Integrated Bioassays in Microfluidic Devices: Botulinum Toxin Assays

Abstract

A microfluidic assay was developed for screening botulinum neurotoxin serotype A (BoNT-A) by using a fluorescent resonance energy transfer (FRET) assay. Molded silicone microdevices with integral valves, pumps, and reagent reservoirs were designed and fabricated. Electrical and pneumatic control hardware were constructed, and software was written to automate the assay protocol and data acquisition. Detection was accomplished by fluorescence microscopy. The system was validated with a peptide inhibitor, running 2 parallel assays, as a feasibility demonstration. The small footprint of each bioreactor cell (0.5 cm2) and scalable fluidic architecture enabled many parallel assays on a single chip. The chip is programmable to run a dilution series in each lane, generating concentration-response data for multiple inhibitors. The assay results showed good agreement with the corresponding experiments done at a macroscale level. Although the system has been developed for BoNT-A screening, a wide variety of assays can be performed on the microfluidic chip with little or no modification.

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

“…Indeed, detection of BoNT on microfluidic chips has been developed [134,135,136]. Assays carried out on a chip are miniaturized versions of the macroscopic assays described above, such as the sandwich type immunoassay [136] or FRET based endopeptidase assay [134,135]. There are clear advantages to microfluidic technology, including a reduction of reagent consumption and the ability to operate in a semiautomatic mode for all steps (e.g., sample preparation, pretreatment, mixing, incubation, washing) carried out on a chip.…”

A fluorescence detection platform using spatial electroluminescent excitation for measuring botulinum neurotoxin A activity

“…Such cleavage activity has been detected by mass spectrometry (Barr et al, 2005;Gaunt et al, 2007;Kalb et al, 2006), with a cantilever micromechanical sensor (Liu et al, 2003;Parpura and Chapman, 2005), using fluorescent sensors to detect FRET-based reactions (Dong et al, 2004), and also a microfluidics device (Mangru et al, 2005). Some of the technical issues concerning these protease-based activity assays, including matrix inhibition and non-specific signals, were recently addressed (Kalb et al, 2006).…”

“…While these activity-based assays are very promising, they are not currently configured in an easy-to-use multi-sample format that is both sensitive and portable. Even the microfluidics application that has been developed requires a microscope for fluorescent detection (Mangru et al, 2005). The EL-CCD detector described in this study was designed with the aim of producing a portable, simple and inexpensive modular-based fluorescence detection system, in a relatively small and compact manner, capable of measuring multiple samples simultaneously.…”

A graphene oxide based fluorescence resonance energy transfer (FRET) biosensor for ultrasensitive detection of botulinum neurotoxin A (BoNT/A) enzymatic activity

“…In order to solve this problem, BoNT activity assays, based on the toxins' ability to cleave specific SNARE proteins, have been developed via various detection approaches such as mass spectroscopy (Kalb et al, 2008), surface plasma resonance (Lévêque et al, 2013), and fluorimetric methods (Schmidt and Stafford, 2003). Among various activity assays, fluorescence resonance energy transfer (FRET) based methods are promising with the advantage of simplicity and direct response (Dong et al, 2004;Gilmore et al, 2011;Mangru et al, 2005;Sun et al, 2009). For FRET assays, the existence of toxin and its enzyme activity can be determined simultaneously by fluorescent signal transduction upon one-step addition of target toxin.…”