One-step DNA Detection through Dual-Color Confocal Analysis of DNA-Assembled Scattering Nanoparticles: Case of a Fragment of Sesame

DNA-based detection methods have many applications in clinical diagnosis, food science, and environmental control. However, most of the analytical techniques currently in use, such as PCR, are expensive, cumbersome, and time... [ view full abstract ]

DNA-based detection methods have many applications in clinical diagnosis, food science, and environmental control. However, most of the analytical techniques currently in use, such as PCR, are expensive, cumbersome, and time consuming. Thus, methods such as enhanced surface plasmon resonance and fluorescence-based devices have been developed, but still present some limitations. Here, we present a novel approach based on Photon Cross Correlation Spectroscopy (PCCS) of metallic nanoparticles (NPs) that provides increased sensitivity, rapid detection and reduced cost in order to open new and breakthrough features for diagnosis.
The detection of DNA relies on the self-assembly of two distinct scattering NPs (silver nanoparticles (AgNPs) and gold nanorods (AuNRs)) mediated via specific-based pair recognition between ss-DNA target and ss-DNA probes anchored to NPs. Upon illumination using two laser beams, the particles passing through the volume of analysis are excited and their resulting scattered lights are collected and analyzed together. In the absence of target, both NPs move independently and their signals are not correlated. However, as the presence of DNA target initiates aggregation of the NPs through hybridization, AgNPs and AuNRs thus diffuse simultaneously. Consequently, their scattering responses are correlated, giving rise to temporal coincidence (Figure 1).
To validate the developed proof-of-concept PCCS, the study focused on the detection of a specific fragment of sesame, an allergenic food ingredient. Here, we have considered several aspects with the aim to improve detection efficiency within the shortest time. The optimization of the hybridization events between a target DNA and its probes, the sensitivity with quantitative analysis and the specificity to particularly distinguish perfectly matched DNA from defective one have been investigated. This new detection method was able to in-situ detect and quantify specific DNA sequence of sesame in a range of concentration from 5 pM to 1.5 nM , with LOD of 1 pM, after 30 min of incubation at 65°C. In addition, we have observed a net decrease in the detection when using one single matched and one deleted base in the sequence of the target and no possible detection in presence of non-complementary DNA