Life-threatening allergic reactions to food allergens, particularly peanut protein Ara h1, are a growing public health concern affecting millions of people worldwide. Thus, accurate and rapid detection is necessary for allergen labeling and dietary guidance and ultimately preventing allergic incidents. Herein, we present a novel ratiometric fluorescence aptasensor based on multivalent aptamer-encoded DNA flowers (Mul-DNFs) for the high-stability and sensitive detection of allergen Ara h1. The flower-shaped Mul-DNFs were spontaneously packaged using ultralong polymeric DNA amplicons driven by a rolling circle amplification reaction, which contains a large number of Ara h1 specific recognition units and has excellent binding properties. Furthermore, dual-color fluorescence-labeled Mul-DNFs probes were developed by hybridizing them with Cy3- and Cy5-labeled complementary DNA (cDNA) to serve as a ratiometric fluorescence aptasensor platform based on fluorescence resonance energy transfer. Benefiting from the combined merits of the extraordinary synergistic multivalent binding ability of Mul-DNFs, the excellent specificity of the aptamer, and the sensitivity of the ratiometric sensor to avoid exogenous interference. The developed ratiometric aptasensor showed excellent linearity (0.05–2000 ng mL–1) with a limit of detection of 0.02 ng mL–1. Additionally, the developed ratiometric fluorescence aptasensor was utilized for quantifying the presence of Ara h1 in milk, infant milk powder, cookies, bread, and chocolate with recoveries of 95.7–106.3%. The proposed ratiometric aptasensor is expected to be a prospective universal aptasensor platform for the rapid, sensitive, and accurate determination of food and environmental hazards.
High cadmium (Cd, 50 µM) caused transcriptional upregulation of Ca2+-dependent protein kinase 21 (CPK21) in roots and leaves of Populus euphratica. The promoter region (1803 bp) of PeCPK21 was engineered to fuse PeCPK21-pro::GUS and transformed in Arabidopsis. The activity of GUS significantly increased in roots, cotyledons, and hypocotyls under 100 μM CdCl2 treatment. The 1599-bp cDNA sequence of PeCPK21 was transferred into Arabidopsis, and overexpression of PeCPK21 increased Cd tolerance as evidenced by less reduction in root length and plant growth under CdCl2 stress conditions (100 µM, 7 d). PeCPK21-interacting proteins were enriched by HaloTag pull-down and corresponding expression profiles after Cd exposure indicate that PeCPK21 interacts with heavy metal stress-associated proteins (HMAPs) to mediate Cd tolerance in transgenic Arabidopsis. PeCPK21 interacted with the Cd-regulated cation/heavy metal transport proteins annexin, OPT3, COPT5, and PDF2.2 to limit Cd uptake and accumulation in transgenic plants. PeCPK21 may also interact with the V-type proton ATPase subunits, VHA-B1, VHA-C, and AVA-P2, which were transcriptionally upregulated by Cd to accelerate vacuolar Cd compartmentalization. Cd-triggered H2O2 production was limited in plants overexpressing PeCPK21, which was due to the activated antioxidant enzymes. The interaction of PeCPK21 with the antioxidant enzymes TAPX, APX1, APX2, TRXM4, GPX3, CDSP32, and PRXQ, which showed transcriptional upregulation by Cd, could enhance antioxidant defense in transgenic Arabidopsis. It is also possible that PeCPK21 interacts with plasma membrane intrinsic proteins, PIP1-1, PIP2A, and PIP2-7, which had an Cd-enhanced transcript, to improve water status in transgenic Arabidopsis. In summary, we hypothesize that the calcium sensor PeCPK21 can attenuate Cd toxicity by interacting with HMAPs to effectively limit Cd accumulation, increase ROS scavenging, and improve water status in transgenic Arabidopsis.
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