Single-Tube Instantaneous Generation of CTAB-Stabilized Gold Nanoparticles for the On-Site Molecular Identification of Foodborne and Multidrug-Resistant Pathogens

This study proposes a highly sensitive and selective modality based on the instantaneous production of cetyltrimethylammonium bromide (CTAB)-stabilized gold nanoparticles (AuNPs) to visualize two loop-mediated isothermal amplification (LAMP)-amplified infectious pathogens, namely, <i>Listeria monocytogenes</i> (a foodborne pathogen) and <i>Streptococcus pneumoniae</i> (a multidrug-resistant pathogen). During the detection process, the LAMP amplicon (DNA) and CTAB formed a DNA-AuCl₄-cetyltrimethylammonium (CTA) ion complex with gold chloride (HAuCl₄). The mild reducing agent, sodium ascorbate, donates a single electron, producing dehydroascorbic acid and DNA-AuCl₂-CTA, which are further reduced at room temperature (RT) to generate wine-red gold nanoparticles (AuNPs) within 1 min. Significantly, the introduced method eliminated the requirement for external heating during LAMP amplicon detection by performing the reaction at RT. Moreover, the developed colorimetric LAMP assay simultaneously performed the amplification and detection processes in a single tube to prevent aerosol contamination and false-positive results. <i>S. pneumoniae</i> was detected in an artificial urine sample with a detection limit of 10 pg/μL, demonstrating the effectiveness of this CTAB-stabilized AuNP-based approach. According to the calibration curve obtained between the relative absorbance values and the DNA concentration, the limits of detection for <i>L. monocytogenes</i> and <i>S. pneumoniae</i> were determined to be 0.19 and 0.25 pg/μL, respectively. This RT-based CTAB-stabilized AuNP production approach provides an energy-efficient, more accessible, and rapid alternative to traditional methods, thereby enabling its integration into facile and sensitive diagnostic platforms.