Carbon Nanotubes Multifunctionalized by Rolling Circle Amplification and Their Application for Highly Sensitive Detection of Cancer Markers

There are still challenges for the development of multifunctional carbon nanotubes (CNTs). Here, a multiwalled carbon nanotube (MWCNT)-based rolling circle amplification system (CRCAS) is reported which allows in situ rolling circle replication of DNA primer on the surface of MWCNTs to create a long single-strand DNA (ssDNA) where a large number of nanoparticles or proteins could be loaded, forming a nano-biohybridized 3D structure with a powerful signal amplification ability. In this strategy, the binding ability of proteins, hybridization, replication ability of DNA, and the catalytical ability of enzymes are integrated on a single carbon nanotube. The CRCAS is then used to develop colorimetric and chemiluminescent assays for the highly sensitive and specific detection of cancer protein markers, alpha-fetoprotein (AFP) and prostate specific antigen (PSA). The colorimetric CRCAS assay is 4000 times more sensitive than a conventional enzyme-linked immunosorbent assay (ELISA), and its concentration range is 10 000 times wider. Control experiments show that as low as 10 pg mL(-1) AFP or PSA could be detected even in the presence of interfering protein markers with a more than 10(5)-fold greater concentration in the sample, demonstrating the high specificity of the CRCAS assay. The limit of detection of the chemiluminescent CRCAS assays for AFP and PSA are 5 fg mL(-1) (70 aM) and 10 fg mL(-1) (0.29 fM), respectively, indicating that the sensitivity is much higher than that of the colorimetric CRCAS assay. Importantly, CRCAS works well with real biological samples.