| Lung cancer is one of the common malignancy, and the morbidity and mortality has leapt to the the head of common tumors, which seriously harm to the human health. With the continuous development of medical science and technology, the level of diagnosis and treatment of lung cancer has been significantly improved, but still far from satisfying the needs of the clinical and human. A large number of studies have shown that the prognosis of lung cancer is closely related to the clinical stage, which the5-year survival rate of lung cancer patients in early-stage can reach70%, and the most effective way to reduce lung cancer mortality is its early diagnosis and treatment. Because of occult onset of lung cancer, most of diagnosed lung cancers are advanced. Therefore, the target-“early detection, early diagnosis, and early treatment”has important clinical value for lung cancer. Currently, early detection of lung cancer in clinical mainly rely on imaging, but it has disadvantages such as high false positive rate and cost. Therefore, identification of benign and malignant lung nodules is an urgent problem to be solved in the detection of early stage for lung cancer. With the continuous development of proteomics, the value of tumor markers has been drawing more attention in the early diagnosis and treatment of lung cancer. Numbers of studies have shown that tumor markers play an important role in differential diagnosis, pathological type, clinical stage, efficacy observation, monitoring recurrence and prognosis assessment of tumor. Ideal tumor markers should be highly sensitive and specific, and easy to be detected. However, there is no marker with good specificity, high sensitivity for lung cancer now. Additionally, it is difficult to evaluate individual with single tumor markers for its limitations.Now joint detection of multiple tumor markers is mostly used in clinical work, but it has cumbersome operations, as well as low sensitivity, thus, it can not be able to meet the purpose of early detection for lung cancer in large scale screening. With the continuous development of the bio-chip technology and nanotechnology, it makes the highly sensitive and multiple tumor markers rapid detection possible.Purpose:To establish a highly sensitive and rapid detection system for multiple tumor markers based on gold nanoparticle probes and protein chip, achieving the early diagnosis of lung cancer.Methods:1. Firstly, gold nanoparticles were marked and charactered. Then, the gold nanoparticle probes and protein chips were made, thus establishing a basic detection system.2. The optimum stable protein quantity of gold nanoparticles and the best added amount of gold nanoparticle probes were optimized through gradient optimization experiments, and then, the dyeing method was screened and optimized. The specific analysis of tumor markers was measured, and the experimental conditions were optimized, such as temperature, time and so on. Finally, the detection system was optimized integrally.3.86lung cancer patients and42healthy persons were detected with the proposed detection system. The results were analyzed, and the standard curves of four tumor markers were drawed. The measure results detected with the proposed detection system were compared with the results tested by electrochemical assay used in clinic and DKKl kit, and the relationship between the measured results and clinical data parameters was analyzed.4. Statistical analysis:statistical analysis such as non-parametric tests, x2test and correlation analysis was performed with SPSS17.0, the test level α=0.05.Results:1. The optimum stable protein quantity of200μL gold nanoparticle solution were2.0μL (antibody concentration of5mg/mL) to CEA,5.0μL (2mg/mL) to CYFRA21-1,14.0μL (1.04mg/mL) to NSE and6.0μL (2mg/mL) to DKK1.2. Gold nanoparticle solution was occurred redshift after marked by detection antibody, and the maximum absorption peak was increased from520nm to530nm.3. The best added amount concentration of gold nanoparticle probes were1.0nM CEA,1.4nM CYFRA21-1,1.8nM NSE and2.0nM DKK1.4. In this study, the the H2O2method gold deposition was chosed to dye, and the optimum concentration were3.0M to H2O2and20mM to gold chloride acid.5. Four tumor markers had no cross-reaction and interference with each other on protein chip, with a good specificity.6. Standard curves of four tumor markers:a good linear relationship was found between40pg/mL to25ng/mL for CEA concentrations, R2=0.99; while70pg/mL to25ng/mL, R2=0.994for CYFRA21-1concentrations;90pg/mL to25ng/mL, R2=0.994for NSE concentration;90pg/mL to25ng/mL, R2=0.996for DKK1concentration. The detection limits were determined through sensitivity test, and the results were lOpg/mL to CEA,15pg/mL to CYFRA21-1,20pg/mL to NSE, and30pg/mL to DKK1.7. The overall consistency of the test results measured by this method and clinical ECLIA assay, DKK1kit was found through the correlation analysis and the correlation coefficients r were0.986to CEA,0.985to CYFRA21-1,0.978to NSE, and0.993to DKK1. The difference of concentration of four tumor markers between the lung cancer group and the healthy group was statistically significant (all P＜0.05), and the concentration in the lung cancer group was significantly higher than the healthy group. In the healthy control group, the difference of concentration of four tumor markers in different gender groups and different age groups (≤60years and＞60years) was not statistically significant (all P＞0.05). In lung cancer group, the difference of concentration of four tumor markers in the different age, sex, and smoking was not statistically significant (all P＞0.05). The difference of concentration of CEA, CYFRA21-1, NSE between different pathological types was statistically significant (all P＜0.05) while DKK1not (P=0.294). The concentration of CEA was highest in lung adenocarcinoma (P＜0.001), while CYFRA21-1in lung squamous cell carcinoma (P=0.004), NSE in small cell lung carcinoma (P=0.002). The difference of four tumor markers in different clinical stages was statistically significant (all P<0.05), and the concentration of Ⅳ stage was significantly higher than the other three stages (all P＜0.05). The95th percentile values of tumor markers for the healthy control group were used as the cutoff values for the following analyses (4.82ng/mL to CEA,3.04ng/mL to CYFRA211,23.7ng/mL to NSE,14.15ng/mL to DKK1). In lung cancer group, the sensitivity was38.37%to CEA,51.16%to CYFRA21-1,26.74%to NSE,52.33%to DKK1, and the sensitivity of four tumor marker joint detection was88.37%, which were greatly improved than any single detection (all P＜0.001). The sensitivity of CEA was highest in adenocarcinoma (54.35%), while CYFRA21-1in squamous cell carcinoma (54.84%), NSE in small cell lung cancer (66.67%).Conclusion:In this study, a highly sensitive protein detection system based on protein chip and gold nanoparticle probes was introduced, with capability of semi-quantitative visual detection. The new technique described herein has been able to detect four kinds of tumor markers and the assay time was less than1.5h. With multiple tumor markers detected rapidly and simultaneously, the target of early diagnosis and assessment for lung cancer was achieved. Additionally, with lots of advantages such as simple instrument and low cost etc, this method could be applied to detect protein markers of tumors and screen high-risk groups, with a great prospect. Meanwhile, it also could provide a theoretical and experimental basis for clinical tumor markers detection,... |
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