3D origami paper-based ratiometric fluorescent microfluidic device for visual point-of-care detection of alkaline phosphatase and butyrylcholinesterase

On-site multiplex enzyme detection is crucial for diagnosis, therapeutics and prognostic. To date, it is still a daunting challenge to develop portable, low-cost, and efficient multi-enzyme detection methods. Herein, a novel sample-in-result-out platform integrating ratiometric fluorescent assays with 3D origami microfluidic paper-based device (μPAD) was developed for simultaneous visual point-of-care testing (POCT) of alkaline phosphatase (ALP) and butyrylcholinesterase (BChE). Cascade catalytic reaction with the same two fluorescent signal indicators was rationally designed to ratiometric fluorescent detection of ALP and BChE: substrate of ALP (pyrophosphate) and product of BChE (thiocholine) can strongly complex with Cu2+, Cu2+ oxidizes o-phenylenediamine to fluorescent 2,3-diaminophenazine (oxOPD) (emission, 565 nm), oxOPD quenches the fluorescence of carbon dots (CDs, emission at 445 nm) via inner filter effect, thus oxOPD/CDs values are relevant to ALP and BChE activities.
Then 3D origami μPAD composing of four layers and two parallel channels was fabricated and simply prepared by one-step plotting with black oil-based marker and specific metal molds. After simple folding and unfolding neighboring layers to sequentially initiate reactions of pre-loaded reagents, fluorescent images on the detection zone can be captured by smartphone and analyzed by red-green-blue software for quantitative analysis. Under optimal conditions, the proposed platform was successfully performed to detect ALP and BChE with activity difference at 3 orders of magnitude in human serum samples without any pretreatment procedures. Excellent selectivity, good precision, favorable linear range, and high accuracy were exhibited. Importantly, the platform opens a promising horizon for high-throughput POCT of multiplex biomarkers.

Fluorescent Determination of Butyrylcholinesterase Activity and Its Application in Biological Imaging and Pesticide Residue Detection

Butyrylcholinesterase (BChE) is an essential human cholinesterase relevant to liver conditions and neurodegenerative diseases, which makes it a pivotal biomarker of health. It therefore remains challenging and highly desired to elaborate efficient chemical tools for BChE with simple operations and satisfactory working performance. In this work, a background-free detection strategy was built by virtue of the judicious coupling of a specific BChE-enzymatic reaction and in situ cyclization. High sensitivity with a low limit of detection (LOD) of 0.075 μg/mL could be readily achieved from the blank background and the as-produced emissive indicators, and the specific reaction site contributed to the high selectivity over other bio-species even acetylcholinesterase (AChE).
In addition to the multifaceted spectral experiments to verify the sensing mechanism, this work assumed comprehensive studies on the application. The bio-investigation ranged from cells to an organism, declaring a noteworthy prospect in disease diagnosis, especially for Alzheimer’s disease (AD), a common neurodegenerative disease with over-expressed BChE. Moreover, its excellent work for inhibition efficacy elucidation was also proved with the accuracy IC50 of tacrine for BChE (8.6 nM), giving rise to an expanded application for trace pesticide determination.

Development of potent reversible selective inhibitors of butyrylcholinesterase as fluorescent probes.

Brain butyrylcholinesterase (BChE) is an attractive target for drugs designed for the treatment of Alzheimer’s disease (AD) in its advanced stages. It also potentially represents a biomarker for progression of this disease. Based on the crystal structure of previously described highly potent, reversible, and selective BChE inhibitors, we have developed the fluorescent probes that are selective towards human BChE. The most promising probes also maintain their inhibition of BChE in the low nanomolar range with high selectivity over acetylcholinesterase.
Kinetic studies of probes reveal a reversible mixed inhibition mechanism, with binding of these fluorescent probes to both the free and acylated enzyme. Probes show environment-sensitive emission, and additionally, one of them also shows significant enhancement of fluorescence intensity upon binding to the active site of BChE. Finally, the crystal structures of probes in complex with human BChE are reported, which offer an excellent base for further development of this library of compounds.

fluorescent probe for butyrylcholinesterase activity in human serum based on a fluorophore with specific binding affinity for human serum albumin.

Non-specific binding of a fluorescent probe to human serum albumin is problematic because it induces signal interference when the probe detects the target biomarker in human serum. To eliminate this problem, we used intrinsically problematic non-specific fluorescence in designing a fluorescent probe for butyrylcholinesterase activity in serum. The probe containing a fluorophore with specific binding affinity for albumin could sensitively detect butyrylcholinesterase activity in serum with high selectivity to acetylcholinesterase and screen the efficiency of butyrylcholinesterase inhibitors.

Redox-Controlled Fluorescent Nanoswitch Based on Reversible Disulfide and Its Application in Butyrylcholinesterase Activity Assay.

Butyrylcholinesterase (BChE) mainly contributing to plasma cholinesterase activity is an important indicator for routinely diagnosing liver function and organophosphorus poisoning in clinical diagnosis, but its current assays are scarce and frequently suffer from some significant interference and instability. Herein, we report a redox-controlled fluorescence nanoswtich based on reversible disulfide bonds, and further develop a fluorometric assay of BChE via thiol-triggered disaggregation-induced emission.
Thiol-functionalized carbon quantum dots (thiol-CQDs) with intense fluorescence is found to be responsive to hydrogen peroxide, and their redox reaction transforms thiol-CQDs to nonfluorescent thiol-CQD assembly. The thiols inverse this process by a thiol-exchange reaction to turn on the fluorescence. The fluorescence can be reversibly switched by the formation and breaking of disulfide bonds caused by external redox stimuli. The specific thiol-triggered disaggregation-induced emission enables us to assay BChE activity in a fluorescence turn-on and real-time way using butyrylthiocholine iodide as the substrate.
As-established BChE assay achieves sufficient sensitivity for practical determination in human serum, and is capable of avoiding the interference from micromolar glutathione and discriminatively quantifying BChE from its sister enzyme acetylcholinesterase. The first design of reversible redox-controlled nanosiwtch based on disulfide expands the application of disulfide chemistry in sensing and clinical diagnostics, and this novel BChE assay enriches the detection methods for cholinesterase activity.

Butyrylcholinesterase Fluorescent Activity Kit (2 Plate)

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Butyrylcholinesterase (BCHE) Antibody

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  • 493.20 EUR
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Native Equine Butyrylcholinesterase

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Rat Butyrylcholinesterase (BCHE) CLIA Kit

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Recombinant Butyrylcholinesterase (BCHE)

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Recombinant Butyrylcholinesterase (BCHE)

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Recombinant Butyrylcholinesterase (BCHE)

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A direct assay of butyrylcholinesterase activity using a fluorescent substrate.

In this study, we report a direct fluorometric assay for butyrylcholinesterase (BChE) activity and screening of its inhibitor, using a fluorescent substrate. 2-(2-(5,6-Dimethoxy-1,3-dioxoisoindolin-2-yl)acetoxy)-N,N,N-trimethylethan-1-ammonium iodide (1) was hydrolyzed by BChE, and its fluorescence was quenched by an intramolecular photoinduced electron transfer process. The resulting change in fluorescence provided a facile method for real-time BChE activity testing.
Remarkably, 1 was selectively hydrolyzed by BChE, even in the presence of excess acetylcholinesterase, thereby facilitating the specific monitoring of BChE activity. This assay method is also useful for screening potential BChE inhibitors. Given its simplicity, selectivity, and higher assay speed, this method may be extended to high-throughput screening of BChE inhibitors and relevant drug discovery.

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