RESUMEN
Loop-mediated isothermal amplification (LAMP) is a powerful gene amplification method, which has many advantages, including high specificity, sensitivity, and simple operation. However, quantitative analysis of the amplified target gene with the LAMP assay is very difficult. To overcome this limitation, we developed a novel biosensing platform for molecular diagnosis by integrating the LAMP method and retroreflective Janus particle (RJP) together. The final amplified products of the LAMP assay are dumbbell-shaped DNA structures, containing a single-stranded loop with two different sequences. Therefore, the concentration of the amplified products can be measured in a manner similar to the sandwich-type immunoassay. To carry out the sandwich-type molecular diagnostics using the LAMP product, two DNA probes, with complementary sequences to the loop-regions, were prepared and immobilized on both the sensing surface and the surface of the RJPs. When the amplified LAMP product was applied to the sensing surface, the surface-immobilized DNA probe hybridized to the loop-region of the LAMP product to form a double-stranded structure. When the DNA probe-conjugated RJPs were injected, the RJPs bound to the unreacted loop-region of the LAMP product. The number of RJPs bound to the loop-region of the LAMP product was proportional to the concentration of the amplified LAMP product, indicating that the concentration of the target gene can be quantitatively analyzed by counting the number of observed RJPs. Using the developed system, a highly sensitive and selective quantification of Salmonella was successfully performed with a detection limit of 102 CFU.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Técnicas Biosensibles/métodos , Materiales Manufacturados , Imagen Óptica/métodos , Salmonella typhimurium/aislamiento & purificación , Aluminio/química , Aluminio/efectos de la radiación , Secuencia de Bases , Sondas de ADN/química , Sondas de ADN/genética , ADN Bacteriano/genética , ADN Complementario/genética , Oro/química , Oro/efectos de la radiación , Luz , Límite de Detección , Microtecnología , Técnicas de Amplificación de Ácido Nucleico , Hibridación de Ácido Nucleico , Fenómenos Ópticos , Dióxido de Silicio/química , Dióxido de Silicio/efectos de la radiación , Succinimidas/químicaRESUMEN
Plasmonic nanomaterials (P-NM) are receiving attention due to their excellent properties, which include surface-enhanced Raman scattering (SERS), localized surface plasmon resonance (LSPR) effects, plasmonic resonance energy transfer (PRET), and magneto optical (MO) effects. To obtain such plasmonic properties, many nanomaterials have been developed, including metal nanoparticles (MNP), bimetallic nanoparticles (bMNP), MNP-decorated carbon nanotubes, (MNP-CNT), and MNP-modified graphene (MNP-GRP). These P-NMs may eventually be applied to optical biosensing systems due to their unique properties. Here, probe biomolecules, such as antibodies (Ab), probe DNA, and probe aptamers, were modified on the surface of plasmonic materials by chemical conjugation and thiol chemistry. The optical property change in the plasmonic nanomaterials was monitored based on the interaction between the probe biomolecules and target virus. After bioconjugation, several optical properties, including fluorescence, plasmonic absorbance, and diffraction angle, were changed to detect the target biomolecules. This review describes several P-NMs as potential candidates of optical sensing platforms and introduces various applications in the optical biosensing field.