A convenient renewable surface plasmon resonance chip for relative quantification of genetically modified soybean in food and feed

Abstract

The cultivation of genetically modified organisms (GMO) continues to expand worldwide. Still, many consumers express concerns about the use of GMO in food or feed, and many countries have legislated on labelling systems to indicate the presence of GMO in commercial products. To deal with the increased number of GMO events and to address related regulations, alternative detection methods for GMO inspection are required. In this work, a genosensor based on Surface Plasmon Resonance under continuous flow was developed for the detection and quantification of a genetically modified soybean (event GTS 40-3-2). In a single chip, the simultaneous detection of the event-specific and the taxon-specific samples were achieved, whose detection limits were 20 pM and 16 pM, respectively. The reproducibility was 1.4%, which supports the use of the chip as a reliable and cost-effective alternative to other DNA-based techniques. The results indicate that the proposed method is a versatile tool for GMO quantification in food and feed samples.

Fig 1

(A) Nomenclature of the DNA sequences used in the SPR-based biosensor construction: (A.I) ssDNA-streptavidin (ssDNA-SA); (A.II) ssDNA (black) linked to (TEG)Biotin (dark green); (A.III) RR or Lec target sequence (red). (B) Representative sensorgram of the novel capture concept used in this study to detect RR event. (C) Schematic illustration of the four steps that occurs in the SPR sensing surface (C.I) Hybridization of the ssDNA-SA with the complementary ssDNA (blue) linked on the chip surface; (C.II) Conjugation with biotinylated DNA probe by electrostatic interaction; (C.III) Hybridization between the analyte and capture probe; (C.IV) Regeneration.

Fig 2. Sensorgram representative of the SCK analysis of the duplex DNA formation on the biosensor.

The SPR experiments were performed at 20°C using 30 μL/min of flow rate and 50 μg/mL of capture probe. The red curve represents the experimental data and the black curve represents the fit of the sensorgram to a Langmuir 1:1 model of interaction. χ² ranged from 84 to 115 (RU²), and the U-Value was 1. k_a and k_d are the association and dissociation rate constants, respectively, and K_D is the dissociation equilibrium constant. The experiments were performed in triplicate (n = 3).

Fig 3

CD spectra of (A) target ssDNA, complement ssDNA and the corresponding duplex; target ssDNA and capture DNA with (B) and without (C) the (TEG)Biotin group and the corresponding duplexes. All spectra were acquired in a 0.1 cm path length quartz cuvette at 25°C, and RR DNA sequences were analyzed at 60 μM in 2× SSPE buffer (pH 7.4).

Fig 4. High resolution melt curve.

(A) dsDNA(Target+capture): T_m = 78 ᵒC; dsDNA(target+capture-(TEG)Biotin): T_m = 82 ᵒC; (B) and dsDNA(target+complement): T_m = 53 ᵒC. All analyzes are compared to the corresponding ssDNA (ssDNA target or complement).

Fig 5

Calibration plots for the (A) event-specific and (B) reference targets. RU values obtained for blank experiment, exogenous DNA (herring sperm), and target (C) RR and (D) Lec DNA sequences at 5 nM.

Fig 6. Analytical responses obtained after dilution of event-specific RR (1:1) and Lec (1:11) amplified DNA of nine real samples.

The quantitative results were expressed as RR percentages determined by RR/LEC. ND: Not Detected.