Lateral flow DNA biosensor for visual detection of nucleic acid with triple-helix DNA functionalized carbon nanotube

We describe a novel lateral flow DNA biosensor (LFDB) based on carbon nanotube (CNT) and triple helix DNA (THD). The carboxylated CNT was first conjugated with amine-modified auxiliary single-stranded DNA probe (P₁) by dehydration reaction and used as signal probe. A main DNA probe (P₀) was introduced to react with the P₁ and formed the THD on the CNT surface. Because of the large spatial effect, P₁ was in an inactive state and cannot hybridize with the capture DNA probe (P₂) fixed on the LFDB test area. When the target DNA was present, P₀ in the triple helix DNA hybridized with the target DNA due to the stronger base action, and the decomposition of the triple helix structure exposed P₁. Therefore, P₁ on CNT surface was activated to hybridize with P₂. The CNT along with P₁ was thus captured at the test area and accumulated to show a black line, which can be observed by naked eye for qualitative analysis and recorded with a portable grayscale reader for quantitative analysis. Single-stranded DNA was used as a target to prove the feasibility of the model. Under the best experimental conditions, the THD-CNT based LFDB was able to detect the lowest DNA concentration of 15 pM, which is 2.67 times better than that of the traditional duplex CNT-based LFDB. It should be noted that the LFDB based on THD functionalized CNT can differentiate between one-base-mismatched DNA and the complementary target DNA, can detected target DNA in 10% human serum, and can be employed as a versatile platform to detect various target (proteins, small molecular) by changing the sequence of P₀. This biosensor platform has enormous potential in the point-of-care detection of a rich diversity of analytes for clinical diagnosis and biomedical research.