Live-Cell Imaging of MicroRNA Expression via Photoinduced Electron Transfer Controlled by Catalytic Hairpin Assembly.

MicroRNAs (miRNAs) serve as emerging biomarkers for a range of diseases, and their quantitative analysis draws increasing attention. Yet, current invasive methods limit continuous tracking within living cells. To overcome this, a nonenzymatic DNA-based nanoprobe is developed for dynamic, noninvasive miRNA tracking via live-cell imaging. This probe features a unique hairpin DNA structure with five guanines that act as internal quenchers, suppressing fluorescence from an attached fluorophore via photoinduced electron transfer. Target miRNA initiates toehold-mediated strand displacement, restoring, and amplifying the fluorescence signal. Additionally, by introducing a single mismatch to the hairpin DNA, the nanoprobe's sensitivity is significantly enhanced, lowering the detection limit to about 60 pM without compromising specificity. To optimize intracellular delivery for prolonged monitoring, the nanoprobe is encapsulated within multilamellar lipid nanovesicles, fluorescently labeled for dual-wavelength ratiometric analysis. The proposed nanoprobe demonstrates a significant advance in live-cell miRNA detection, promising enhanced in situ analysis for a better understanding of miRNAs' pathophysiological function.

Target-Catalyzed Self-Assembly of DNA-Streptavidin Nanogel for Enzyme-Free miRNA Assay.

Rapid, sensitive, specific, and user-friendly microRNA (miRNA) assays are in high demand for point-of-care diagnosis. Target-catalyzed toehold-mediated strand displacement (TMSD) has received increasing attention as an enzyme-free molecular tool for DNA detection. However, the application of TMSD to miRNA targets is challenging because relatively weak DNA/RNA hybridization leads to failure in the subtle kinetic control of multiple hybridization steps. Here, a simple method is presented for miRNA assay based on the one-pot self-assembly of Y-shaped DNAs with streptavidin via an miRNA-catalyzed TMSD cascade reaction. A single miRNA catalyzes the opening cycle of DNA hairpin loops to generate multiple Y-shaped DNAs carrying biotin and a quencher at the end of their arms. Introducing a single base-pair mismatch near the toehold facilitates RNA-triggered strand displacement while barely disturbing nonspecific reactions. The Y-shaped DNAs are self-assembled with fluorescently labeled streptavidin (sAv), which produces nanoscale DNA-sAv nanogel particles mediating efficient Förster resonance energy transfer in their 3D network. The enhancing effect dramatically reduces the detection limit from the nanomolar level to the picomolar level. This work proves that TMSD-based DNA nanogel with a base-pair mismatch incorporated to a hairpin structure is a promising approach towards sensitive and accurate miRNA assay.

Chitosan-coated mesoporous silica particles as a plastic-free platform for photochemical suppression and stabilization of organic ultraviolet filters.

Photochemical instability and reactivity of organic ultraviolet (UV) filters not only degrade the performance of sunscreen formulations but also generate toxic photodegradation products and reactive oxygen species (ROS). Although the encapsulation of organic UV filters into synthetic polymer particles has been widely investigated, synthetic plastics were recently banned for personal care and cosmetic products due to marine and coastal pollution issues. Here we present a plastic-free, photochemically stable and inactive UV filter platform based on chitosan-coated mesoporous silica microparticles, denoted 'mSOCPs', incorporating octyl methoxycinnamate (OMC) as a sunscreen agent. Sunlight induced the degradation of ∼80% free OMC in artificial sweat in 1 h at room temperature, while only 20% of OMC degraded for 3 h when encapsulated within mSOCPs. Moreover, mSOCPs efficiently suppressed the photochemical generation of ROS by about 99% through the combined effects of the mesoporous silica structure and chitosan coating. Accordingly, mSOCPs substantially increased the cell viability of fibroblasts exposed to UV irradiation. This work demonstrates that the biopolymer coatings of mesoporous inorganic particles can be a promising approach to the plastic-free encapsulation of organic UV filters for suppressing their photochemical reactivity and degradation.

Short DNA-catalyzed formation of quantum dot-DNA hydrogel for enzyme-free femtomolar specific DNA assay.

Fast, sensitive, specific, and user-friendly DNA assay is a key technique for the next generation point-of-care molecular diagnosis. However, high-cost, time-consuming, and complicated enzyme-based DNA amplification step is essential to achieve high sensitivity. Herein, a short target DNA-catalyzed formation of quantum dot (QD)-DNA hydrogel is proposed as a new DNA assay platform satisfying the above requirements. A single-stranded target DNA catalyzes the opening cycle of DNA hairpin loops, which are quickly self-assembled with DNA-functionalized QDs to generate QD-DNA hydrogel. The three-dimensional hydrogel network allows efficient resonance energy transfer, dramatically lowering the limit of detection down to ~6 fM without enzymatic DNA amplification. The QD-DNA hydrogel also enables a rapid detection (1 h) with high specificity even for a single-base mismatch. The clinical applicability of the QD-DNA hydrogel is demonstrated for the Klebsiella pneumoniae carbapenemase gene, one of the key targets of drug-resistant pathogenic bacteria.

Subnanomolar FRET-Based DNA Assay Using Thermally Stable Phosphorothioated DNA-Functionalized Quantum Dots.

Quantum dots (QDs) can serve as an attractive Förster resonance energy transfer (FRET) donor for DNA assay due to their excellent optical properties. However, the specificity and sensitivity of QD-based FRET analysis are prominently reduced by nonspecific DNA adsorption and poor colloidal stability during DNA hybridization, which hinders the practical applications of QDs as a biosensing platform. Here, we report subnanomolar FRET assay of DNA through the stabilization of DNA/QD interface using DNA-functionalized QDs with phosphorothioated single-stranded DNA (pt-ssDNA) as a multivalent ligand in an aqueous solution. In situ DNA functionalization was achieved during the aqueous synthesis of CdTe/CdS QDs, resulting in the maximum photoluminescence quantum yields of 76.9% at an emission wavelength of 732 nm. Conventional monothiolated ssDNA-capped QDs exhibited particle aggregation and photoluminescence (PL) quenching during DNA hybridization at 70 °C due to the dissociation of surface ligands. Such colloidal instability induced the nonspecific adsorption of DNA, resulting in false-positive signal and decreased sensitivity with the limit of detection (LOD) of 16.1 nM. In contrast, the pt-ssDNA-functionalized QDs maintained their colloidal stability and PL properties at elevated temperatures. The LOD of the pt-ssDNA-functionalized QDs was >30 times lower (0.47 nM) while maintaining the high specificity to a target sequence because the strong multivalent binding of pt-ssDNA to the surface of QDs prevents the detachment of pt-ssDNA and nonspecific adsorption of DNA. The study suggests that the ligand design to stabilize the surface of QDs in an aqueous milieu is critically important for the high performance of QDs for specific DNA assay.