Carbon nanotubes multifunctionalized by rolling circle amplification and their application for highly sensitive detection of cancer markers

Abstract

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 10⁵-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.

Keywords: cancer markers; carbon nanotubes; detection systems; enzymes; rolling circle amplification.