Multifuntional Gold Nanoparticles for the SERS Detection of Pathogens Combined with a LAMP–in–Microdroplets Approach

Abstract

We developed a droplet-based optofluidic system for the detection of foodborne pathogens. Specifically, the loop-mediated isothermal amplification (LAMP) technique was combined with surface-enhanced Raman scattering (SERS), which offers an excellent method for DNA ultradetection. However, the direct SERS detection of DNA compromises the simplicity of data interpretation due to the variability of its SERS fingerprints. Therefore, we designed an indirect SERS detection method using multifunctional gold nanoparticles (AuNPs) based on the formation of pyrophosphate generated during the DNA amplification by LAMP. Towards this goal, we prepared multifunctional AuNPs involving three components with key roles: (1) thiolated poly(ethylene glycol) as stabilizing agent, (2) 1-naphthalenethiol as Raman reporter, and (3) glutathione as a bioinspired chelating agent of magnesium (II) ions. Thus, the variation in the SERS signal of 1-naphthalenethiol was controlled by the aggregation of AuNPs triggered by the complexation of pyrophosphate and glutathione with free magnesium ions. Using this strategy, we detected Listeria monocytogenes, not only in buffer, but also in a food matrix (i.e., ultra-high temperaturemilk) enabled by the massive production of hotspots as a result of the self-assemblies that enhanced the SERS signal. This allowed the development of a microdroplet-LAMP-SERS platform with isothermal amplification and real-time identification capabilities.

A SERS-based 3D nanobiosensor: towards cell metabolite monitoring

“…[29][30][31] In particular, AuNSs were selected due to the extra SERS enhancement offered by their tips, and the Ag-coated AuNRs were selected (Au@AgNRs) due to the better SERS enhancement of silver vs. gold, as previously demonstrated. 23,32 In this work, in order to obtain a good dispersion and distribution of the NPs within the GG-SLH, the NPs were added in situ before the gelation reaction of the precursor hydrogels. 15 In order to test the loading efficiency into the GG-SLH, as a function of the initial concentration of NPs added, the extinction spectra of the nanostructures were obtained and the maximum absorbance was identified (560 nm for Au@AgNRs and 830 nm for AuNSs).…”

Naked‐eye detection strategies coupled with isothermal nucleic acid amplification techniques for the detection of human pathogens

“…Rapid and sensitive detection of shrimp infectious myonecrosis virus using a reverse transcription loop-mediated isothermal amplification and visual colorogenic nanogold hybridization probe assay. Journal of Virological Methods,193(2), 542-547. https://doi.org/10.1016/j.jviromet.2013.07.017 Azinheiro, S., Carvalho, J., Prado, M., & Garrido-Maestu, A. (2020).…”

Accessible, fast and easy fabrication of hydrophilic-in-hydrophobic microdroplet arrays

“…The new fabrication method removed this obstacle, rendering the MDA technology, and hence many MDA-based assay techniques, accessible to more labs. In fact, in addition to the digital ELISA method described here, there are several other applications that could be performed on the MDA platform, such as the loop-mediated isothermal amplification (LAMP) for the detection and quantification of DNA [26,27]. LAMP could be easily performed on the described MDA device because it is a technique based on isothermal nucleic acid amplification which, in contrast to polymerase chain reaction methods, does not require sophisticated instrumentation like the thermal cycler for alternating temperature steps.…”