Controlled Self-Assembly of a Close-Packed Gold Octahedra Array for SERS Sensing Exosomal MicroRNAs.

MicroRNAs (miRNAs) in exosomes can be transferred from parental cells to recipient cells by trafficking exosomes, and they are effective in regulating the gene expression of the recipient cells. Therefore, exosomal miRNAs play a vital role in cancer biology and could be potential biomarkers for cancer diagnosis and therapeutic responses. However, accurate detection of exosomal miRNAs is still challenging due to the low abundance of any given miRNA in exosomes. Herein, a surface-enhanced Raman scattering (SERS)-based sensor was developed for the quantitative determination of let-7a miRNAs in MCF-7 cell-derived exosomes (MCF-7 exosomes) using a close-packed and ordered Au octahedral array as a sensing platform. Au octahedra in the array uniformly stand on their triangular face. This kind of orientation produces "hot surfaces" rather than "hot spots" and greatly improves the detection sensitivity and uniformity. Let-7a detection with single-base specificity was thus achieved from the SERS intensity change induced by the structural switch of the probing DNA from a hairpin to a duplex in the presence of the target. The sensor showed a broad linear range (10 aM to 10 nM) and a low detection limit (5.3 aM) without using any signal amplification strategy. Moreover, this sensor could accurately detect target let-7a in MCF-7 exosomes and further value the impact of drug treatment on exosomal let-7a expression, indicating promising applications of the developed sensor for cancer diagnostics and therapy.

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.

Coral-shaped Au nanostructures for selective apoptosis induction during photothermal therapy.

Apoptosis and necrosis are major cell death pathways that are induced by plasmonic nanoparticle (NP)-mediated photothermal therapy (PPTT). Apoptosis is commonly regarded as a 'cleaner' method of killing cells than necrosis because apoptotic cells maintain plasma membrane integrity, which prevents inflammation caused by the leakage of cytoplasmic contents. Here we report the use of PPTT employing coral-shaped Au nanostructures (Au NCs) to specifically induce apoptosis in human breast cancer MCF-7 cells. Au NCs have high efficacy in photothermal conversion owing to their strong adsorption in the near-infrared (NIR) region and high surface-to-volume ratio. The in vitro experiments showed that laser irradiation with low power density (0.5 W cm-2) for 15 min selectively induced apoptosis, which is mediated by the activation of nuclear encoded proteins Bak and suppression of Bcl-2 protein. Moreover, the use of Au NCs as heaters can effectively ablate MCF-7 xenograft tumors and prevent the return of cancer. The in vivo apoptotic pathway of MCF-7 cells was further confirmed to be selectively induced via immunohistochemistry analysis. These results offer a feasible protocol to selectively induce apoptotic cell death, which benefits the efficacy of PPTT, to promote the clinical use of PPTT.

Label-Free Raman Observation of TET1 Protein-Mediated Epigenetic Alterations in DNA.

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), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (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.

Raman observation of a molecular signaling pathway of apoptotic cells induced by photothermal therapy.

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.