An Ultrasensitive Gold Nanoparticle-based Lateral Flow Test for the Detection of Active Botulinum Neurotoxin Type A

Abstract

Botulism is a severe and potentially lethal paralytic disease caused by several botulinum neurotoxin-producing Clostridia spp. In China, the majority of the cases caused by botulism were from less-developed rural areas. Here, we designed specific substrate peptides and reconfigured gold nanoparticle-based lateral flow test strip (LFTS) to develop an endopeptidase-based lateral flow assay for the diagnosis of botulism. We performed this lateral flow assay on botulinum neurotoxin-spiked human serum samples. The as-prepared LFTS had excellent performance in the detection of botulinum neurotoxin using only 1 μL of simulated serum, and its sensitivity and specificity were comparable to that of mouse lethality assay. Moreover, the assay takes only half a day and does not require highly trained laboratory staff, specialized facility, or equipment. Finally, our LFTS can be potentially extended to other serotypes of BoNTs by designing specific substrate peptides against the different types of BoNTs. Overall, we demonstrate a strategy by which LFTS and endopeptidase activity assays can be integrated to achieve facile and economic diagnosis of botulism in resource-limited settings.

A Rapid, Sensitive, and Portable Biosensor Assay for the Detection of Botulinum Neurotoxin Serotype A in Complex Food Matrices

“…In this study, we have established that CANARY ® can detect BoNT/A holotoxin with a LOD of 10.0 ± 2.5 ng/mL. This LOD is within the range of lateral flow devices but not as sensitive as other assays [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ]. An ultrasensitive gold nanoparticle endopeptidase-based lateral flow has been developed and has a LOD ≈ 20.0 pg/mL in sera comparable to the mouse bioassay; however, this assay requires 12 h of digestion but food and beverage matrices were not evaluated [ 28 ].…”

“…BoNT detection assays utilize multiple methods including antibody-based, mass-spectrometry, nucleic acid-based, cell-based, and enzymatic assays, as well as in vivo and ex vivo mouse assays in buffer and some matrices [ 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. All of the current technologies have advantages and disadvantages.…”

“…Additionally, newer assays have combined different technologies to improve the sensitivity of detection. Depending on the assay, the detection limits range from sub-picogram to nanogram per mL or attomolar to pM for buffer and some food matrices [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ].…”

Rapid Detection of Botulinum Neurotoxins—A Review

“…A key requirement of MBA replacements in relation to their usage in the pharmaceutical production sectors is the ability to quantify active toxin levels, so the inability to do this presents a significant hurdle. In 2017 Liu et al achieved an increase in sensitivity for a LFA system, their system was capable of detecting as low as 20pg/mL for BoNT/A using a very small sample size (1 μL) [70]. To achieve the improvement, Liu et al reconfigured gold nanoparticle-based lateral flow strips with specific substrate peptides that integrate endopeptidase activity to the assay [70].…”

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

“…However, this method requires long assay times (typically, 48 h), is expensive and laborious, and introduces an ethical dilemma regarding the use of laboratory animals. Alternative methods, such as mass spectrometric assays [8], enzyme-linked immunosorbent assays (ELISAs) [9,10,11], surface plasmon resonance [12], lateral flow immunoassay [13,14,15,16], high-performance liquid chromatography [17], and fluorescence resonance energy transfer [18] have successfully aimed at rapidity (within 20 min) and sensitivity (15–150 pg/mL). However, further research is still required to fulfill optimal criteria, such as real-time and label-free detection with rapidity, simplicity, and sensitivity including quantitative analysis and transportability.…”