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.
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.
Quantum dots-based fluorescent probes for turn-on and turn-off sensing of butyrylcholinesterase.
A novel turn-on and turn-off sensor based on the fluorescence change of quantum dots (QDs) has been developed to detect the activity of butyrylcholinesterase (BChE). In the turn-on sensing system, we realized the detection of BChE with just one enzyme. A linear calibration plot of the activity of BChE was obtained in the wide amounts range from 10 to 1000 U/L and the detection limit was 10 U/L. In the turn-off sensing system, we realized the sensing of BChE with a wide linear relationship of 10-2000 U/L which was much wider than many other detection methods. We also studied the application in serum sample detection.
The BChE sensor shows great performances. Furthermore, the turn-on and turn-off sensing mechanisms were studied extensively. These results showed that our strategy would most probably be applicable in assembling diagnostic micro-device for realizing the rapid clinical analysis of BChE.
Butyrylcholinesterase Fluorescent Activity Kit (2 Plate) |
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| K016-F1 | Arbor Assays | 2x96 well plates | 411 EUR |
Multi-Species Butyrylcholinesterase Fluorescent Detection Kit |
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| IMLBCHEKTF | Innovative research | each | 538 EUR |
Multi-Species Butyrylcholinesterase Fluorescent Detection Kit |
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| MBS8420174-1Kit | MyBiosource | 1Kit | 730 EUR |
Multi-Species Butyrylcholinesterase Fluorescent Detection Kit |
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| MBS8420174-5x1Kit | MyBiosource | 5x1Kit | 3330 EUR |
Butyrylcholinesterase |
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| 44-0120 | Fitzgerald | 1 kU | 264 EUR |
Butyrylcholinesterase (BCHE) |
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| THP-0279 | Creative BioMart | 1 vial | 3198.4 EUR |
Human Butyrylcholinesterase |
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| PRV-461 | Creative BioMart | 1 vial | 1598.4 EUR |
Butyrylcholinesterase assay kit |
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| BC121-50T24S | ELK Biotech | 50T/24S | 110 EUR |
Butyrylcholinesterase Assay Kit |
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| SH0215 | Jiaxing Korain Biotech Ltd (BT Labs) | 50T/24Sample | 200 EUR |
Butyrylcholinesterase Rabbit mAb |
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| A8861 | Abclonal | 100μL | 88.56 EUR |
Butyrylcholinesterase Rabbit pAb |
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| A1460 | Abclonal | 100μL | 1246.96 EUR |
Butyrylcholinesterase Rabbit mAb |
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| E45R11427N | EnoGene | 50 ul | 346.5 EUR |
Butyrylcholinesterase Rabbit mAb |
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| E2R381915 | EnoGene | 100ul | 275 EUR |
Butyrylcholinesterase Rabbit pAb |
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| E45R21157N | EnoGene | 50 ul | 297.75 EUR |
Amplite® Fluorimetric Butyrylcholinesterase Activity Assay Kit |
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| 11407-100Tests | AAT Bioquest | 100 Tests | 558 EUR |
Butyrylcholinesterase, Human Plasma |
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| 16-16-020305-100U | Athens Research & Technology | 100units | 900 EUR |
Butyrylcholinesterase, Human Plasma |
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| 16-16-020305-10U | Athens Research & Technology | 10units | 200 EUR |
Butyrylcholinesterase, Human Plasma |
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| 16-16-020305-1KU | Athens Research & Technology | 1000units | 1200 EUR |
Rat Butyrylcholinesterase ELISA Kit |
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| MBS066252-10x96StripWells | MyBiosource | 10x96-Strip-Wells | 6725 EUR |
Rat Butyrylcholinesterase ELISA Kit |
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| MBS066252-48StripWells | MyBiosource | 48-Strip-Wells | 550 EUR |
Rat Butyrylcholinesterase ELISA Kit |
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| MBS066252-5x96StripWells | MyBiosource | 5x96-Strip-Wells | 3420 EUR |
Rat Butyrylcholinesterase ELISA Kit |
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| MBS066252-96StripWells | MyBiosource | 96-Strip-Wells | 765 EUR |
Intrathecal delivery of fluorescent labeled butyrylcholinesterase to the brains of butyrylcholinesterase knock-out mice: visualization and quantification of enzyme distribution in the brain.
- Exogenously delivered butyrylcholinesterase (BChE) has proven to be an efficient bioscavenger against highly toxic organophosphorus poisons and nerve agents. The scavenger properties of BChE when delivered via intramuscular, intravenous, subcutaneous, or intraperitoneal routes are limited to the body’s peripheral sites because the 340 kDa enzyme does not cross the blood-brain barrier (BBB). Overcoming the BBB is an important step toward evaluating the neuroprotective properties of BChE within the central nervous system (CNS). This study examines the feasibility of delivering BChE to the brain and spinal cord by intrathecal (IT) injection. Mice completely devoid of BChE were injected intrathecally with either BChE (80 units) that was labeled with near-infrared fluorescent dye (BChE/IRDye) or a molar equivalent amount of carboxylate dye.
- The BChE/IRDye and carboxylate dye were tracked using an in vivo imaging system demonstrating the real-time distribution of BChE in the brain and the residence time in the brain and spinal cord through 25 h post-dosing. BChE/IRdye levels in the brain peaked at 6h post-dosing. BChE enzyme activity was quantified in plasma and brain sections by BChE activity assays of plasma and of perfused tissues.
- Average BChE activity levels were 0.6 units/g in the brains of mice treated with BChE/IRDye at 4h post-dosing. Intense fluorescent signal in the cortex, dentate gyrus and ventricles of the brain at 25 h post-dosing was visualized by confocal microscopy and the presence of BChE was confirmed with activity assays of frozen sections. This procedure proved to be an efficient, safe and rapid method to deliver BChE to the CNS of mice, providing a research tool for determining neural protection by BChE following OP exposure.