ABSTRACT
We propose a CRISPR/Cas12a-mediated recombinase polymerase amplification (RPA) detection method that combines RPA with Cas12a cleavage for the detection of halal food adulteration, which is of global concern, particularly for Muslim consumers. We optimized the reagent concentrations for the Cas12a cleavage steps and designed and screened gRNA targeting a conserved area of the mitochondrial cytochrome C oxidase subunit I (COX1) gene. This procedure successfully detected the presence of porcine components as low as 5 pg/µL in the linear range of 5-1000 pg/µL. The assay's detection limit was 500 times lower than CRISPR-based approaches that exclude a preamplification step, allowing the detection of trace porcine DNA in food samples. The assay additionally showed no cross-reaction with nontarget species. Therefore, this detection platform shows tremendous potential as a method for the quick, sensitive, and specific detection of porcine-derived components.
ABSTRACT
A molybdenum sulfide/zirconium oxide/Nafion (MoS2/ZrO2/Naf) based electrochemiluminescence (ECL) aptasensor for the selective and ultrasensitive detection of ApoA1 is proposed, with Ru(bpy)3 2+ as the luminophore. The chitosan (CS) modification on the nanocomposite layer allowed glutaraldehyde (GLUT) cross-linking, resulting in the immobilization of ApoA1 aptamers. Scanning electron microscopy, tunneling electron microscopy, and energy dispersive X-ray spectroscopy were used to characterize the nanocomposite, while electrochemiluminescence (ECL), cyclic voltammetry, and electrochemical impedance spectroscopy were used to analyze the aptasensor assembly. The nanocomposite was used as an electrode modifier, which increased the intensity of the ECL signal. Due to the anionic environment produced on the sensor surface following the specific interaction of the ApoA1 biomarker with the sensor, more Ru(bpy)3 2+ were able to be electrostatically attached to the aptamer-ApoA1 complex, resulting in enhanced ECL signal. The ECL aptasensor demonstrated outstanding sensitivity for ApoA1 under optimal experimental conditions, with a detection limit of 53 fg/mL and a wide linear dynamic range of 0.1-1000 pg/mL. The potential practical applicability of this aptasensor was validated by analyzing ApoA1 in human serum samples, with recovery rates of 94-108% (n = 3). The proposed assay was found to be substantially better compared to the commercially available enzyme-linked immunosorbent assay method, as reflected from over 1500 times improvement in the detection limit for ApoA1.
ABSTRACT
Rac1 GTPase is hyperactivated in tumors and contributes to malignancy. Rac1 disruption of junctions requires its effector PAK1, but the precise mechanisms are unknown. Here, we show that E-cadherin is internalized via micropinocytosis in a PAK1-dependent manner without catenin dissociation and degradation. In addition to internalization, PAK1 regulates E-cadherin transport by fine-tuning Rab small GTPase function. PAK1 phosphorylates a core Rab regulator, RabGDIß, but not RabGDIα. Phosphorylated RabGDIß preferentially associates with Rab5 and Rab11, which is predicted to promote Rab retrieval from membranes. Consistent with this hypothesis, Rab11 is activated by Rac1, and inhibition of Rab11 function partially rescues E-cadherin destabilization. Thus, Rac1 activation reduces surface cadherin levels as a net result of higher bulk flow of membrane uptake that counteracts Rab11-dependent E-cadherin delivery to junctions (recycling and/or exocytosis). This unique small GTPase crosstalk has an impact on Rac1 and PAK1 regulation of membrane remodeling during epithelial dedifferentiation, adhesion, and motility.
