Large Number Hexagonal Cavities Microfluidic Digital Chip for Gene Mutation Ultrasensitive Analysis in Lung Cancer

Abstract

Lung cancer is the highest incidence of malignant tumors in the world. Targeted therapy based on gene mutation detection including epidermal growth factor receptor (EGFR) gene mutation is one of the first-line treatments. In this work, took EGFR gene G719S mutation as a representative object, a large number hexagonal cavities microfluidic chip (LHMC) platform based on digital PCR (dPCR) for the absolute quantification of gene mutation in lung cancer was established. In this chip, 113,137 cavities chip was designed to increase the number of absolute quantitative, which was 2~5 times higher than the traditional one. The hexagonal shape of cavities elevates the filling rate and filling speed. A set of primers and probes for G719S with high sensitivity, high specificity and high reliability were designed and screened. Then the PCR parameters were optimized. The results demonstrated that the chip platform shows good performance. The minimum detection concentration of the gene mutant was 3.01 copies/μL, and a good correlation (Y= 0.725X- 0.581, R2= 0.984) was noted between the measured value and the expected value. This chip possesses sensitively detect positive mutations in G719S and completely negative when detecting other substances. The developed LHMC-dPCR chip is a rapid and accurate gene mutation analysis platform, which has faster speed and lower price than classic detection methods, for instance, droplet dPCR, DNA sequencing method. Moreover, LHMC-dPCR is not limited by the number of nucleic acids and droplets and there is no need to estimate the nucleic acid concentration of the sample. This chip platform could also detect other gene mutations, for example, L858R, exon 19 deletions, in other tumors including lung cancer.