CRISPR/Cas12a-derived ratiometric fluorescence sensor for high-sensitive Pb2+ detection based on CDs@ZIF-8 and DNAzyme.

Benefiting from specific target recognition and trans-cleavage capabilities, the CRISPR/Cas12a system has great application prospects in the design of highly sensitive and rapid fluorescence biosensors. The CRISPR/Cas12a-based fluorophore-quencher molecular beacons exhibit single-color emission and are easily exposed to interference from environmental factors. Herein, we design a CRISPR/Cas12a-derived ratiometric fluorescence sensor for Pb2+ detection based on embedded carbon dots@zeolitic imidazolate framework-8 (CDs@ZIF-8) composites and DNAzyme. The functions of ZIF-8 about encapsulating red emissive CDs in the inner cavity and adsorbing DNA on the outer surface are integrated to establish dual fluorescence signals, thereby reducing the possibility of interference and improving sensing accuracy. The presence of Pb2+ is converted into the change of activator by the GR5 DNAzyme to activate the CRISPR/Cas12a system, which provides signal amplification through multiple turnovers of side branch cutting, achieving highly sensitive detection of Pb2+ with a low detection limit of 18 pM. This method has the advantages of simplicity, universality, and excellent quantitative ability, and has broad prospects in sensing applications.

Facile and sensitive detection of mercury ions based on fluorescent structure-switching aptamer probe and exonuclease â ¢-assisted signal amplification.

Hg2+ is highly toxic to human health and ecosystem. In this work, based on the unique fluorescent property of 2-Aminopurine (2-AP), the formation of T-Hg2+-T mismatch structure and the signal amplification of exonuclease III (Exo III) assisted target cycle, a fluorescent probe for facile and sensitive detection of Hg2+ is constructed. The hairpin-looped DNA probe is rationally designed with 2-AP embedded in the stem and thymine-rich recognition overhangs extended at the termini. The cleavage of the double stranded DNA stem with stable T-Hg2+-T pairs catalyzed by Exo III is prompted to happen upon recognition of trace Hg2+. Under the optimal reaction conditions, there is an excellent linear relationship between Hg2+ concentration and fluorescence intensity in the range of 7.5-200 nM with a detection limit of 0.38 nM. In addition, the detection results of Hg2+ in Songhua River water and fish samples are satisfactory. The fluorescent probe avoids labeling additional quenchers or quenching materials and has strong anti-interference ability. Thus, the fluorescent probe has a broad prospect in practical application.

Gold nanoclusters-manganese dioxide composite-based fluorescence immunoassay for sensitive monitoring of fenitrothion degradation in Chinese cabbage.

Understanding the residues and degradation of organophosphorus pesticides (OPs) in crops has attracted increasing attention. Herein, we designed a sensitive fluorescence immunoassay (FIA) by employing nanobody-linked alkaline phosphatase (Nb-ALP) and gold nanoclusters anchored manganese dioxide (AuNCs-MnO2) composite. In immunoassay protocol, Nb-ALP is used to competitively recognize the coating antigen and pesticide. After competitive immunoreaction, alkaline phosphatase catalyzes l-ascorbic acid-2-phosphate to produce ascorbic acid that can trigger the decomposition of the AuNCs-MnO2 composite, regulating the fluorescence response. As a proof-of-concept, fenitrothion (FNT) is chosen as the target analyte. As a result, the developed FIA exhibits high detection sensitivity (IC10 = 5.78 pg/mL), which is about 56-times higher than that of the conventional enzyme-linked immunosorbent assay. The developed FIA has been successfully applied for precisely monitoring the degradation of FNT in Chinese cabbage with excellent anti-interference ability and reproducibility, paving the way for the determination of pesticide residues in real food samples.

Engineering DNAzyme strategies for fluorescent detection of lead ions based on RNA cleavage-propelled signal amplification.

Based on the high recognition ability and flexible programmability of GR5 DNAzyme, two fluorescent biosensors were engineered for amplified detection of Pb2+ via incorporating Ti3C2TX MXenes and embedding 2-aminopurine (2-AP), respectively. The quencher-required approach relied on the DNA affinity and fluorescence quenching ability of Ti3C2TX MXenes. Benefiting from the low background signal modulated by Ti3C2TX MXenes, the sensitive determination of Pb2+ was achieved in the linear range of 0.2-10 ng mL-1 with the limit of detection (LOD) of 0.05 ng mL-1. The quencher-free approach combined the fluorescent trait of 2-AP embedded in DNA structure, and the RNA cleavage-propelled digestion process of Exonuclease I (Exo I) for signal amplification, indicating the sensitive detection of Pb2+ with the LOD as low as 0.02 ng mL-1 in the linear range of 0.1-10 ng mL-1. Both DNAzyme assays exhibited simple procedures, favorable specificity, rapid analysis, and satisfactory application in standard reference materials (lead in drinking water) and spiked water samples. The two fluorescent biosensors established in this work would not only provide theoretic fundament for DNA adsorption of Ti3C2TX MXenes and the design of 2-AP-embedded DNAzyme assays, but also hold a great potential for on-site monitoring of lead pollution in water samples.