(J-383) Microfluidic Nano-biosensor for Detection of Botulinum Neurotoxin

Introduction:: Botulinum neurotoxin (BoNT) is a potent foodborne toxin that can cause muscle paralysis and death. It is produced by anaerobic spore-forming bacteria that belong to the Clostridium genus. Currently, there are seven identified serotypes of BoNT, serotypes A to G. Serotypes A, B, E, and F can cause human botulism through contaminated foods, wounds, and improper injections for medical/cosmetic reasons. Due to its lethality and ease of dissemination, BoNT has been identified as one of the top bioterrorism agents.

The “gold standard” method of detection is the mouse bioassay (MBA). Its main limitations are that it takes several days to receive results and that it requires the use of laboratory animals. Other detection methods such as enzyme-linked immunoassays (ELISA), immunological-PCR assays, and mass-spectrometry (MS)-endopeptidase assays have been developed. However, immunoassays cannot provide information on endopeptidase activity, which is the key to BoNT toxication, while MS-based assays require expensive instruments and trained personnel.

We developed a novel microfluidic nano-biosensor utilizing luminescent semiconductor nanocrystals, quantum dots (QDs), to detect biologically active BoNT and differentiate BoNT serotypes. QDs are tethered to the surface of microfluidic channels through serotype-specific peptides that can be cleaved due to BoNT endopeptidase activity. In the presence of the toxin, the QDs were released from the channels due to the cleavage. The photoluminescence of the QDs was measured to indicate the detection. This biosensor developed offers a low-cost and easy-to-operate rapid method that is capable of detecting biologically active BoNT within an hour.

(Preferred Sub-Track: Biosensors)

Materials and Methods:: The principle of the microfluidic nano-biosensor is presented in Figure 1. The microfluidic channels feature the capability of droplet-based passive pumping, which only needs pipetting to ensure simple operation. Serotype-specific peptides for serotypes A (BoNT/A) or F (BoNT/F) are attached to polydimethylsiloxane (PDMS) microfluidic channels through the linkage by polyethylene glycol (PEG) spacers. After incubation and rinsing of the channels, QDs are introduced to the PDMS surface. After one hour of incubation and water rinsing, the channels with tethered QDs are ready for the detection.

To demonstrate the BoNT detection, peptides specific to BoNT/A and /F were paired with QD 525 (QDs with an emission maximum at 525 nm) and QD 655, respectively. Catalytic domains (light chains, Lc) of BoNT/A and /F, which represent the endopeptidase activity of the toxin, were used as the detection targets. Upon the existence of BoNT/A light chain (LcA), peptides specific to BoNT/A were cleaved and the QD 525 were removed from the channel area after rinsing, while the QDs would stay in the area when LcA was absent. Using the other pair of BoNT/F specific peptides and QD 655, serotype F can be detected. The photoluminescence of the QDs was observed using a UV lamp and measured using a fluorescence spectrometer.

Results, Conclusions, and Discussions:: To characterize and prove the construction of the biosensor, we observed and measured the photoluminescence of microfluidic channels with different treatments as: 1) without any surface modification, 2) without peptide or PEG, and 3) with all modifications as described in the Materials and Methods section. After introducing QDs to these channels followed by water rinsing, QDs were only observed in the channels with all modifications, showing a photoluminescence significantly higher than others. The results indicated that the QDs were tethered to the channels through the peptides. Then we demonstrated the detection using the type A and F light chains by introducing them to the channels with their respective peptides and QDs. Compared to negative control channels without target light chains, lower photoluminescence was observed after rinsing in the channels receiving specific light chains, indicating the detachment of the QDs after rinsing due to the cleavage of the peptides by BoNT.

In conclusion, we developed a simple, rapid and inexpensive biosensor that can be used to detect the presence of biologically active BoNT. Our next goal is to optimize the biosensor to detect both serotypes A and F with high sensitivity and specificity. Since this biosensor detection principle can be applied to other BoNT serotypes by using different peptide and QD pairs, it can be potentially applied to detect multiple serotypes at the same time. With a detection time of one hour, while also being easy to operate, this biosensor can be potentially employed as a quick field-deployable screening method.

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