Development Of Lung Cancer Detection Strip Based On Nucleic Acid Aptamer

Lung cancer is a malignant tumor originating from the bronchial mucosa or glands of the lung.The incidence rate and mortality increase rapidly.At present,the diagnostic methods of lung cancer mainly include X-ray,spiral CT,fiber bronchoscopy,etc.,but they are all based on morphological standards.These standards are not sensitive to early detection,because the number of tumor cells in early stage and early stage of cancer is small,there is a lack of specific molecular markers,the detection rate is low,and early diagnosis is limited.Therefore,searching for molecular markers of malignant tumors with strong specificity and easy early diagnosis has become a research hotspot.Adapters have attracted widespread attention due to their high specificity and affinity.This project is a colloidal gold test strip developed based on nucleic acid aptamers that can detect lung cancer.The test strip was prepared using a lung cancer adapter selected by our research group in the early stage,and was modified,designed,synthesized,validated for lung cancer serum specificity,isothermal amplification,and optimized through a series of steps.This study used thermal precipitation method to prepare Fe3O4,tetraethyl silicate,and 3-aminopropyltriethoxysilane coating,succinic anhydride method to couple carboxyl groups,and performance analysis through protein adsorption.The results showed that the carboxyl magnetic beads prepared by this method（Fe₃O₄@Si O₂@COOH）has good adsorption performance and has a good adsorption effect on bovine serum albumin（12.5mg/g）.Ten aptamers were designed,and q-PCR was used to verify the specificity.One aptamer with better specificity to lung cancer serum was selected.Isothermal amplification was used to amplify signals,and nucleic acid electrophoresis and nucleic acid protein analyzer were used to detect the amplification results.The isothermal amplification system was optimized,and the optimal amplification time and temperature and the amount of various enzymes in the amplification system were determined.A colloidal gold test strip utilizing the principle of nucleic acid hybridization was designed.Due to the tendency of colloidal gold to agglomerate with high salt,it is difficult for colloidal gold to agglomerate with nucleic acid in a high salt state after coupling.Therefore,the dosage ratio of colloidal gold to thiol nucleic acid has been determined;Optimized probe usage for detection and quality control lines.This study used the principle of nucleic acid hybridization to prepare colloidal gold test strips and conducted serum testing on 8 lung cancer patients and 8 healthy individuals.The detection steps included binding the adapter to the patient’s serum,magnetic bead separation,heating elution,isothermal amplification of the signal,and colloidal gold test strip detection.The results showed that early lung cancer patients had a clearly colored red strip on both the quality control line and the detection line;There is a clear red strip on the quality control line for serum testing of healthy individuals.Five healthy individuals on the testing line have a very light red strip,while three healthy individuals have no strip on the testing line.This suggests that the detection method of this test strip has certain reference significance for early detection of lung cancer.The advantage of this test strip is that it can undergo isothermal amplification（without the need for a PCR instrument）,which increases the convenience and sensitivity of detection,and has the advantages of simplicity,sensitivity,and low price.Currently,there is no test strip or method for tumor detection using isothermal amplification technology in the domestic and foreign markets.Therefore,the successful development of this test strip provides a beneficial reference for the development of home based tumor detection reagents.