Establishment Of Diagnositic Model With Protein Chip Diognostic System And Development Novel Electrochemical Immunoassay For Tumor Markers

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.Objective1. 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 Methods1. The clinical significance and expression of C-12 multiple tumor marker protein chip detective system in tumorsA 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> softwareThe 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 labelsElectrochemical 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 modelPrussian 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 enolasePB-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 enhancerThe 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.Results1. The clinical significance and expression of C-12 multiple tumor marker protein chip detective system in tumors1) 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> software1) 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 labels1) 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 model1) 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 enolase1) 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 enhancer1) 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.Conclusion1. The clinical significance and expression of C-12 multiple tumor marker protein chip detective system in tumorsC-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> softwareDiagnostic 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 labelsa 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 modelWe 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 enhancerThe 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.