Terminal Protection of Small-Molecule-Linked DNA for Sensitive Electrochemical Detection of Protein Binding via Selective Carbon Nanotube Assembly

Abstract

Small-molecule-linked DNA has emerged as a versatile tool for the interaction assay between small organic molecules and their protein receptors. We report herein the proof-of-principle of a terminal protection assay of small-molecule-linked DNA. This assay is based on our new finding that single-stranded DNA (ssDNA) terminally tethered to a small molecule is protected from the degradation by exonuclease I (Exo I) when the small molecule moiety is bound to its protein target. This finding translates the binding of small molecules to proteins into the presence of a specific DNA sequence, which enables us to probe the interaction between small organic molecules and their protein targets using various DNA sequence amplification and detection technologies. On the basis of selective assembly of single-walled carbon nanotubes (SWNTs) with surface-tethered small-molecule-linked ssDNA not protected by protein binding, a novel electrochemical strategy for terminal protection assay has been developed. Through detecting the redox signal mediated by SWNT assembly on a 16-mercaptohexadecanoic acid-blocked electrode, this strategy is able to ensure substantial signal amplification and a low background current. This strategy is demonstrated for quantitative analysis of the interaction of folate with a tumor biomarker of folate receptor (FR), and a detection limit of 3 pM FR is readily achieved with desirable specificity and sensitivity, indicating that the terminal protection assay can offer a promising platform for small molecule-protein interaction studies.

Biofunctionalization of Nanomaterials

“…A quasilinear response is obtained in a logarithmic concentration scale within a four-order-of-magnitude concentration range from 5 pg mL −1 to 50 ng mL −1 , with a readily achieved detection limit of 3 pg mL −1 [121]. Similarly, this strategy is demonstrated for the quantitative analysis of the interaction of folate with a tumor biomarker of folate receptor, and a detection limit of 3 pM of folate receptor is readily achieved with a desirable specificity and sensitivity [122]. …”

Nucleic Acid Fluorescent Probes for Biological Sensing

“…55,56 Based on these properties of SWNTs, a series of sensing systems has been developed for nucleic acid analysis 55 and protein recognition. 57,58 Graphene oxides offer even better fluorescence quenching properties than SWNTs due to their planar structure. 59 Reduced graphene oxide (rGO) can form a complex with acridine orange (AO) and effectively quench the fluorescence of AO, but the AO-rGO complex can be reversibly destroyed by a G-quadruplex structure aptamer (PS2.M).…”

Electrochemical Biosensing Based on Carbon Nanotubes

“…Because of strong electrostatic and hydration repulsions between the DNA-SWCNTs and the negatively charged SAM, the SWCNTs wrapped by the protected ssDNA cannot be adsorbed on the MHA SAM, keeping the MHA SAM isolated with no background signal. According to the mechanism, a tumor biomarker of folate receptor is detected within the range 10 pM-5.0 nM and a detection limit of 3 pM [60].…”