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A simple, label-free and sensitive fluorescence protein assay has been developed on the basis of aptamer exonuclease protection and exonuclease III (Exo III)-assisted recycling amplification-responsive cascade ZnPPIX/G-quadruplex supramolecular fluorescent labels. In the sensing system, a special aptamer probe containing the aptamer sequence at the 3′-terminus and the DNAzyme sequence at the 5′-terminus was applied, which has the capacity to recognize a protein target with high affinity and specificity. Exonuclease I (Exo I) can efficiently catalyze the degradation of free single stranded DNA probes in the 3′ to 5′ direction. In the presence of the target protein, the strong binding between the target protein and its aptamer can protect aptamer probes from degradation. Subsequently, the protected aptamer probes act as catalysators to trigger hybridization with the hairpin DNA probe that contains a partially “caged” G-quadruplex sequence. Upon interaction with the protected aptamer probes, the hairpin opens to yield the active G-quadruplex structure. In the presence of exonuclease III (Exo III), Exo III-assisted recycling amplification occurs generating numerous G-quadruplex supramolecular structures. The zinc(ii)-protoporphyrin IX (ZnPPIX) fluorophore binds to the G-quadruplexes and this results in the enhanced fluorescence of the fluorophore. The resulting fluorescence of the ZnPPIX/G-quadruplex provides the readout signal for the sensing event. Thrombin is used as the model analyte in the current proof-of-concept. The developed method was demonstrated to have very high sensitivity for the detection of proteins with a limit of detection of 0.2 pM without using washes or separations. In addition, this new method for protein detection is simple and inherits all the advantages of aptamers. The mechanism, moreover, may be generalized and used for other forms of protein analysis. [ABSTRACT FROM AUTHOR]

In the present work, gold nanocluster (GNC) induced by bovine serum albumin (BSA) was synthesized as a novel fluorescence probe to detect nitrite (NO) sensitively and selectively. The fluorescence of GNC was found to be quenched effectively by NO. Under the optimum conditions, it was found that the change of fluorescence intensity was proportional with the concentration of NO in the linear range of 0.1-50 μM ( R = 0.9990), with a detection limit (S/N = 3) of 30 nM. The absorption spectroscopy, circular dichroism (CD), and X-ray photoelectron spectroscopy (XPS) studies were employed to discuss the quenching mechanism. In addition, the present approach was successfully applied in real water samples. [ABSTRACT FROM AUTHOR]

Site-specific identification of DNA methylation and assay of MTase activity are imperative for determining specific cancer types, provide insights into the mechanism of gene repression, and develop novel drugs to treat methylation-related diseases. Herein, we developed a highly sensitive fluorescence assay of DNA methyltransferase by methylation-sensitive cleavage-based primer generation exponential isothermal amplification (PG-EXPA) coupled with supramolecular fluorescent Zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex. In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The cleaved hairpin probe then functions as a signal primer to initiate the exponential isothermal amplification reaction (EXPAR) by hybridizing with a unimolecular DNA containing three functional domains as the amplification template, producing a large number of G-quadruplex nanostructures by utilizing polymerases and nicking enzymes as mechanical activators. The G-quadruplex nanostructures act as host for ZnPPIX that lead to supramolecular complexes ZnPPIX/G-quadruplex, which provides optical labels for amplified fluorescence detection of Dam MTase. While in the absence of Dam MTase, neither methylation/cleavage nor PG-EXPA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a wide dynamic range from 0.0002 to 20 U/mL and an extremely low detection limit of 8.6×10 −5 U/mL, which is superior to most conventional approaches for the MTase assay. Owing to the specific site recognition of MTase toward its substrate, the proposed sensing system was able to readily discriminate Dam MTase from other MTase such as M.SssI and even detect the target in a complex biological matrix. Furthermore, the application of the proposed sensing strategy for screening Dam MTase inhibitors was also demonstrated with satisfactory results. This novel method not only provides a promising platform for monitoring activity and inhibition of DNA MTases, but also shows great potentials in biological process researches, drugs discovery and clinical diagnostics. [ABSTRACT FROM AUTHOR]

A cascadic sensing system was developed for detection of DNA target at ultralow concentration by a combination of magnetic nanoparticles (MNPs) and exonuclease III (Exo III)-induced cascade two-stage isothermal amplification in the study. An ingeniously designed capture hairpin probe (CHP) that integrates target-binding and signal transduction sequences within one multifunctional design was assembled on MNPs. Upon sensing of the analyte nucleic acid, the hairpin probe on MNPs could be opened and stepwise removed by Exo III accompanied by the releasing of target DNA for the successive hybridization and cleavage process and the generation of bare signal transduction sequences of CHP as a new trigger for next circular reaction. The new DNA triggers initiate hybridizing with hairpin DNA probe that contains a partially "caged" G-quadruplex sequence (GHP), forming a duplex structure and liberating the active G-quadruplex structure. Then, Exo III digests the resulting duplex domain, leading to the recycling of new DNA trigger and simultaneously generating numerous ZnPPIX/G-quadruplex supramolecular complexes with the help of the zinc (II)-protoporphyrin IX (ZnPPIX), as an optical label for amplified fluorescence sensing event. Finally, numerous liberated cascade ZnPPIX/G-quadruplex supramolecular complexes give a remarkable fluorescence response. Because of two-stage autocatalytic recycling amplification and the specifically catalyzed formation of ZnPPIX/G-quadruplex supramolecular complexes, this newly designed protocol provides a high sensitivity with a detection limit of 0.75 fM, can discriminate mismatched DNA from perfectly matched target DNA, and gives low matrix effect due to using MNPs as the separation and amplification elements in the real samples. Therefore, it holds great potential for early diagnosis in gene-related diseases. [ABSTRACT FROM AUTHOR]