IntroductionFood safety is gaining prominence as a global issue, driving the demand for rapid, simple, on-site and low-cost biosensor technologies. Biosensors have emerged as a cheap and quick alternative to traditional... [ view full abstract ]
Introduction
Food safety is gaining prominence as a global issue, driving the demand for rapid, simple, on-site and low-cost biosensor technologies. Biosensors have emerged as a cheap and quick alternative to traditional chromatographic methods. Gold nanoparticles (AuNPs) exhibit optical properties (surface plasmon resonance) offering the prospect for label-free and real-time measurements. AuNPs can be biofunctionalized with oligonucleotides (probes) that are specific for recognition of food pathogens, giving rise to nano-bioensors (genosensors) with applications in food quality and safety assurance. Optical excitation of plasmons is responsible for their large absorption and scattering properties, which can be controlled by modifying the behavior of the nanoparticles in solution (aggregation/dispersion).
Methods
AuNPs were chemically synthesized by the citrate reduction method. The nanoparticles were functionalized by oligonucleotides hybridizing genomic DNA of Mycobacterium bovis (bovine tuberculosis), which correspond to a segment of the RD4 gene (5'-CGCCTTCCTAACCAGAATTG-3'). The alkanothiol-modified probe was reduced with DTT to form the thiol group needed for chemisorption of the oligonucleotides onto the AuNP surface, and was chromatographically purified (Sephadex G-50). The genosensor was built by functionalizing AuNPs with the thiolated probe. Detection of M. bovis genomic DNA was performed previous denaturation of the DNA (95°C for 5 min). The hybridation was carried out at 55°C, and color change was followed spectrophotometrically upon addition of HCl 0.1 N.
Results
AuNPs were analysed spectrophotometrically (Figure 1) and by dynamic light scattering, showing homogeneous dispersion of nanoparticles with an average diameter of 10 nm (Figure 2). Optical detection of M. bovis was demonstrated using samples contaminated with different amounts of genomic DNA. A shift was observed on the AuNPs absorption spectrum, which was associated to a color change from red to purple (λmax = 520 nm to λmax = 620 nm). A standard curve was obtained by correlating the color change (A520/A620) with the DNA concentration (Figure 3).
Discussion
AuNPs aggregate in the absence of genomic DNA. Under this condition, the color change (red to purple) is attributable to the absence of hybridization between the genosensor aptamers and DNA that stabilize the nanoparticles, while color red gets more intense in the presence of the DNA.
