Facile electrochemical detection of botulinum neurotoxin type E using a two-step proteolytic cleavage

Park, Seonhwa; Shin, Yu Mi; Song, Ji‐Joon; Yang, Haesik

doi:10.1016/j.bios.2015.05.016

Cited by 17 publications

(13 citation statements)

References 37 publications

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“…The SPR assay offers the advantage of rapidity as BoNT/E activity at 1 LD 50 /ml can be easily detected in less than 15 minutes. Extension of the cleavage period to 5h increased detection sensitivity for BoNT/E to 0.01 LD 50 /ml, a value lower than those previously reported for BoNT/E activity-based assays 15 17 19 . As LD 50 data can depend on assay conditions 24 we confirmed our assay sensitivity using two different sources of BoNT/E.…”

Section: Discussioncontrasting

confidence: 58%

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An optical biosensor assay for rapid dual detection of Botulinum neurotoxins A and E

Lévêque

Ferracci

Maulet

et al. 2015

Sci Rep

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The enzymatic activity of the pathogenic botulinum neurotoxins type A and E (BoNT/A and E) leads to potentially lethal paralytic symptoms in humans and their prompt detection is of crucial importance. A chip assay based on Surface Plasmon Resonance monitoring of the cleavage products is a simple method that we have previously established to detect BoNT/A activity. We have now developed a similar format assay to measure BoNT/E activity. A monoclonal antibody specifically recognizing SNAP25 cleaved by BoNT/E was generated and used to measure the appearance of the neo-epitope following injection of BoNT/E over SNAP-25 immobilized on a chip. This assay detects BoNT/E activity at 1 LD50/ml within minutes and linear dose-responses curves were obtained using a multiplexed biosensor. A threshold of 0.01 LD50/ml was achieved after 5 h of cleavage. This assay is 10-fold more sensitive than the in vivo assay for direct detection of BoNT/E in serum samples. The SNAP25 chip assay is able to discriminate in an automated manner the presence of BoNT/E, BoNT/A or a combination of both toxins.

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Section: Discussioncontrasting

confidence: 58%

“…Measurement of BoNT/E enzymatic activity constitutes the most sensitive approach to specifically detect this toxin. A variety of in vitro activity assays have already been published including methods using capillary gel electrophoresis 14 , ELISA 15 , mass spectrometry 16 17 or fluorescent and electrochemical readouts 8 18 19 .…”

mentioning

confidence: 99%

An optical biosensor assay for rapid dual detection of Botulinum neurotoxins A and E

Lévêque

Ferracci

Maulet

et al. 2015

Sci Rep

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“…The type E light chain of BoNT (BoNT/E-Lc) and antibody BoNT/E-Lc (Anti-BoNT/E-Lc) were obtained from the Department of Biological Sciences and Laboratory of Immunology and Infectious Diseases in KAIST [26]. BoNT/E-Lc (50 kDa) was in solution form at 7.4 μM, dissolved in 50 mM Tris-buffer solution (pH 7), whereas the antibody was a monoclonal immunoglobulin G (mAb IgG, 160 kDa) in solution form at 8.6 μM in 10 mM phosphate-buffered saline (PBS).…”

Section: Methodsmentioning

confidence: 99%

Real-Time Monitoring of a Botulinum Neurotoxin Using All-Carbon Nanotube-Based Field-Effect Transistor Devices

Lee

Nahm

Choi

2018

Sensors

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The possibility of exposure to botulinum neurotoxin (BoNT), a powerful and potential bioterrorism agent, is considered to be ever increasing. The current gold-standard assay, live-mouse lethality, exhibits high sensitivity but has limitations including long assay times, whereas other assays evince rapidity but lack factors such as real-time monitoring or portability. In this study, we aimed to devise a novel detection system that could detect BoNT at below-nanomolar concentrations in the form of a stretchable biosensor. We used a field-effect transistor with a p-type channel and electrodes, along with a channel comprising aligned carbon nanotube layers to detect the type E light chain of BoNT (BoNT/E-Lc). The detection of BoNT/E-Lc entailed observing the cleavage of a unique peptide and the specific bonding between BoNT/E-Lc and antibody BoNT/E-Lc (Anti-BoNT/E-Lc). The unique peptide was cleaved by 60 pM BoNT/E-Lc; notably, 52 fM BoNT/E-Lc was detected within 1 min in the device with the antibody in the bent state. These results demonstrated that an all-carbon nanotube-based device (all-CNT-based device) could be produced without a complicated fabrication process and could be used as a biosensor with high sensitivity, suggesting its potential development as a wearable BoNT biosensor.

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“…Importantly, in electrochemical detection, it is possible to obtain additional high signal amplification via redox cycling, leading to a facile achievement of low detection limits. − In the presence of redox cycling, signaling species are chemically or catalytically regenerated after electrochemical oxidation or reduction, contributing to increased electrochemical signals. Even in protease detection (based on proteolytic reaction) that requires a long incubation period, highly sensitive protease detection was obtained with a short incubation period when a redox cycling reaction was employed. − …”

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confidence: 99%

Combined Signal Amplification Using a Propagating Cascade Reaction and a Redox Cycling Reaction for Sensitive Thyroid-Stimulating Hormone Detection

Park

Kim

et al. 2019

Anal. Chem.

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Propagating cascade reactions based on two proteases are promising for obtaining high signal amplification. However, in many cases, biosensors that use cascade reactions do not have low detection limits because of the inherent slowness of proteolytic reactions. Here, we report a sensitive electrochemical immunosensor using a high-signal-amplification method that combines a propagating cascade reaction and a redox cycling reaction. The cascade reaction uses ecarin and prothrombin: the ecarin label proteolytically converts inactive prothrombin into active thrombin, which then proteolytically liberates electroactive p-aminophenol (AP) from an AP-conjugated peptide. The liberated AP is electrochemically oxidized to p-benzoquinone imine (QI), regenerated by the reduction of QI by NADH, and then electrochemically reoxidized. This electrochemical–chemical (EC) redox cycling reaction significantly increases the electrochemical signal. The developed immunosensor is also compared with an immunosensor that uses only a propagating cascade reaction and an immunosensor that uses a single proteolytic reaction and an EC redox cycling reaction. The detection limits for thyroid-stimulating hormone (TSH) obtained using the three immunosensors are 3 pg/mL, 2 ng/mL, and 4 ng/mL, respectively, indicating that the newly developed immunosensor is more sensitive than the other two. The measured concentrations of TSH in clinical serum are found to agree well with those determined using a commercial instrument.

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Facile electrochemical detection of botulinum neurotoxin type E using a two-step proteolytic cleavage

Cited by 17 publications

References 37 publications

An optical biosensor assay for rapid dual detection of Botulinum neurotoxins A and E

An optical biosensor assay for rapid dual detection of Botulinum neurotoxins A and E

Real-Time Monitoring of a Botulinum Neurotoxin Using All-Carbon Nanotube-Based Field-Effect Transistor Devices

Combined Signal Amplification Using a Propagating Cascade Reaction and a Redox Cycling Reaction for Sensitive Thyroid-Stimulating Hormone Detection

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