ABSTRACT
To enable rapid and accurate point-of-care DNA detection, we have developed a single-step, amplification-free nucleic acid detection platform, a DNA substrate-mediated autocatalysis of CRISPR/Cas12a (DSAC). DSAC makes use of the trans-cleavage activity of Cas12a and target template-activated DNA substrate for dual signal amplifications. DSAC employs two distinct DNA substrate types: one that enhances signal amplification and the other that negatively modulates fluorescent signals. The positive inducer utilizes nicked- or loop-based DNA substrates to activate CRISPR/Cas12a, initiating trans-cleavage activity in a positive feedback loop, ultimately amplifying the fluorescent signals. The negative modulator, which involves competitor-based DNA substrates, competes with the probes for trans-cleaving, resulting in a signal decline in the presence of target DNA. These DNA substrate-based DSAC systems were adapted to fluorescence-based and paper-based lateral flow strip detection platforms. Our DSAC system accurately detected African swine fever virus (ASFV) in swine's blood samples at femtomolar sensitivity within 20 min. In contrast to the existing amplification-free CRISPR/Dx platforms, DSAC offers a cost-effective and straightforward detection method, requiring only the addition of a rationally designed DNA oligonucleotide. Notably, a common ASFV sequence-encoded DNA substrate can be directly applied to detect human nucleic acids through a dual crRNA targeting system. Consequently, our single-step DSAC system presents an alternative point-of-care diagnostic tool for the sensitive, accurate, and timely diagnosis of viral infections with potential applicability to human disease detection.
ABSTRACT
Physicochemical properties of nanoparticles, such as particle size, surface charge, and particle shape, have a significant impact on cell activities. However, the effects of surface functionalization of nanoparticles with small chemical groups on stem cell behavior and function remain understudied. Herein, we incorporated different chemical functional groups (amino, DETA, hydroxyl, phosphate, and sulfonate with charges of +9.5, + 21.7, -14.1, -25.6, and -37.7, respectively) to the surface of inorganic silica nanoparticles. To trace their effects on mesenchymal stem cells (MSCs) of rat bone marrow, these functionalized silica nanoparticles were used to encapsulate Rhodamine B fluorophore dye. We found that surface functionalization with positively charged and short-chain chemical groups facilitates cell internalization and retention of nanoparticles in MSCs. The endocytic pathway differed among functionalized nanoparticles when tested with ion-channel inhibitors. Negatively charged nanoparticles mainly use lysosomal exocytosis to exit cells, while positively charged nanoparticles can undergo endosomal escape to avoid scavenging. The cytotoxic profiles of these functionalized silica nanoparticles are still within acceptable limits and tolerable. They exerted subtle effects on the actin cytoskeleton and migration ability. Last, phosphate-functionalized nanoparticles upregulate osteogenesis-related genes and induce osteoblast-like morphology, implying that it can direct MSCs lineage specification for bone tissue engineering. Our study provides insights into the rational design of biomaterials for effective drug delivery and regenerative medicine.
