Surface Acoustic Wave Immunosensor for Detection of Botulinum Neurotoxin

Abstract

A Love-type acoustic wave sensor (AT-cut quartz substrate, SiO₂ guiding layer) with a center frequency of approximately 120 MHz was used to detect a simulant of pathogenic botulinum neurotoxin type A-recombinant of BoNT-A light chain-in liquid samples. The sensor was prepared by immobilizing monoclonal antibodies specific for botulinum neurotoxin via a thiol monolayer deposited on a gold substrate. Studies have shown that the sensor enables selective analyte detection within a few minutes. In addition, the sensor can be used several times (regeneration of the sensor is possible using a low pH buffer). Nevertheless, the detectability of the analyte is relatively low compared to other analytical techniques that can be used for rapid detection of botulinum neurotoxin. The obtained results confirm the operation of the proposed sensor and give hope for further development of this label-free technique for detecting botulinum neurotoxin.

Keywords: SAW; biological aerosol; biological warfare agents (BWA); botulinum neurotoxin; label-free detection; on-site detection of BWA.

Figure 1

Botulinum neurotoxin type A (BoNT-A) crystal structure and schematic diagram of functional domains. The image of the crystal structure of the toxin was taken from the RCSB PDB (RCSB.org) of PBD ID 3BTA [1].

Figure 2

Scheme of the measurement system used in the work.

Figure 3

Home-made functionalization cell in (a) disassembled and (b) folded state. (c) Screenshot of the control application during measurement with BoNT-A LC (visible changes in phase—red line, and frequency—blue line, due to BoNT-A LC dosing and sensor regeneration).

Figure 4

Sensor fabrication steps: (a) substrate preparation, (b) mixed thiols self-assembled monolayer—MIX SAM formation, (c) MAb immobilization, (d) layer blocking to minimize non-selective interactions.

Figure 5

(a) Exemplary frequency characteristics of a bare SAW device recorded in air. (b) Amplitude and (c) phase characteristics of the SAW device recorded in air after successive stages of functionalization (color markings are common to (b,c)).

Figure 6

Phase versus time plot of sensor recorded during three subsequent measurement cycles. The initial dosing times of individual solutions are marked with arrows (the times have been increased by the time resulting from the dead volume of the microfluidic system at a given flow rate). The beginning of a given solution dosing also means the end of the dosing of the previous solution. Black color indicates the flow of running buffer PBS, red—BoNT-A LC dosing in PBS at a concentration of 0.5 ug/mL, and green—Gly-HCl regeneration buffer flow.

Figure 7

Phase versus time plot of the sensor exposed to a non-complementary protein solution to the immobilized antibodies and then to a BoNT-A LC solution.

Figure 8

Phase versus time plot of the sensor with immobilized BSA exposed to BoNT-A LC.

Figure 9

Sensor response values to the solution of BoNT-A LC in PBS at a concentration of 0.5 μg/mL recorded 10 min after starting dosing (blue triangles) and steady-state values (red squares) in the successive five measurement cycles of the sensor.