An aptasensor using DNA aptamer and white light common-path SPR spectral interferometry to detect cytochrome-c for anti-cancer drug screening

摘要

Liver cancer is a major health hazard and more than 6 million new cases are reported worldwide every year. It is a difficult cancer to treat because multi-drug resistance (MDR) is frequently developed during chemotherapy. Therefore, identifying new drugs to overcome MDR becomes an urgent need to cure the disease. It has been known for a while that cytochrome c (cyto-c) is a crucial death regulator that triggers apoptosis in cancer cells when released from the mitochondria. This makes cyto-c a good biomarker to report drug effect after treatment. In this study, we developed an aptasensor by using an aptamer against cyto-c and a common-path phase surface plasmon resonance (CPSPR) sensing technique for drug screening. Our cyto-c aptamer is a small (76-mer) single-stranded DNA selected from a synthetic DNA library by virtue of its high binding affinity and specificity based on its unique DNA sequence and 3D configuration after folding. The CPSPR is a novel technique that employs a white-light source (600-800 nm) for SPR excitation across the visible to near infrared spectrum with a fixed light incidence angle. It measures phase change in SPR at an optimized coupling wavelength in a label-free manner. Compared to other conventional laser based phase detecting schemes, this system offers an extended dynamic range of measurement (80 nM to 80 pM) and ultra-sensitivity (detection limit ≥50 pM). After setting the groundwork, we used this aptasensor to test a number of agents (atractyloside, capsaicin, cordycepin, polyphyllin D (PD), veratridine) from the Chinese herbs for killing cancer. Among all agents tested, PD was found to release more cyto-c from a liver cancer line with MDR than its counterpart without MDR. The same was found from the isolated mitochondria indicating that our aptasensor can also be used for mechanistic studies. Since the detection process can be completed within minutes, our approach provides a simple, rapid and sensitive solution for the selection and development of novel anti-cancer agents.