Huanshun Yin,Xinxu Wang,Yunlong Guo,Yunlei Zhou,Shiyun Ai
An effective assay method for monitoring protein kinase activity and screening inhibitors is greatly beneficial to kinase-related drug discovery, early diagnosis of diseases, and therapeutic effect evaluation. Herein, we develop a simple electrochemical method for detecting the activity of casein kinase II (CK2) based on phosphorylation against carboxypeptidase Y (CPY) digestion triggered signal amplification , where CK2 catalyzed phosphorylation event protects the substrate peptide from the digestion of CPY, maintains the repulsive force of the substrate peptide towards the redox probe, and results in a weak electrochemical signal. Whereas, without phosphorylation, the substrate peptide is digested by CPY and a strong electrochemical signal is obtained. The detection feasibility is demonstrated for the assay of CK2 activity with low detection limit of 0.047 unit/mL. Moreover, the biosensor was used for the analysis of kinase inhibition. Based on the electrochemical signal dependent inhibitor concentration, the IC 50 value of ellagic acid was estimated to be 39.77 nM. The proposed method is also successfully applied to analyze CK2 activity in cell lysates, proving the applicability in complex biological samples.
A metal–organic-framework (MOF) fluorescent sensor is reported based on NH 2 -MIL-101(Fe) propelled pesticide and alkaline phosphatase (ALP) catalytic reaction. Different from previous reports, a cascade reaction system combined with MOF structural changes to generate fluorescence was employed. The rationale is that ALP can hydrolyze L-ascorbic acid 2-phosphate (AAP) into L-ascorbic acid (AA), which can reduce Fe 3+ to decompose structurally NH 2 -MIL-101(Fe), resulting in 2-aminoterephthalic acid (NH 2 -BDC) with intense fluorescence. The fluorescence can be decreased to different degrees due to inhibition of organophosphate pesticides (OPPs) on the activity of ALP. By taking chlorpyrifos (CPY) as the model compound of an OPP pesticide and adding ALP and CPY into the NH 2 -MIL-101(Fe) framework, the resulting cascade reaction fluorescence sensors exhibit a good sensitivity for CPY and ALP sensing. The working ranges are 0.02–2 μg/L and 0.2–20 mU/mL with detection limits (LOD) of 5.31 ng/L and 0.05 mU/mL, respectively. The proposed sensor has been actually applied to satisfactory detection of CPY and ALP in food and serum samples. This fluorescence-based assay may extend the application of MOF-based biosensors. Graphical Abstract
The fluorophores with long-lived fluorescent emission are highly desirable for time-resolved fluorescence imaging (TRFI) in monitoring target fluorescence. By embedding the aggregates of a thermally activated delayed fluorescence (TADF) dye, 2,3,5,6-tetracarbazole-4-cyano-pyridine (CPy), in distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG2000) matrix, CPy-based organic dots (CPy-Odots) with a long fluorescence lifetime of 9.3 μs (in water at ambient condition) and high brightness (with an absolute fluorescence quantum efficiency of 38.3%) are fabricated. CPy-Odots are employed in time-resolved and confocal fluorescence imaging in living Hela cells and in vivo. The green emission from the CPy-Odots is readily differentiated from the cellular autofluorescence background because of their stronger emission intensities and longer lifetimes. Unlike other widely studied DSPE-PEG2000 encapsulated Odots which are always distributed in cytoplasm, CPy-Odots are located mainly in plasma membrane. In addition, the application of CPy-Odots as a bright microangiography agent for TRFI in zebrafish is also demonstrated. Much broader application of CPy-Odots is also prospected after further surface functionalization. Given its simplicity, high fluorescence intensity, and wide availability of TADF materials, the method can be extended to develop more excellent TADF Odots for accomplishing the challenges in future bioimaging applications.
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