Surface-enhanced Raman spectroscopy (SERS), which utilizes nanogaps between noble-metal nanostructures as hot spots to yield ultrasensitive SERS signals, is an outstanding label-free and straightforward tool for DNA methylation analysis. Herein, a plasmonic gold nanohole array (PGNA) with well-controlled hot spots and an open surface was designed as a SERS substrate for DNA methylation detection. A finite-difference time-domain (FDTD) simulation was first employed to investigate the electric field distributions of the PGNA as a function of the geometric parameters. The plasmonic response was tuned to 785 cm −1 to match the ring breathing vibrational band of cytosine, the intensity change of which was revealed to be a marker of DNA methylation. Then, guided by the FDTD simulation results, the PGNA was fabricated via the electron beam lithography (EBL) technique. The fabricated PGNA had an open and easily accessible surface topology, a SERS enhancement factor of ∼10 6 , and a relative standard deviation (RSD) of 7.1% for 500 repetitions over an area of 20 × 20 μm 2 using 1 μM Rhodamine 6G as the Raman reporter. The fabricated PGNA was further used as a platform for determining DNA methylation. The proposed method exhibited a sensitivity for detecting 1% of methylation changes. Moreover, insight into the dynamic information on methylation events was obtained by combining principal component analysis (PCA) with 2D correlation spectroscopy analysis. Finally, clear discrimination of the different methylation sites, such as 5-methylcytosine and N6-methyladenine, was demonstrated.

Epigenetic modifications of DNA are known to modulate gene activity and expression and are believed to result in genetic diseases, such as cancer. Four modified cytosines were discovered in mammalian genomes: 5-methycytoine (5mC), 5hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5carboxycytosine (5caC). They are regarded as DNA epigenetic markers and play key roles in the regulation of the dynamic balance between DNA methylation and demethylation. Although detection approaches toward 5mC are ubiquitous, few assays have reported the simultaneous determination of all four modified cytosines as well as monitoring of their dynamic alterations. Here, we developed a label-free surface enhanced Raman spectroscopy (SERS)-based method for directly sensing the four DNA modifications by using a plasmonic gold nanohole array (PGNA) with well-controlled hot spots and an open surface as the substrate. This method is based on identifying SERS spectral features resulting from DNA base modifications.Our study shows that 5mC, 5hmC, 5fC, and 5caC exhibit distinct Raman spectroscopic signatures at 785, 660, 1450, and 1680 cm −1 , respectively. Moreover, the developed method can be used for tracking of the dynamic alterations among these four modified cytosines in DNA mediated by the ten-eleven translocation (TET) protein. The dynamic stepwise conversion from 5mC into 5hmC, 5fC, and 5caC is further demonstrated to be a typical three-step consecutive reaction with rate constants of 0.6, 0.25, and 0.15 min −1 , respectively, which has not been achieved before via a SERS-based method.

Plasmonic nanoparticle (NP)-mediated photothermal therapy (PPTT) has been explored as a minimally invasive approach to cancer therapy and has progressed from concept to the early stage of clinical trials. Better understanding of the cellular and molecular response to PPTT is crucial for improvement of therapy efficacy and advancement of clinical application. However, the molecular mechanism underlying PPTT-induced apoptosis is still unclear and under dispute. In this work, we used nuclear-targeting Au nanostars (Au NSs) as both a photothermal agent to specifically induce apoptosis in cancer cells and as a surface enhanced Raman spectroscopy (SERS) probe to monitor the time-dependent SERS spectra of MCF-7 cells which are undergoing apoptosis. Through SERS spectra and their synchronous and asynchronous SERS correlation maps, the occurrence and dynamics of a cascade of molecular events have been investigated, and a molecular signaling pathway of PPTT-induced apoptosis, including release of cytochrome c, protein degradation, and DNA fragmentation, was revealed, which was also demonstrated by metabolomics, agarose gel electrophoresis, and western blot analysis, respectively.These results indicated that PPTT-induced apoptosis undergoes an intrinsic mitochondria-mediated apoptosis pathway. Combined with western blot results, this intrinsic mitochondria-mediated apoptosis pathway was further demonstrated to be initiated by a BH3-only protein, BID. This work is beneficial for not only improving the fundamental understanding of the molecular mechanism of apoptosis induced by PPTT but also for guiding the modulation of PPTT to drive forward its clinical application.

The use of coral-shaped Au nanostructures as a heater to selectively induce apoptosis in photothermal therapy of cancer is reported.