Asymmetric PCR Combined With Surface-enhanced Raman Spectroscopy For The Detection Of Liquid Biopsy Specimen In Lung Cancer

BackgroundThe targeted therapy against EGFR mutation gene is the most important development in lung cancer in recent years.It is very important for targeted therapy to accurately detect the details of driver genes and timely monitor the real-time status of drug-resistant genes.However,the low acquisition rate of lung cancer tissue specimens in clinical practice makes it difficult to carry out multiple,repeated and accurate mutation genes detection.In recent years,liquid biopsy specimens have attracted more and more attention from researchers and doctors,among which circulating tumor DNA（ct DNA）has gradually become an excellent substitute for tumor histological specimens due to its unique advantages.However,the detection rate and specificity of EGFR mutation using peripheral blood ct DNA are low,and the detection technology developed on the basis of PCR cannot meet the demand of trace nucleic acid detection,so it is urgent to develop a more efficient nucleic acid detection technology.In this study,based on the previous research of our group,we further combined the hypersensitive surface-enhanced Raman spectroscopy（SERS）and asymmetric PCR to detect the EGFR mutation genes in peripheral blood ct DNA of NSCLC patients.Materials and Methods1.Two methods were used to synthesize silver seeds and the properties of silver seed particles were compared.The sea urchin like Au NCs nanoparticles were prepared by seed crystal growth method,and then the physicochemical properties of the granules were exzamined.The surface morphology and topological structure of the nanoparticles were observed by scanning electron microscope（SEM）,and the element composition of the nanoparticles was analyzed by energy spectrum analysis（EDS）.The nanoparticle size and morphological structure were observed by transmission electron microscopy（TEM）.The maximum absorption peak of ultraviolet visible spectrum of nanoparticles was measured.The particle size distribution and Zeta potential of nanoparticles were also detected.The SERS signal of sea urchin like Au NCs nanoparticles was tested and the enhancement factor of Au NCs nanoparticles was calculated using crystal violet（CV）as the presentation molecule.Finally,finite difference time domain（FDTD）method was used to simulate the electromagnetic field distribution of Au NCs particles.2.The Au NCs nanoparticles were firstly prepared,and then Au NCs probes were constructed by linking EGFR mutation-specific molecule beacons（MBs）on the surface of Au NCs nanoparticles.Au NCs probe morphological characteristics were observed by SEM and TEM.The maximum absorption peak and strength of Au NCs probe were measured.The hydration particle size distribution and Zeta potential of Au NCs probe were tested.The optimum SERS signal was determined by comparing the volume ratios of different Au NCs nanoparticles and MBs.The kinetics characteristics of time and concentration of Au NCs probe were studied.The specificity against target sequence and enzyme stability of the of Au NCs probe were also studied.3.The Au NCs probe combined with asymmetric PCR technology was used to construct an novel detection method.Human lung adenocarcinoma cell lines were used to extract DNA from the cells,and blood/tissue paired samples were collected from 22patients with stage IV lung adenocarcinoma.The EGFR mutation genes of the above specimens was detected using the asymmetric PCR-SERS method,and the sensitivity,specificity and detection threshold of this novel technology were calculated.Results1.In this study,by comparing two synthetic technology of silver seed nanoparticles,the silver nanoparticles with an average diameter of 30 nm were more regular and uniform after adding glycerol into the reaction system.The synthesized Au NCs with a particle size of about 120 nm were covered with 79 nano-spines of about 20 nm in length.The element composition of Au NCs analyzed by EDS showed gold（58.36%）,silicon（41.56%）and silver（0.08%）.The maximum absorption peak of Au NCs nanoparticles in uv-vis spectrum was located at 835 nm.The crystal violet molecule was used as the reporter molecule,and the single-particle enhancement factor was calculated as 3.44×10⁷.FDTD was used to calculate the electromagnetic field distribution of a single Au NCs particle at the incident laser wavelength of 633 nm.The“hot spots”or maximum values of|E|were located between the tips of adjacent spikes and in the nano-gaps of Au NCs particles.2.The EGFR mutant nucleic acid probes were prepared using Au NCs particle and MBs in this study.The molecular layer of the MBs was about 5-7 nm thick.The maximum uv-visible light absorption peak of the Au NCs probes was 794 nm.the average particle size was 150±20nm,and the zeta potential was-20±5m V.The SERS signal of Cy3molecule at the peak of 1466 cm^-1 was set as the standard peak of this experiment.The minimum detection limit of Au NCs probe was 5.8 n M,which was obtained from the time/concentration dynamics experiment of Au NCs probe.The specificity and enzyme stability of Au NCs probes showed that the probes had good target sequence specificity and enzyme stability.3.After combining with asymmetric PCR technology and Au NCs probe,asymmetric PCR-SERS detection system was constructed.In the cytological model of lung cancer,the minimum threshold of asymmetric PCR-SERS method for detecting EGFR mutantion genes reached 4.24 f M.In the detection of 22 patients with stage IV lung adenocarcinoma tissue/blood paired samples,the sensitivity of EGFR mutation in peripheral blood ct DNA samples detected by asymmetric PCR-SERS method was 73.3%（i.e.the sensitivity of del E746-A750del was 75%;the sensitivity of L858R was 71.4%）,and specificity for detecting wild-type EGFR reached 100%（7/7）.ConclusionIn this study,a novel asymmetric PCR-SERS method was established by preparing nano-nucleic acid probes with good performance and combining SERS technology and asymmetric PCR technology.In the detection of lung cancer cytological models and tissue/blood paired samples of lung cancer patients,the asymmetric PCR-SERS method showed high sensitivity and specificity,and the lower limit of detection reached 4.24 f M.The asymmetric PCR-SERS method showed a good clinical application prospect.