Rapid SERS Detection of Botulinum Neurotoxin Type A

Abstract

Surface-enhanced Raman scattering (SERS) is a powerful technique for decoding of 2-5-component mixes of analytes. Low concentrations of analytes and complex biological media are usually non-decodable with SERS. Recognition molecules, such as antibodies and aptamers, provide an opportunity for a specific binding of ultra-low contents of analyte dissolved in complex biological media. Different approaches have been proposed to provide changes in SERS intensity of an external label upon binding of ultra-low contents of the analytes. In this paper, we propose a SERS-based sensor for the rapid and sensitive detection of botulinum toxin type A. The silver nanoisland SERS substrate was functionalized using an aptamer conjugated with a Raman label. The binding of the target affects the orientation of the label, providing changes in an analytical signal. This trick allowed detecting botulinum toxin type A in a one-stage manner without additional staining with a monotonous dose dependence and a limit of detection of 2.4 ng/mL. The proposed sensor architecture is consistent with the multiarray detection systems for multiplex analyses.

Keywords: SERS; aptamer; aptasensor; biosensor; botulinum neurotoxin.

Figure 1

Structure of botulinum neurotoxin serotype A. Receptor-binding domain is shown in green color; the translocating domain is shown in blue color; the catalytic domain is shown in orange color. The structure was built using PyMol software (2.4.1 version) from the crystal structure with pdb id 3BTA [7].

Figure 2

Affinity of DNA aptamers to BoNT serotype A (Xeomin) estimated with biolayer interferometry. The association stage corresponds to 0–200 s of the experiment; the dissociation stage corresponds to 0–200 s of the experiment.

Figure 3

Thermal stability of aptamer Apt_2.22 estimated with CD and UV spectroscopies. CD (A) and UV spectra (B) are provided at different temperatures. The predicted structure of the aptamer is shown in part (C). Melting curves are shown for the UV melting experiment at a wavelength of 260 nm (D) and CD melting experiment at a wavelength of 247 nm (E).

Figure 4

SERS spectra of the substrates obtained with different concentrations of Apt 2.22_mod solution.

Figure 5

Distribution of SERS intensity at Apt_2.22_mod aptamer concentration of 35 nM (A), 17.5 nM (B), and 8.8 nM (C). Part (D)—SEM image of SERS substrate.

Figure 6

(A) The dependence of SERS signal on BoNT A concentration. SERS substrates were functionalized using Apt_2.22_mod at the concentration of 17.5 nM. (B) Distribution of SERS signal intensities on experimental and control zones during BoNT A (2.4 ng/mL) detection.

Figure 7

Dependence of SERS signal on the analyte exposure time (BoNT A).

Figure 8

SERS intensity distributions of experiment and control while detecting IgGHum (2.4 ng/mL) using Apt_2.22_mod aptamer at a concentration of 17.5 nM.

Figure 9

SERS intensity distributions in the samples with 2.4 ng/mL of BoNT A (experiment) and the control samples. SERS substrates were functionalized with aptamer of RSV at a concentration of 17.5 nM.

Figure 10

SERS intensity distributions in the samples of blood serum with 2.4 ng/mL BoNT A (experiment) and toxin-free blood serum (control).