Detection Of EGFR Mutation Gene In Lung Cancer By Using Surface Enhanced Raman Spectroscopy

Background:The targeted therapy against EGFR mutation gene is one of the most effective treatments in the patients with advanced lung adenocarcinomas.It is important to monitor the EGFR mutation gene and drug resistance gene in the whole therapy.However,the detection sensitivity and specificity of the current techniques,such as sARMS?ddPCR and NGS,can not satisfy the demand in pleural effusion and liquid biopsy specimen.Otherwise,these techniques are time-consuming and high-cost so that they are not commonly applied in the clinical practice.Hence,it has a remarkable significance for the precise treatment of lung cancer to explore a novel detection technique.Materials and Methods1.In this research,EGFR mutation status was analyzed by DNA sequencing in 153 lung adenocarcinoma tissues.Then,30 samples including 10 with wild type?wt?-EGFR,10 with L858 R and 10 with E19 del mutations were selected into Raman and IHC analysis.PCA and SVM were applied for final prediction.2.We synthesized the sea-urchin like Au nanocluster?AuNC?and capped crystal violet?CV?,polyethylene glycol,and EGFR mutation specific antibody on its surface.By combining the PCA/SVM algorithm,the nanoprobe was used to detect EGFR mutation gene in cancer cells and exfoliative cells of malignant pleural effusion?MPE?in 35 patients with lung cancer.3.We synthesized the sea-urchin like Au nanocluster?AuNC?and capped molecular beacon targeted EGFR mutation gene on its surface.The nanoprobe was used to detect EGFR mutation gene in ssDNA and ctDNA of 28 patients with lung cancer.Results1.EGFR mutation status was analyzed by DNA sequencing in 153 lung adenocarcinoma tissues.Of these,75 samples carried the EGFR mutation,including 29 with E19 del,33 with L858 R mutation,7 with T790 M mutation,and 6 with multiple mutations.Then,30 samples including 10 with wild type?wt?-EGFR,10 with L858 R and 10 with E19 del mutations were selected into Raman and immunohistochemistry?IHC?analysis.After removing the spectra from normal and non-mutation regions,441 spectra were appropriate for Raman analysis: 149 from wt-EGFR,135 from L858 R mutation and 157 from of E19 del.The Raman peaks at 675,1107,1127 and 1582 cm-1 were significantly increased in wt-EGFR tissues which can be attributable to specific amino acids and DNA.The Raman peaks at 1085,1175 and 1632 cm-1 which assigned to arginine were slightly increased in L858 R tissues.The overall intensity of E19 del tissues was weaker than others due to the exon 19 deletion that removes residues 746-750 of the expressed protein.The PCA/SVM algorithm yielded an overall accuracy of 87.8% for diagnosing L858 R or E19 del from wt-EGFR tissues.2.We synthesized the sea-urchin like Au nanocluster?Au NC?with an average diameter of 92.4 nm composed of 15 nm nanopricks.By introducing abundant sharp nanopricks,the enhancement factor of AuNC reached at 1.94 × 107.After capped with crystal violet?CV?,polyethylene glycol,and EGFR mutation specific antibody,the AuNC-EGFR had excellent SERS activity and EGFR mutation targeted recognition capability in lung cancer cells.Characteristic SERS signal at 1617cm-1 of CV was linear correlation with number of H1650 cells,demonstrating the minimum detection limit as 25 cells in 1 m L suspension.The gold mass in single H1650 cells exposed to AuNC-E746₇50 for 2 hours ranged from 208.6 to 231.4pg,which approximately corresponded to 56 to 62 AuNCs per cell.Furthermore,SERS was pre-clinically utilized to test EGFR mutation status in MPE samples from 35 patients with lung adenocarcinoma.Principal component analysis and support vector mechine algorithm were constructed for EGFR mutation diagnostic analysis,yielding an overall accuracy of 90.7%.3.Molecular beacon targeted EGFR mutation gene was capped on the sea-urchin like Au nanocluster.This biosensor had excellent SERS activity and EGFR mutation gene recognition.The target DNA sequence could be detected at a lowest concentration of 1×10-8 mol/L,and also this method could detect the DNA of at least 100 cells.To obtain more ssDNA,we combined the biosensor with the asymmetrical PCR.And this novel technique was used to detect the EGFR mutation gene in ctDNA of the patients with lung cancer,with a sensitivity of 61.1% and specificity of 100%.Conclusion:In this study,we used the SERS signal induced by gold nanoparticles to detect the EGFR mutation of lung cancer.We constructed a novel method and two nanoprobes for three kinds of specimen,including lung cancer tissues,MPE,and peripheral blood ctDNA.The diagnostic method of Raman spectroscopy and PCA/SVM supplied a simple,rapid and low-cost procedure for predicting EGFR mutation status in cancer tissues.The nanoprobes for lung cancer cells and ssDNA had a high sensitivity and specificity,and showed great potential in applications such as predicting gene typing of clinical lung cancer in near future.