Plasmonic ELISA based on DNA-directed gold nanoparticle growth for Cronobacter detection in powdered infant formula samples

Abstract

The traditional gold nanoparticle (AuNP) growth-based plasmonic ELISA (pELISA) strictly and directly controlled by reducing reagents can achieve high sensitivity, but it remains fragile toward the surrounding environment. This work developed a sandwich pELISA for Cronobacter detection in powdered infant formula samples by mediating AuNP growth through DNA. In this assay, DNA adsorbed on the surface of gold nanoseeds guided the anisotropic crystal growth with hydroxylamine as a reducing reagent, and the catalase-hydrogen peroxide (Cat-H₂O₂) system was introduced to bridge the DNA-directed AuNP growth and pELISA, as such DNA can be cleaved into fragments by the hydroxyl radical generated from oxidation of H₂O₂ through Fenton reagents. Under optimized conditions, the proposed pELISA can qualitatively detect Cronobacter species (Cronobacter muytjensii ATCC 51329) by the naked eye with a cut-off limit of 3 × 10⁵ cfu/mL. This method also revealed a good linear range (3 × 10² to 3 × 10⁷ cfu/mL) for quantitative detection of C. muytjensii ATCC 51329 with a limit of detection of 1.6 × 10² cfu/mL, which is approximately 162.5 times lower than that of horseradish peroxidase-based conventional ELISA (2.6 × 10⁴ cfu/mL). By taking advantage of highly stable DNA-directed AuNP growth, the proposed method shows a good performance in powdered infant formula samples spiked with different concentrations of C. muytjensii ATCC 51329 with average recoveries ranging from 90.79 to 119.09% and coefficient of variation ranging from 4.24 to 9.55%. These values corresponded to an acceptable accuracy and precision for the proposed method. In brief, this work shows potential for screening other analytes in food safety, clinical diagnostics, and environmental monitoring.

Keywords: Cronobacter; DNA-directed; gold nanoparticle growth; plasmonic enzyme-linked immunosorbent assay; powdered infant formula.