DNA nanoassembly based turn-on amplification probe for sensitive colorimetric CRISPR/Cas12a-mediated detection of pathogen DNA.

Clustered regularly interspaced short palindromic repeat (CRISPR) system has been explored as an efficient tool for nucleic acid diagnostics. However, it normally needs instrumentation or produces turn-off signals. Herein, a bulged Y-shape DNA (Y-DNA) nanoassembly was designed and synthesized as a novel turn-on probe. A CRISPR/Cas12a and Y-DNA probe mediated colorimetric assay (named as CYMCOA) strategy was developed for visual detection of pathogen DNA. Upon activating Cas12a with pathogen DNA, the Y-DNA bulge is catalytically trans-cleaved, releasing the G-quadruplex sequence embedded in the Y-DNA nanoassembly as a peroxidase-like DNAzyme. Visible signals with chromogen substrates are thus produced. The CYMCOA strategy was combined with recombinase polymerase amplification (RPA), an isothermal amplification technique, in detecting Helicobacter pylori (Hp) bacteria and SARS-CoV-2 N plasmids as two model pathogens. The bioassay has very excellent detection sensitivity and specificity, owing to the triple cascade amplification reactions and the very low mismatch tolerance. The lower limit of detection values were 0.16 cfu⋅mL-1, 1.5 copies⋅µL-1, and 0.17 copies⋅µL-1 for Hp bacteria, Hp plasmids, and SARS-CoV-2 N plasmids respectively. The detection is fast and accurate. The colorimetric bioassay strategy provides to be a simple, accurate, fast and instrumentation-free platform for nucleic acids detections in various settings, including crude and emergent situations.

O2-Generation-Enhanced Responsive Starvation/Photothermal Synergistic Tumor Therapy Based on the AuNRs@MnO2@SiO2 Nanocarrier and Thermosensitive Biomimetic Camouflaging.

Cancer starvation/photothermal combined tumor therapy (CST/PTT) has attracted great interest attributed to their mutual compensation and synergistically enhanced effect. However, the very low O2 supply in the tumor microenvironment (TME) greatly limits the CST efficiency of glucose oxidase (GOx). Additionally, the easy degradation in blood circulation and significant off-target effects are big challenges for clinical applications of the GOx-based CST. In this study, a drug delivery system (DDS) with specific tumor-targeted GOx delivery, near-infrared (NIR) light and TME responsive O2 generation, NIR-responsive glucose consumption, high GOx loading, and efficient NIR photothermia was developed. Positively charged AuNRs@MnO2@SiO2 nanoparticles (named AMS+ NPs) were synthesized. GOx was covalently loaded with a high loading ratio of 36.0%. Finally, a thermosensitive biomimetic hybrid membrane composed of a thermosensitive lipid (TSL) membrane, red blood cell membrane (RBCM), and 4T1 cancer cell membrane (CCM) was coated on the NPs through a double-layer strategy. The AMS+-G@TSL@[RBC-CC-TSL]M NPs consumed 32.7 times glucose at 50 °C as that at 37 °C and generated 4.9 times O2 upon NIR laser irradiation. The thermosensitive biomimetic NPs showed an efficient targeting capability to the homotypic 4T1 cancer cells/tumors accompanied by good biocompatibility, macrophage evading capability, high cancer cell cytotoxicity, and excellent antitumor efficacy. The tumor growth inhibition ratio with NIR laser irradiation reached 92.8%. The AMS+-GOx@TSL@[RBC-CC-TSL]M NPs provide a smart, efficient, safe, PTT/CST combined DDS for highly efficient tumor therapy.

Red fluorescent nanoprobe based on Ag@Au nanoparticles and graphene quantum dots for H2O2 determination and living cell imaging.

A sensitive and turn-on fluorescence nanoprobe based on core-shell Ag@Au nanoparticles (Ag@AuNPs) as a fluorescence receptor and red emissive graphene quantum dots (GQDs) as a donor was fabricated. They were conjugated together through π-π stacking between the GQDs and single-strand DNA modified at the Ag@AuNPs surface. The absorption spectrum of the receptor significantly overlapped with the donor emission spectrum, leading to a strong Förster resonance energy transfer (FRET) and thus a dramatic quenching. The sensing mechanism relies on fluorescence recovery following DNA cleavage by •OH produced from Fenton-like reaction between the peroxidase-like Ag nanocore and H2O2. The red emissive feature (Ex/Em, 520 nm/560 nm) provides low background in physiological samples. The •OH production, great spectrum overlapping, and red emission together contributes to good sensitivity and living cell imaging capability. The fluorescence assay (intensity at 560 nm) achieves a low detection limit of 0.49 µM H2O2 and a wide linear range from 5 to 200 µM, superior to most of the reported fluorescent probes. The RSD value for 100 µM H2O2 was 1.4%. The nanoprobe exhibits excellent anti-interferences and shows low cytotoxicity. The recovery of 100 µM standard H2O2 in a cancer cell lysate was 85.8%. Most satisfactorily, it can realize monitoring and imaging H2O2 in living cells. This study not only presents a sensitive H2O2 probe but also provides a platform for detecting other types of reactive oxygen species.