Detection Technology of MicroRNA-21 for Non-small Cell Lung Cancer
|Keywords||lung cancer microRNA real-time fluorescence quantitative PCR electrochemical impedance microelectrode|
The current clinical used tumor biomarkers have their advantages and disadvantages. On one hand, they promote the development of lung cancer diagnosis. On the other hand, they have large traumatic effects on patients, and its clinical application is limited. Circulating microRNA biomarkers in cancer treatment has a very important potential value. Blood-based microRNAs as molecular markers for tumor diagnosis and prognosis can be accurate, stable, convenient and with less trauma. The traditional microRNAs detection techniques require tedious multi-step operation, so the large number of operational errors must affect the accuracy of detection results. It’s necessary to develop new microRNAs detection technologys in order to simplify operations and reduce operator errors and improve the detection accuracy. Electrochemical impedance detection technique has good specificity, high sensitivity and big convenience. We can use this method combined with other detection technologies to improve the level of microRNA detection.This paper carried out the following several aspects:（1） Firstly, we applied real-time fluorescence quantitative PCR method to detect miR-21 expression levels in blood from lung cancer patients and the normal population, and according to clinical and pathological features the statistics were analyzed.（2） Sencondly, microelectrodes were prepared based on electrochemical etching technology. The analysis and characterization of these microelectrodes were realized by scanning electron microscopy and cyclic voltammetry method.（3） Lastly, we used thiol-based self-assembly technology to build impedance biosensor for microRNAs detection. First of all, the thiol labeled nucleic acid probe self-assembled onto gold electrode surface by strong electrostatic adsorption of the Au-S, formed RISC-miR-21 complexs recognition layer. Through measuring the sensor surface electron transfer resistance Ret increments before and after the hybrid capture reaction to achieve the microRNAs detection rapidly. Secondly, we used the sensor to capture RISC-miR-21 complexs. Finally, the detection conditions of the sensor were optimized. The main conclusions are following:（1） The results of real-time fluorescence quantitative PCR detection on miR-21 expression levels in blood from lung cancer patients and the normal population indicated that miR-21 expression levels in the blood of patients with lung cancer were significantly higher than the normal population（P<0.01）. Among the 20 cases of lung cancer patients, 85% （17/20） of them showed miR-21-upregulated. Statistics showed that the abnormal expression of miR-21 and the clinical characteristics of the sample were independent （P>0.05）. Receiver operating characteristic curve analysis showed that the size of the area under the curve （AUC） was about 0.912±0.045, suggested 78.80% sensitivity and 100% specificity of miR-21 as a potential molecular marker had better diagnostic performance. Circulating miR-21 would be one of the potential biomarkers of lung cancer.（2） Alloy microelectrodes were prepared by electrochemical etching technology. Characterization of micro-electrode surface morphology and the effective radius were obtained by scanning electron microscope （SEM）. The cyclic voltammetry analysis on microelectrodes was achieved by electrochemical workstation. Characterization results showed that: under the conditions of 0.1 mol/L of KCl solution and 5 V voltage, prepared microelectrodes had the best performance, the effective radius of those can be below 5μm. Cyclic voltammetry response curves showed that the conductivity and reversibility and stability of the prepared microelectrodes were both good.（3） We used thiol-based self-assembly technology to build impedance biosensor successfully. By optimized the impedance biosensor detection conditions, concluded that the sensor fostered in 36℃for 30 min would be the best. Cyclic voltammetry results indicated that the electrode surface can form a stable film by layer self-assembly method, therefore realized effective combination with RISC-miR-21 complex. AC impedance test results showed that thiol-based probe had fixed on the electrode surface by self-assembly, and there were obstacles for [Fe （CN）6]3-/4- electronic balance on the electrode surface. Electrode surface would be closed with dodecanethiol treatment, meanwhile increased the electron transfer resistance Ret. Probe with the RISC-miR-21 binding complex further hindered [Fe （CN）6]3-/4- activities, electron transfer resistance Ret continued to increase.