Detection and Characterization of Nodularin by Using Label-Free Surface-Enhanced Spectroscopic Techniques

Abstract

Nodularin (NOD) is a potent toxin produced by Nodularia spumigena cyanobacteria. Usually, NOD co-exists with other microcystins in environmental waters, a class of cyanotoxins secreted by certain cyanobacteria species, which makes identification difficult in the case of mixed toxins. Herein we report a complete theoretical DFT-vibrational Raman characterization of NOD along with the experimental drop-coating deposition Raman (DCDR) technique. In addition, we used the vibrational characterization to probe SERS analysis of NOD using colloidal silver nanoparticles (AgNPs), commercial nanopatterned substrates with periodic inverted pyramids (Klarite^TM substrate), hydrophobic Tienta^® SpecTrim^TM slides, and in-house fabricated periodic nanotrenches by nanoimprint lithography (NIL). The 532 nm excitation source provided more well-defined bands even at LOD levels, as well as the best performance in terms of SERS intensity. This was reflected by the results obtained with the Klarite^TM substrate and the silver-based colloidal system, which were the most promising detection approaches, providing the lowest limits of detection. A detection limit of 8.4 × 10^-8 M was achieved for NOD in solution by using AgNPs. Theoretical computation of the complex vibrational modes of NOD was used for the first time to unambiguously assign all the specific vibrational Raman bands.

Keywords: Raman; SERS; cyanotoxin; microcystins; nodularin.

Figure 1

Main algal toxins correlated with blooming, and the most relevant techniques used for their detection as reported between 2017 and 2022 (source—Web of Science Collection). A table summarizing the lowest LODs obtained by using label-based approaches or elaborated platforms designed for trace-level detection [30,31,32,36,48,49,50,54,57,63,64,65,67].

Figure 2

Raman spectrum of NOD (grey) calculated at AFPD/6-311+G(2d,p) level of theory as compared to experimental spectrum (orange) registered with 633 nm laser line under ambient conditions.

Figure 3

DCDR spectra recorded on Tienta^® SpectRIM^TM substrate for NOD in ethanol with a final concentration of 10⁻³ M (A) and 10⁻⁴ M (B), respectively by using the 785 nm, 532 nm and 633 nm laser lines. Inset with optical image by using 20× objective.

Figure 4

Optical images showing the samples for NOD on Klarite^TM substrate by using 100× (A) and 20× (B) magnification. Arrows show selected points for laser irradiation. SERS spectra recorded on Klarite^TM substrate for NOD in ethanol at different concentration using 532 nm laser line (C).

Figure 4

Optical images showing the samples for NOD on Klarite^TM substrate by using 100× (A) and 20× (B) magnification. Arrows show selected points for laser irradiation. SERS spectra recorded on Klarite^TM substrate for NOD in ethanol at different concentration using 532 nm laser line (C).

Figure 5

(A) SERS spectra on of NOD/ethanol samples at different concentrations using a 532 nm laser line and citrate-reduced AgNPs. (B) Linear fit of the relative intensity ratio of the SERS bands at 1647 cm⁻¹ and 879 cm⁻¹ as a function of NOD concentration. Error bars indicate standard deviation R2 = 0.955.

Figure 6

SERS spectra on of NOD/ethanol samples at 10⁻³ M concentration using the 532 nm laser line (a) and 633 nm laser line (b) using 25 nm Ag covered nanotrenches on plastic.

Figure 7

Optimized structure of NOD in gas phase at APFD/6-311+G(2d,p) level of theory (A) with the three main chemical components marked (B) in red—the main ring, blue—Adda, and green—L-Arg. The atom labels used in DFT calculations are shown on Nodularin’s chemical structure (C).