGGN surface (HRP-anti-CEA-NGGN).
5. The signal-enhanced label-free immunosensor based on assembly of Prussian blue-SiO2 nanocomposite for amperometric measurement of Neuron-specific enolase
PB-SiO2 nanocomposite which was produced by using a microemulsion method was used to obtain a nanostructural monolayer on a glassy carbon electrode (GCE) surface. Then 3-aminopropyltriethoxy silane (APTES) was allowing assembly of PB-SiO2 monolayer to form a protective layer and functionalize the interface with -NH2. Then chitosan stabled gold nanoparticle (CS-nanoAu) was subsequently attached, while the entire surface was finally loaded with neuron-specific enolase antibody.
6. An electrochemical immunosensor for simultaneous multiplexed detection for NSE and pro-GRP using liposomes as enhancer
The liposomes contained ascorbic acid (AA) or Uric acid (UA) as entrapped marker were prepared respectively. Then the resulting liposomes were bound to the sencondery antibodies: pro-gastrin-releasing peptide antibody (anti-ProGRP) or neuron-specific enolase antibody (anti-NSE). With the employment of sandwich type immunoreactions format, the liposomes containing entrapped marker labeled sencondery antibodies were employed to form the immune complexes, which was then destroyed by the addition of surfactant and the entrapped marker was detected electrochemically using MWNT modified electrodes.
Results
1. The clinical significance and expression of C-12 multiple tumor marker protein chip detective system in tumors
1) At least one kind of tumor marker was found positive in 128 cases , with positive rate of 10.59%, which was significantly lower than that of tumor groups. The accuracy rate in examiners was 89.66% by C-12 multiple tumor marker protein chip detective system, while 62.77% in tumors. The sensitivity was 66.11%, the specificity was 48.68%.
2) The positive rates of CEA, CA125 and CA242 were 40.93%, 29.78% and 13.48%, separately, which were significant different from their counterpart of lung binign disease group and health control. Moreover CEA was significance rather than CA125 and CA242 in lung cancer diagnosis. Grade of efficacy and status was closely related to CA19-9 , CEA , CA242 , AFP and CA125 in adenocarcinoma , to CA125 in squamous cell carcinoma ( SCC) , and to CA19-9 and CA125 in small cell lung cancer ( SCLC) .
3) It was 71.97% positive ratio in collrectal carcinoma which was significantly higher than the other two groups. The sensitivity, specificity, accuracy, positive predict value and negative value of C-12 system were 71.97%, 55.7%, 83.09%, 39.64%. The positive ratio of combined detection was higher than everyone of C-12 system only (P<0.05). The expression of tumor markers CA19-9, CEA, CA242, AFP,β-HCG,HGH and CA125 in carcinoma group was markedly stronger than other two groups, meanwhile the sensitivity and validity of combined detection of CA19-9, CEA, CA242 was proved. The level of CA19-9, CEA, CA242 and CA125 was related to prognosis in colorectal carcinoma.
4) The positive rate of pancreatic cancer is significantly higher than that of pancreatic begin patients and normal persons (P<0.05). CA19-9, CEA and CA242 have a clinical significance in diagnosis. Combined detection can significantly increase the diagnostic sensitivity, but the specificity has been decreased.
5) The positive rate of gastric cancer (50.00%) was significantly higher than that of gastric begin patients (34.82%) and normal persons (10.59%) (P<0.05). The CA19-9, CEA, CA242, CA125 and HGH had a clinical significance (P<0. 05) .
6) In urinary system tumor, positive value is 93.33%,negative valve is 23.68%,specificity is 81.81%,Sensitivity is 49.12%. In male genital system tumor, positive value is 64.86%,negative valve is 46.15%,specificity is 48.00%,Sensitivity is 62.16%. In prostate carcinoma, combined measurement of PSA and f-PSA can increase diagnostic specificity to 90% (P<0.01), which will not decrease diagnostic sensitivity, The Combining diagnosis of multiple serum tumor markers using C-12 protein chip has a great significance with diagnosis of the single serum tumor marker in both urinary system tumor and male genital system tumor .
2. Establishment of diagnostic function by Bayesian methods and Development of < Multiple Tumor Marker Analysis System V1.0> software
1) Three grades of diagnostic functions were successfully established in C-12 multiple tumor marker.
2) The specificity, sensitivity and accuracy rate were 70.11%, 66.10% and 68.07% by C-12 multiple tumor marker while 82.11%, 71.28% and 83.97% by the first grade diagnostic function.
3) The accuracy rate, sensitivity and specificity were higher than C-12 multiple tumor marker chip in all the 10 types of malignant tumors by the second diagnostic function.
4) The third diagnostic function could identify part of the type of tumor, such as breast cancer, prostate cancer, lung cancer, liver cancer, ovarian cancer, endometrial cancer and pancreatic cancer.
5) The < multiple tumor marker analysis system V1.0> software was developed successfully. The functions of this software include data read, query, print 10 tumor scorings can be calculated and printed by this software.
3. Signal amplification of electrochemical immunosensor for the detection of human serum IgG using double-codified nanosilica particles as labels
1) With the sandwich-type immunoassay format, the linear range of the developed immunosensor by using anti-IgG-SiO2-HRP as tracer and hydrogen peroxide (H2O2) as enzyme substrate is 0.01-15 nmol/L IgG with a detection limit of 5.0 pmol/L, while the assay sensitivity by directly using HRP-labeled anti-IgG as secondary antibodies is 1.0-10 nmol/L with a detection limit of 0.1 nmol/L IgG..
2) The reproducibility, stability and specificity of the proposed immunoassay method were acceptable.
3) The IgG concentrations of the clinical serum specimens assayed by the developed immunosensor show consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay (ELISA) method.
4. Nanogold-enwrapped graphene nanocomposites as trace labels for sensitivity enhancement of electrochemical immunosensors in clinical immunoassays: Carcinoembryonic antigen as a model
1) The method using HRP-anti-CEA-NGGNs as detection antibodies shows high signal amplification, and exhibits a dynamic working range of 0.05-350 ng/mL with a low detection limit of 0.01 ng/mL CEA (at 3s).
2) The reproducibility, stability and specificity of the proposed immunoassay method were acceptable.
3) The nanogold-enwrapped graphene nanocomposites facilitate the electron transfer between the analyte and the base electrode in contrast with that of using silica nanoparticles previously reported by us.
5. The signal-enhanced label-free immunosensor based on assembly of Prussian blue-SiO2 nanocomposite for amperometric measurement of Neuron-specific enolase
1) As the PB-SiO2 nanostructural sensing film provides plenty of active sites for the direct catalysis of H2O2, the proposed immunosensor exhibited excellent performance with high sensitivity.
2) The immunosensor exhibited good linear behavior in the concentration range from 0.25-5.0 and 5.0-75 ng/mL for the quantitative analysis of neuron-specific enolase (NSE), a putative serum marker of small cell lung carcinoma (SCLC), with a limit of detection of 0.08 ng/mL in the presence of 0.75 mmol/L H2O2.
3) No significant difference between the results obtained using the immunosensor and the ELISA, which showed an acceptable degree of agreement, especially at NSE concentrations in human serum with the immunosensor.
6. An electrochemical immunosensor for simultaneous multiplexed detection of NSE and pro-GRP using liposomes as enhancer
1) A sensitive immunosensor was construced based on a new signal amplification strategy was developed for the simultaneous multiplexed quantitative analysis using the liposomes contained different electrchemical active molecule as singal enhancer.
2) A electrochemical immunosensor was developed for the simultaneous multiplexed quantitative analysis of ProGRP in the concentration range from 100-1000 pg/mL and NSE in the concentration range from 5-50 ng/mL, respectively.
3) The concentrations of NSE and ProGRP in human serum obtained by the developed immunosensor show consistent results in comparison with those obtained by commercially available enzyme linked immunosorbent assay (ELISA) method.
Conclusion
1. The clinical significance and expression of C-12 multiple tumor marker protein chip detective system in tumors
C-12 multiple tumor marker protein chip detective system can be been used in the tumor screen, diagnosis, evaluation of efficacy and the follow-up of high-risk groups. It can’t be wildly used with low specificity, sensitivity, accuracy and difficulty analysis in clinical practice.
2. Establishment of diagnostic function by Bayesian methods and Development of < Multiple Tumor Marker Analysis System V1.0> software
Diagnostic function established by Bayesian methods has advantages of high sensitivity, specificity and accuracy and is of great clinical value in early detection of the cancers. The software can automatic diagnosis the sort of tumor, which is a scientific, convenient, efficient tool for clinician.
3. Signal amplification of electrochemical immunosensor for the detection of human serum IgG using double-codified nanosilica particles as labels
a newly double-codified immunoassay method based on HRP-doped nanosilica particles modified with anti-IgG antibodies has been introduced for electrochemical detection of human IgG in a sandwich-type immunoassay format. The electrochemical signal is amplified by using double-codified nanosilica particles as tracer and hydrogen peroxide as enzyme substrate. Highlight of the developed immunoassay method is the intrinsic electrochemical properties of the double-codified nanosilica particles that being proportional to the protein concentration can be directly quantified by cyclic voltammetry. This method has many desirable merits including sensitivity, accuracy, and little required instrumentation.
4. Nanogold enwrapped graphene nanocomposites as trace labels for sensitivity enhancement of electrochemical immunosensors in clinical immunoassays: Carcinoembryonic antigen as a model
We develop a new conductive nanolabel with highly amplified properties and high sensitivity for the sandwich-type electrochemical immunoassays. The assayed results of serum samples with the sensor received an acceptable agreement with the reference values. The convenient operation and ultrasensitivity of the developed methodology provides a promising potential in clinical diagnosis.
5. The signal-enhanced label-free immunosensor based on assembly of Prussian blue-SiO2 nanocomposite for amperometric measurement of Neuron-specific enolase Tests result indicated this strategy could provide a promising approach of the sensor preparation and a practical method with high sensitivity, shorter detection time and border linear dynamic range for clinical immunoassay in dignosis of NSE.
6. An electrochemical immunosensor for simultaneous multiplexed detection for NSE and pro-GRP using liposomes as enhancer
The multiplex immunoassay can offer higher sample throughput, less sample consumption, shorter assay time and lower cost than the traditional parallel single analyte immunoassay. The multiplex immunoassay provides a clinical serum diagnostic solution in small cell lung carcinoma.
发展高灵敏的免疫分析技术对于肿瘤标志物的检测有着至关重要的意义。电化学免疫传感器利用抗原和抗体间的高度特异性结合,将免疫分析法和电化学传感器相结合,该传感器具有灵敏度高,分析速度快,操作简便、价格低廉及选择性好等优点,被广泛应用于临床诊断领域。纳米生物技术是纳米技术与生物技术交叉渗透形成的新技术,是生物医学领域的一个重要发展方向。纳米粒子具有优良的电学、磁学、光学性质以及优越的生物相容性,可用于固定抗体,在免疫分析中已得到广泛的应用。现今,免疫分析法和免疫传感器的研制成为检测肿瘤标志物的研究新兴技术。大量研究用于研发操作简便易行的传感器。尽管这个领域的技术具有不少优点,但是寻求一种更加简便,灵敏度更高,选择性更好的免疫方法仍然是现代医学检验和生物医学研究领域的研究热点。
本研究用C-12蛋白芯片法检测2177例恶性肿瘤患者(肺癌836例,肝癌318例,胰腺癌84例,胃癌123例,肠癌338例,乳腺癌206例,卵巢癌47例,子宫内膜癌28例,食管癌197例)和2111例正常及良性疾病的非肿瘤患者的12项常见肿瘤标志物,并结合临床资料进行回顾性研究。进而应用Bayes法建立肿瘤判别诊断函数,提高C-12蛋白芯片对肿瘤诊断的准确率并探讨多肿瘤标志物蛋白芯片检测系统对恶性肿瘤诊断的临床意义。采用HTA、结合VBscript及Javascript语言编程,研制《多蛋白芯片肿瘤判别》软件。同时将纳米技术、生物传感技术和电化学分析技术结合用于免疫分析。应用纳米二氧化硅颗粒、纳米金包裹石墨烯纳米复合物、PB- SiO2纳米复合物和包裹了抗坏血酸(AA)和尿酸(UA)的脂质体等纳米材料,采用不同方法构建功能化生物分子固定界面,制备了一系列高灵敏的电化学免疫传感器。研究目的
1.探讨多肿瘤标志物在10种常见肿瘤中的表达及其临床意义。
2.通过Bayes法建立多肿瘤标志物判别函数,以明确肿瘤类型,提高肿瘤诊断准确率,比较多肿瘤标志物判别函数与C-12多肿瘤标志物蛋白芯片检测系统的差异和对肿瘤诊断的临床意义。进而研制基于多肿瘤蛋白芯片检测结果的肿瘤判别工具软件系统。
3.将IgG抗体标记到辣根过氧化物酶(HRP)掺杂二氧化硅纳米颗粒表面(anti-IgG-SiO2-HRP),建立一种简单又灵敏的电化学免疫分析方法检测人血清中IgG的水平。
4.制备纳米金包裹的石墨烯纳米复合物(NGGN)标记二抗,基于夹心免疫分析模式,建立一种新型高灵敏电化学免疫免疫传感器,用于检测血清中癌胚抗原(CEA)含量。
5.使用PB-SiO2复合纳米粒子,壳聚糖-金纳米粒子复合物(CS-nanoAu)制备检测小细胞肺癌的性能优良的新型电化学免疫传感器。
6.采用分别包裹了抗坏血酸(AA)和尿酸(UA)的脂质体作为信号放大的载体,构建了一种可同时检测小细胞肺癌血清标志物ProGRP和NSE的生物传感技术。
研究内容和方法
1.多肿瘤标志物在恶性肿瘤中的表达及其临床意义用蛋白芯片法检测2177例恶性肿瘤患者(肺癌836例,肝癌318例,胰腺癌84例,
胃癌123例,肠癌338例,乳腺癌206例,卵巢癌47例,子宫内膜癌28例,食管癌197例)和2111例正常及患良性疾病的非肿瘤患者的12项常见肿瘤标志物,并结合临床资料进行回顾性研究。建立肿瘤蛋白芯片数据库和临床数据库。进而分析各肿瘤标志物在常见十种肿瘤中的表达及其临床意义。
2.多肿瘤标志物判别函数的建立及诊断软件的研制
应用Bayes法建立肿瘤判别诊断函数,比较12种肿瘤标志物和判别诊断函数对肿瘤诊断的准确率差异。并采用HTA、结合VBscript及Javascript语言编程。
3.双重标记纳米二氧化硅颗粒电化学免疫传感器检测血清中IgG
合成HRP掺杂纳米二氧化硅,然后将IgG抗体键合到纳米二氧化硅的表面,制得anti-IgG-SiO2-HRP并作为检测抗体,使用夹心免疫分析方法,在酶底物过氧化氢存在的条件下,免疫反应前后电流的变化与抗原的浓度成正比。
4.新型纳米金包裹石墨烯纳米复合物检测癌胚抗原高灵敏电化学免疫传感器的研究。
电沉积普鲁士蓝修饰玻碳电极,并在其表面电沉积一层纳米金,固载癌胚抗体蛋白。制备辣根过氧化物酶(HRP)交联的二抗标记的NGGN颗粒,利用夹心免疫分析方法实现对癌胚抗原的高灵敏检测。
5.基于PB- SiO2纳米复合物的信号增强的免标记电化学免疫传感器用于检测神经化烯醇酶(NSE)
利用层层自组装技术将PB-SiO2复合纳米粒子,壳聚糖-金纳米粒子复合物(CS-nanoAu)依次组装到玻碳电极表面形成具有高表面能和良好氧化还原活性的夹心结构纳米复合膜。
6.基于脂质体信号增强的电化学免疫传感器同时检测血清中ProGRP和NSE
采用分别包裹了抗坏血酸(AA)和尿酸(UA)的脂质体作为信号放大的载体,利用免疫反应的高特异性,结合电化学的高灵敏性,构建了一种可同时检测ProGRP和NSE的生物传感技术。采用“三明治”夹心形式,当固载的捕获抗体与待测物发生特异性的结合,再用表面固定了检测抗体并同时内部包埋了UA或者AA的脂质体夹心检测,最后用表面活性剂破乳,释放出电活性分子,通过检测UA和AA的线性扫描伏安(LSV)峰电流信号强度来检测样品中对应的ProGRP和NSE的浓度。
研究结果
1.多肿瘤标志物在恶性肿瘤中的表达及其临床意义
1) 1209例体检者中,128例结果异常,阳性率为10.59%,显著低于肿瘤组(P<0.01)。12项肿瘤标志物检测结果显示其在体检组的诊断准确率为89.66%,肿瘤患者的诊断准确率为62.77%,灵敏度为66.11%,特异度为48.68%。
2)单项指标中CEA、CA125和CA242阳性率分别为40.93%、29.78%和13.48%,与肺部良性疾病以及正常人群阳性率有统计学意义(P<0.05),其中CEA对肺癌诊断有统计学差异。肺腺癌的临床疗效病情分级与CA19-9、CEA、CA242、AFP以及CA125有相关性,鳞癌只与CA125具有相关性,小细胞肺癌与CA19-9、CA125有相关性。
3)结直肠癌组蛋白芯片的阳性率为71.97%,显著高于结直肠良性病变和正常体检组。C-12芯片检测结直肠癌的灵敏度是71.97%,特异度是55.7%,阳性预测值是83.09%,阴性预测值是39.64%;联合检测的阳性率显著高于单项肿瘤标志物的检测(P<0.05)。CA19-9、CEA、CA242、AFP、β-HCG,HGH以及CA125阳性率明显高于良性病变组和正常体检组(P<0.05),同时CA19-9、CEA、CA242和CA125联合检测的灵敏度和准确率明显高于单项检测指标。CA19-9、CEA、CA242以及CA125表达强度和肿瘤预后相关。
4)胰腺癌患者的阳性率(81.82%)显著高于胰腺良性病变患者(48.72%)和健康体检者(10.59%)(P<0.05)。CA19-9,CEA和CA242,其指标两两联合或三项联合检测较单项指标检测的敏感度和准确率都有所升高,但特异度有所下降。联合检测可提高敏感度和准确率,对诊断更有价值。
5)用C-12芯片检测胃癌的灵敏度是50% ,显著高于胃良性病变患者(34.82%)和健康体检者(10.59%) ( P<0.05) ,有诊断意义的指标是CA19-9、CEA、CA125、CA242和HGH ( P<0.05) ,前面3项指标联合检测的敏感度为44.12 % ,准确率为59.22 %。
6)用C-12芯片检测泌尿系肿瘤的阳性预测值是93.33%,阴性预测值是23.68%,特异度是81.81%,灵敏度是49.12%;男性生殖系肿瘤的阳性预测值是64.86%,阴性预测值是46.15%,特异度是48%,灵敏度是62.16%。在前列腺癌中,PSA和f-PSA联合检测时在其灵敏度不变的情况下,特异度可达到90%,并与前列腺良性病变之间有显著性差异(P<0.01)。对于泌尿系肿瘤和男性生殖系肿瘤,C-12芯片的联合检测的阳性率与单项肿瘤标志物检测有明显差异(P<0.05)。
2.多肿瘤标志物判别函数的建立及诊断软件的研制
1)成功建立了多肿瘤标志物的三级诊断判别函数。
2)一级判别函数诊断的特异度为82.11%,灵敏度为71.28%,准确率为83.97%。而C-12蛋白芯片法检测12项肿瘤标志物的特异度为70.11%,灵敏度为66.10%,准确率为68.07%。
3)二级判别函数能显著提高10种常规肿瘤诊断的灵敏度、特异度和准确率。
4)三级诊断判别函数对确定部分肿瘤的类型有意义。
5)成功编制出《多蛋白芯片肿瘤判别》软件,该软件功能主要包括:肿瘤蛋白芯片数据的录入,十种常见肿瘤判别结果提示,数据查询及报告打印等功能。
3.双重标记纳米二氧化硅颗粒电化学免疫传感器检测血清中IgG
1)该免疫传感器的线性范围为0.01-15 nmol/L IgG,检测限为5.0 pmol/L。而使用HRP标记的IgG抗体(HRP-anti-IgG)作为检测抗体,线性范围为1.0-10 nmol/L,检测限为0.1 nmol/L IgG。
2)该免疫传感器具有良好的重现性、稳定性和特异性。
3)使用该免疫传感器对人血清中IgG的检测,其结果和酶联免疫分析方法检测的结果一致。
4.新型纳米金包裹石墨烯纳米复合物检测癌胚抗原高灵敏电化学免疫传感器的研究
1)与传统的HRP-anti-CEA和纳米金标记的HRP-anti-CEA相比,其灵敏度能提高100倍,检测线可达到0.01 ng/mL CEA。
2)该免疫传感器具有良好的重现性、稳定性和特异性。
3)与前期工作相比,本研究中石墨烯能改变该传感器的导电性,有利于电子的传输,可用于临床诊断。
5.基于PB- SiO2纳米复合物的信号增强的免标记电化学免疫传感器用于检测神经化烯醇酶(NSE)
1)利用PB-SiO2纳米复合物对H2O2明显的电催化作用,构建信号增强的电流型免疫传感器,提高传感器的灵敏度。
2)在含有0.75 mM H2O2的测试底液中,NSE的浓度与还原峰电流的变化值成正比。该免疫传感器对NSE抗原的标准血清样品的线性响应范围为0.25-5.0 ng/mL和5.0-75 ng/mL,检测限为0.08ng/mL。
3)免疫传感器与ELISA法的测定NSE结果一致,表明该免疫传感器可较好的应用于人血清中NSE的直接检测。
6.基于脂质体信号增强的电化学免疫传感器同时检测血清中ProGRP和NSE
1)通过包裹了不同电活性分子的脂质体作为信号放大的载体,可有效增强免疫反应电流响应信号,提高传感器的灵敏度。
2)成功研制同时检测NSE和ProGRP的免疫传感器,ProGRP检测限是100-1000 pg/mL,NSE检测限是5-50 ng/mL。
3)将该免疫传感器用于人血清中NSE和ProGRP的同时检测,并与ELISA法的测定结果比较,结果表明该免疫传感器与经典的ELISA法的测定结果具有良好相关性。
结论
1.多肿瘤标志物在恶性肿瘤中的表达及其临床意义
C-12蛋白芯片在肿瘤普查、诊断、判断预后、评价疗效和高危人群随访观察等方面都具有较大的实用价值。但其具有诊断灵敏度低、特异度低,分析困难,无法准确定位等缺点,限制其临床的进一步应用。
2.多肿瘤标志物判别函数的建立及诊断软件的研制
Bayes法建立多肿瘤标志物判别函数具有较高灵敏度、特异度和准确率,对肿瘤的诊断具有重要临床价值。研制的软件自动完成肿瘤诊断判别方程计算,为肿瘤临床医师提供了一种科学、便捷、高效的工具。
3.双重标记纳米二氧化硅颗粒电化学免疫传感器检测血清中IgG
成功研制一种灵敏度高、制备简单的电化学免疫传感器,通过使用HRP掺杂的二氧化硅作为标记,使用夹心免疫分析模式,实现对人体血清中IgG的检测。该方法的优点就在于使用了双重标记纳米颗粒作为检测抗体,改善了夹心免疫分析方法,提高了该免疫分析的灵敏度。
4.新型纳米金包裹石墨烯纳米复合物检测癌胚抗原高灵敏电化学免疫传感器的研究。
成功研制了一种新型的高灵敏的导电的纳米标记物用于构建电化学免疫传感器,该免疫传感器可用于对痕量血清中CEA的检测,为临床肿瘤的诊断提供了一个新的方法。
5.基于PB- SiO2纳米复合物的信号增强的免标记电化学免疫传感器用于检测神经化烯醇酶(NSE)
成功构建了检测NSE电化学传感器免疫传感器,具有灵敏度高、响应时间快、线性范围宽等特点,实现了微量NSE的定量检测,表明该传感器在NSE的诊断方面具有一定的应用前景。
6.基于脂质体信号增强的电化学免疫传感器同时检测血清中ProGRP和NSE成功构建了同时检测NSE和ProGRP的电化学传感器,具有敏感性高、特异性强、检测快速、成本低廉的特点,建立了血清学诊断小细胞肺癌的临床快速检测方法。
The World Health Organization pointed out, 95 percent cancer can be cured with early diagnosis and timely treatment. Up to now, tumor marker detective technology is almost the only non-invasive effective way to find out malignant tumor. Tumor markers have been used in the tumor screen, diagnosis, prognosis, evaluation of efficacy and the follow-up of high-risk groups. With recent advances in biotechnology such as proteomics, FISH, ELISA, RT-PCR, and SELDI-TOF, many promising biomarkers have been identified and are currently under investigation and validation. More than twenty tumor marker were used in clinic, no one can exactly predict tumor with low specificity and low sensitivity. Domestic and foreign research indicated that recombination of multiple tumor marker would be a realistic way to improve diagnostic accurate. At present, the C-12 protein chip diognostic system including carbohydrate antigen19-9, neuron specific enolase, carcinoembryonic antigen, carbohydrate antigen242, carbohydrate antigen125,carbohydrate antigen153,alpha-fetoprotein,Ferritin, free prostate specific antigen,prostate specific antigen,human growth hormone andβ-HCG tumor marker were used to diagnosis in normally 10 tumor . It can be been used in the tumor screen, diagnosis, evaluation of efficacy and the follow-up of high-risk groups. It can’t be wildly used with low specificity, sensitivity, accuracy and difficulty analysis in clinical practice.
Developing of sensitive immunoassay technology is crucial for realizing the detection of tumor markers. The electrochemical immunosensor uses specificity of the reaction of antigen and antibody to combine electroanalysis with immunoassay, has been broadly applied in clinical diagnosis owing to the advantages of their high sensitivity, fast analytical time, simple measurement, low cost and high selectivity. Nanometer biotechnology developed from nanometer technique and biology is an novel technique of interdisciplinary studies and is the most advanced research program in the field of biomedicine. Nanoparticles have excellent properties in electricity, magnetism, optics and predominant biocompatibility, it can be used as a platform to immobilize antibodies by adsorption which exert crucial functions in the fields of biochemical immunoanalysis. In recent years, many immunoassay methods and immunosensors as currently popular techniques are being developed for the detection of tumor-related biomarkers. And great efforts have been made worldwide to develop and improve immunoassays for the detection of biomarkers with the aim of making portable and affordable devices. Despite of many advances in this field, it is still a challenge to exploit new approaches that can improve the simplicity, selectivity, and sensitivity of clinical immunoassay, to meet the requirements of modern medical diagnostics and biomedical research applications.
A retrospective study was done on data of 2177 patients with malignant tumors (836 with lung cancer, 318 with liver cancer, 84 with pancreatic cancer, 123 with gastric cancer, 338 with colorectal cancer, 206 with breast cancer, 47 with ovarian cancer, 28 with endometrial cancer and 197 with esophageal cancer) and 2111 normal and benign lesion (control group), in whom 12 tumor markers were detected by protein chip technology. Furthermore, the diagnostic function was established to evaluate the accuracy rate in cancer by bayesian methods and evaluate the clinical values of C-12 multiple tumor marker protein chip detective system in diagnosis of cancer. The < multiple tumor marker analysis system V1.0> was programmed by HTA based on VBscript and Javascript language. Ten tumor scorings can be calculated and printed by this software. We also focuse on designing and fabricating novel biomimetic interface combining some kinds of nanoparticles with double-codified nanosilica particles, nanogold-enwrapped graphene nanocomposites, Prussian blue-SiO2 nanocomposite and the liposomes containing ascorbic acid (AA) or Uric acid (UA) for the immobilization of biomolecules.
Objective
1. To research the expression of 12 tumor markers in 10 common tumors, also evaluate its role in tumor diagnosis, treatment and patient’s prognosis.
2. To establish the diagnostic function by bayesian methods to identify the type of tumor and evaluate the accuracy rate. The difference of the diagnostic function and the C-12 multiple tumor marker protein chip detective system was compared to evaluate their clinical value in diagnosis of tumor. Furthermore, to develop a simple, convenient tumor analysis software with detective results of multiple tumor marker.
3. A simple and sensitive method for in situ amplified electrochemical immunoassay of human serum IgG has been developed by using double-codified nanosilica particles as labels based on horseradish peroxidase-doped nanosilica particles (HRP-SiO2) with the conjugation of anti-IgG antibodies (anti-IgG-SiO2-HRP).
4. A new, highly sensitive electrochemical immunosensor with a sandwich-type immunoassay format was designed to quantify carcinoembryonic antigen (CEA) in serum, as a model tumor marker, using nanogold-enwrapped graphene nanocomposites (NGGNs) as trace labels in clinical immunoassays.
5. A signal-enhanced label-free electrochemical immunosensor was constructed to quantify neuron-specific enolase (NSE), a putative serum marker of small-cell lung carcinoma (SCLC), by the employment of Prussian blue doped silica dioxide (PB-SiO2) nanocomposite and chitosan stabled gold nanoparticle (CS-nanoAu).
6. A multiplex immunoassay with high diagnostic sensitivity and specificity was constructed to quantify neuron-specific enolase (NSE) and pro-gastrin-releasing peptide (ProGRP), with the amplification strategy of the liposomes containing ascorbic acid (AA) or Uric acid (UA) as entrapped marker.
Materials and Methods
1. The clinical significance and expression of C-12 multiple tumor marker protein chip detective system in tumors
A retrospective study was done on data of 2177 patients with malignant tumors (836 with lung cancer, 318 with liver cancer, 84 with pancreatic cancer, 123 with gastric cancer, 338 with colorectal cancer, 206 with breast cancer, 47 with ovarian cancer, 28 with endometrial cancer and 197 with esophageal cancer) and 2111 normal and benign lesion (control group), in whom 12 tumor markers were detected by protein chip technology. And established tumor markers database and clinical database. To analysis 12 tumor marker expressed in ten common malignant tumor and it’s Clinical Significance.
2. Establishment of diagnostic function by Bayesian methods and Development of < Multiple Tumor Marker Analysis System V1.0> software
The diagnostic function was established by Bayesian method to compare the accuracy difference in diagnosis of tumors with 12 tumor markers. And the < multiple tumor marker analysis system V1.0> was programmed by HTA based on VBscript and Javascript language.
3. Signal amplification of electrochemical immunosensor for the detection of human serum IgG using double-codified nanosilica particles as labels
Electrochemical immunoassay method for the detection of human serum IgG by using double-codified nanosilica particles as labels based on HRP-doped nanosilica particles modified with anti-IgG antibodies. The detection is based on the catalytic reduction of the carried HRP in the nanosilica particles relative to the H2O2 system with a sandwich-type immunoassay format.
4. Nanogold-enwrapped graphene nanocomposites as trace labels for sensitivity enhancement of electrochemical immunosensors in clinical immunoassays: Carcinoembryonic antigen as a model
Prussian Blue (PB) was first modified on a glassy carbon electrode, then gold nanoparticles monolayer were obtained by electrochemically deposition for the anti-CEA antibodies loading. The immunoassay was performed using horseradish peroxidase (HRP)-conjugated anti-CEA as secondary antibodies attached on the NGGN surface (HRP-anti-CEA-NGGN).
5. The signal-enhanced label-free immunosensor based on assembly of Prussian blue-SiO2 nanocomposite for amperometric measurement of Neuron-specific enolase
PB-SiO2 nanocomposite which was produced by using a microemulsion method was used to obtain a nanostructural monolayer on a glassy carbon electrode (GCE) surface. Then 3-aminopropyltriethoxy silane (APTES) was allowing assembly of PB-SiO2 monolayer to form a protective layer and functionalize the interface with -NH2. Then chitosan stabled gold nanoparticle (CS-nanoAu) was subsequently attached, while the entire surface was finally loaded with neuron-specific enolase antibody.
6. An electrochemical immunosensor for simultaneous multiplexed detection for NSE and pro-GRP using liposomes as enhancer
The liposomes contained ascorbic acid (AA) or Uric acid (UA) as entrapped marker were prepared respectively. Then the resulting liposomes were bound to the sencondery antibodies: pro-gastrin-releasing peptide antibody (anti-ProGRP) or neuron-specific enolase antibody (anti-NSE). With the employment of sandwich type immunoreactions format, the liposomes containing entrapped marker labeled sencondery antibodies were employed to form the immune complexes, which was then destroyed by the addition of surfactant and the entrapped marker was detected electrochemically using MWNT modified electrodes.
Results
1. The clinical significance and expression of C-12 multiple tumor marker protein chip detective system in tumors
1) At least one kind of tumor marker was found positive in 128 cases , with positive rate of 10.59%, which was significantly lower than that of tumor groups. The accuracy rate in examiners was 89.66% by C-12 multiple tumor marker protein chip detective system, while 62.77% in tumors. The sensitivity was 66.11%, the specificity was 48.68%.
2) The positive rates of CEA, CA125 and CA242 were 40.93%, 29.78% and 13.48%, separately, which were significant different from their counterpart of lung binign disease group and health control. Moreover CEA was significance rather than CA125 and CA242 in lung cancer diagnosis. Grade of efficacy and status was closely related to CA19-9 , CEA , CA242 , AFP and CA125 in adenocarcinoma , to CA125 in squamous cell carcinoma ( SCC) , and to CA19-9 and CA125 in small cell lung cancer ( SCLC) .
3) It was 71.97% positive ratio in collrectal carcinoma which was significantly higher than the other two groups. The sensitivity, specificity, accuracy, positive predict value and negative value of C-12 system were 71.97%, 55.7%, 83.09%, 39.64%. The positive ratio of combined detection was higher than everyone of C-12 system only (P<0.05). The expression of tumor markers CA19-9, CEA, CA242, AFP,β-HCG,HGH and CA125 in carcinoma group was markedly stronger than other two groups, meanwhile the sensitivity and validity of combined detection of CA19-9, CEA, CA242 was proved. The level of CA19-9, CEA, CA242 and CA125 was related to prognosis in colorectal carcinoma.
4) The positive rate of pancreatic cancer is significantly higher than that of pancreatic begin patients and normal persons (P<0.05). CA19-9, CEA and CA242 have a clinical significance in diagnosis. Combined detection can significantly increase the diagnostic sensitivity, but the specificity has been decreased.
5) The positive rate of gastric cancer (50.00%) was significantly higher than that of gastric begin patients (34.82%) and normal persons (10.59%) (P<0.05). The CA19-9, CEA, CA242, CA125 and HGH had a clinical significance (P<0. 05) .
6) In urinary system tumor, positive value is 93.33%,negative valve is 23.68%,specificity is 81.81%,Sensitivity is 49.12%. In male genital system tumor, positive value is 64.86%,negative valve is 46.15%,specificity is 48.00%,Sensitivity is 62.16%. In prostate carcinoma, combined measurement of PSA and f-PSA can increase diagnostic specificity to 90% (P<0.01), which will not decrease diagnostic sensitivity, The Combining diagnosis of multiple serum tumor markers using C-12 protein chip has a great significance with diagnosis of the single serum tumor marker in both urinary system tumor and male genital system tumor .
2. Establishment of diagnostic function by Bayesian methods and Development of < Multiple Tumor Marker Analysis System V1.0> software
1) Three grades of diagnostic functions were successfully established in C-12 multiple tumor marker.
2) The specificity, sensitivity and accuracy rate were 70.11%, 66.10% and 68.07% by C-12 multiple tumor marker while 82.11%, 71.28% and 83.97% by the first grade diagnostic function.
3) The accuracy rate, sensitivity and specificity were higher than C-12 multiple tumor marker chip in all the 10 types of malignant tumors by the second diagnostic function.
4) The third diagnostic function could identify part of the type of tumor, such as breast cancer, prostate cancer, lung cancer, liver cancer, ovarian cancer, endometrial cancer and pancreatic cancer.
5) The < multiple tumor marker analysis system V1.0> software was developed successfully. The functions of this software include data read, query, print 10 tumor scorings can be calculated and printed by this software.
3. Signal amplification of electrochemical immunosensor for the detection of human serum IgG using double-codified nanosilica particles as labels
1) With the sandwich-type immunoassay format, the linear range of the developed immunosensor by using anti-IgG-SiO2-HRP as tracer and hydrogen peroxide (H2O2) as enzyme substrate is 0.01-15 nmol/L IgG with a detection limit of 5.0 pmol/L, while the assay sensitivity by directly using HRP-labeled anti-IgG as secondary antibodies is 1.0-10 nmol/L with a detection limit of 0.1 nmol/L IgG..
2) The reproducibility, stability and specificity of the proposed immunoassay method were acceptable.
3) The IgG concentrations of the clinical serum specimens assayed by the developed immunosensor show consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay (ELISA) method.
4. Nanogold-enwrapped graphene nanocomposites as trace labels for sensitivity enhancement of electrochemical immunosensors in clinical immunoassays: Carcinoembryonic antigen as a model
1) The method using HRP-anti-CEA-NGGNs as detection antibodies shows high signal amplification, and exhibits a dynamic working range of 0.05-350 ng/mL with a low detection limit of 0.01 ng/mL CEA (at 3s).
2) The reproducibility, stability and specificity of the proposed immunoassay method were acceptable.
3) The nanogold-enwrapped graphene nanocomposites facilitate the electron transfer between the analyte and the base electrode in contrast with that of using silica nanoparticles previously reported by us.
5. The signal-enhanced label-free immunosensor based on assembly of Prussian blue-SiO2 nanocomposite for amperometric measurement of Neuron-specific enolase
1) As the PB-SiO2 nanostructural sensing film provides plenty of active sites for the direct catalysis of H2O2, the proposed immunosensor exhibited excellent performance with high sensitivity.
2) The immunosensor exhibited good linear behavior in the concentration range from 0.25-5.0 and 5.0-75 ng/mL for the quantitative analysis of neuron-specific enolase (NSE), a putative serum marker of small cell lung carcinoma (SCLC), with a limit of detection of 0.08 ng/mL in the presence of 0.75 mmol/L H2O2.
3) No significant difference between the results obtained using the immunosensor and the ELISA, which showed an acceptable degree of agreement, especially at NSE concentrations in human serum with the immunosensor.
6. An electrochemical immunosensor for simultaneous multiplexed detection of NSE and pro-GRP using liposomes as enhancer
1) A sensitive immunosensor was construced based on a new signal amplification strategy was developed for the simultaneous multiplexed quantitative analysis using the liposomes contained different electrchemical active molecule as singal enhancer.
2) A electrochemical immunosensor was developed for the simultaneous multiplexed quantitative analysis of ProGRP in the concentration range from 100-1000 pg/mL and NSE in the concentration range from 5-50 ng/mL, respectively.
3) The concentrations of NSE and ProGRP in human serum obtained by the developed immunosensor show consistent results in comparison with those obtained by commercially available enzyme linked immunosorbent assay (ELISA) method.
Conclusion
1. The clinical significance and expression of C-12 multiple tumor marker protein chip detective system in tumors
C-12 multiple tumor marker protein chip detective system can be been used in the tumor screen, diagnosis, evaluation of efficacy and the follow-up of high-risk groups. It can’t be wildly used with low specificity, sensitivity, accuracy and difficulty analysis in clinical practice.
2. Establishment of diagnostic function by Bayesian methods and Development of < Multiple Tumor Marker Analysis System V1.0> software
Diagnostic function established by Bayesian methods has advantages of high sensitivity, specificity and accuracy and is of great clinical value in early detection of the cancers. The software can automatic diagnosis the sort of tumor, which is a scientific, convenient, efficient tool for clinician.
3. Signal amplification of electrochemical immunosensor for the detection of human serum IgG using double-codified nanosilica particles as labels
a newly double-codified immunoassay method based on HRP-doped nanosilica particles modified with anti-IgG antibodies has been introduced for electrochemical detection of human IgG in a sandwich-type immunoassay format. The electrochemical signal is amplified by using double-codified nanosilica particles as tracer and hydrogen peroxide as enzyme substrate. Highlight of the developed immunoassay method is the intrinsic electrochemical properties of the double-codified nanosilica particles that being proportional to the protein concentration can be directly quantified by cyclic voltammetry. This method has many desirable merits including sensitivity, accuracy, and little required instrumentation.
4. Nanogold enwrapped graphene nanocomposites as trace labels for sensitivity enhancement of electrochemical immunosensors in clinical immunoassays: Carcinoembryonic antigen as a model
We develop a new conductive nanolabel with highly amplified properties and high sensitivity for the sandwich-type electrochemical immunoassays. The assayed results of serum samples with the sensor received an acceptable agreement with the reference values. The convenient operation and ultrasensitivity of the developed methodology provides a promising potential in clinical diagnosis.
5. The signal-enhanced label-free immunosensor based on assembly of Prussian blue-SiO2 nanocomposite for amperometric measurement of Neuron-specific enolase Tests result indicated this strategy could provide a promising approach of the sensor preparation and a practical method with high sensitivity, shorter detection time and border linear dynamic range for clinical immunoassay in dignosis of NSE.
6. An electrochemical immunosensor for simultaneous multiplexed detection for NSE and pro-GRP using liposomes as enhancer
The multiplex immunoassay can offer higher sample throughput, less sample consumption, shorter assay time and lower cost than the traditional parallel single analyte immunoassay. The multiplex immunoassay provides a clinical serum diagnostic solution in small cell lung carcinoma.
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