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BACKGROUND Prostate cancer cells have very high PCA3 messenger RNA levels, which turns it into one of the new biomarkers for prostate cancer prognosis and diagnosis. OBJECTIVE Our goal here is to development a new aptasensor to detect PCA3 release by cancer cell. METHODS DNA hairpin containing PCA3 aptamer was thiolated, conjugated to methylene blue (MB) redox probe and immobilized on gold electrode through self-assembly to detect label-free cancer cells. RESULTS Our data have evidenced stable and sensitive sensor presenting wide linear detection range (0-150 ng/mL). In addition, monitoring PCA3 released by a different type of prostate cells can provide in-depth knowledge about prostate cancer dynamics; therefore, it is a powerful platform for earlier clinical diagnostic. The released PCA3 can vary depending on the type of adopted prostate cells. CONCLUSION PCA3 release was monitored in a group of cells for 2 h; it showed significantly higher expression in both LNCaP and PC-3 cells. This strategy provides unique and simple methodology to achieve more sensitive and specific PCA3 detection; thus, it emerged as promising tool for early cost-effective diagnosis.