Sensitivity-Improved SERS Detection of Methyltransferase Assisted by Plasmonically Engineered Nanoholes Array and Hybridization Chain Reaction

Detection of methyltransferase (MTase) activity is of great significance in methylation-related disease diagnosis and drug screening. Herein, we present a dual-amplification sensing strategy that is assisted by plasmonically enhanced Raman intensity at engineered nanoholes array, along with signal amplification by the hybridization chain reaction (HCR) for the ultrasensitive detection of M.SssI MTase activity and inhibitor screening. An engineered surface-enhanced Raman scattering (SERS) substrate, namely, a structured nanoholes array (NHA) with wavelength-matched surface plasmon resonance (SPR) at the wavelength of laser excitation (785 nm), was rationally designed through finite-difference time-domain (FDTD) simulations, precisely fabricated through master-assisted replication, and then used as a sensing platform. Uniform and intense SERS signals were achieved by turning on the plasmonic enhancement under the excitation of SPR. Probe DNA was designed to hybridize with target DNA (a BRCA1 gene fragment), and the formed dsDNA with the recognition site of M.SssI was assembled on the NHA. In the presence of M.SssI, the HCR process was triggered upon adding DNAs labeled with the Raman reporter Cy5, leading to an amplified SERS signal of Cy5. The intensity of Cy5 increases with increasing M.SssI activity, which establishes the basis of the assay for M.SssI. The developed assay displays an ultrasensitivity that has a broad linear range (0.002-200 U/mL) and a low detection limit (2 × 10^-4 U/mL), which is superior to that of the reported SERS-based detection methods. Moreover, it can selectively detect M.SssI in human serum samples and evaluate the efficiency of M.SssI inhibitors.