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.

Structure-switching aptamer triggering signal amplification strategy for tobramycin detection based on hybridization chain reaction and fluorescence synergism.

Based on hybridization chain reaction (HCR) and fluorescence synergism, a novel aptasensor for tobramycin was successfully constructed. Tobramycin competed with cDNA-FAM to bind aptamers immobilized on magnetic beads. After magnetic separation, the released cDNA-FAM acted as initiator to trigger HCR amplification, thus the fluorescence was significantly enhanced due to binding of SYBR Green Ⅰ (SGI) to the formed long double-stranded DNA and the synergistic fluorescence of FAM. In the absence of tobramycin, the initiator was magnetically separated and no HCR occurred, more importantly, graphene oxide can quench the fluorescence of excessive hairpins/SGI and cDNA-FAM, so almost no background signal was detected. This aptasensor can monitor tobramycin in the range of 0.3-50 µM with low detection limit of 17.37 nM. Due to the potential generality of structure-switching aptamers and effectiveness of fluorescence synergism, this enzyme-free amplification strategy can be extended to other applications by rational design of nucleic acid sequences.

A signal-on fluorescent aptasensor by sensitized Tb3+ luminescence for detection of melamine in milk.

A fluorescent aptasensor based on sensitized terbium(III) luminescence was constructed to detect melamine in milk. Tb3+ as the fluorescence probe can be sensitized by a guanine-rich single-stranded DNA sequence, so the complementary sequence of the polythymidine aptamer (cDNA) was modified with six consecutive guanine bases (G6). In the absence of melamine, melamine aptamer combined with cDNA to form a double helix structure, and G6 hybridized with the extended cytosine bases in the aptamer, resulting in low fluorescence intensity of Tb3+. In the presence of melamine, cDNA was released due to the specific recognition of melamine to the aptamer, resulting in stronger sensitized fluorescence intensity of Tb3+. Under the optimum conditions, the linear concentration of melamine in the milk ranged from 1.0 µg/mL to 10.0 µg/mL. This aptasensor can be used for the accurate and rapid detection of melamine in milk with a detection limit of 0.02 µg/mL, and has the advantages of high sensitivity, high efficiency, simple operation and low cost.