Gold Nanomaterial-Based Microfluidic Paper Analytical Device for Simultaneous Quantification of Gram-Negative Bacteria and Nitrite Ions in Water Samples

This study presents a rapid microfluidic paper-based analytical device (μPAD) capable of simultaneously monitoring Gram-negative bacteria and nitrite ions (NO₂^-) for water quality monitoring. We utilize gold nanoparticles (AuNPs) functionalized with polymyxin molecules (AuNPs@polymyxin) to cause color change due to aggregation for the detection of Gram-negative bacteria, and antiaggregation in the presence of o-phenylenediamine (OPD) for NO₂^- detection. In this study, Escherichia coli (E. coli) serves as the model of a Gram-negative bacterium. Using the developed μPADs, the color changes resulting from aggregation and antiaggregation reactions are measured using a smartphone application. The linear detection ranges from 5.0 × 10² to 5.0 × 10⁵ CFU/mL (R² = 0.9961) for E. coli and 0.20 to 2.0 μmol/L (R² = 0.995) for NO₂^-. The detection limits were determined as 2.0 × 10² CFU/mL for E. coli and 0.18 μmol/L for NO₂^-. Notably, the newly developed assay exhibited high selectivity with no interference from Gram-positive bacteria. Additionally, we obtained acceptable recovery for monitoring E. coli and NO₂^- in drinking water samples with no significant difference between our method and a commercial assay by t test validation. The sensor was also employed for assessing the quality of the pond and environmental water source. Notably, this approach can also be applied to human urine samples with satisfactory accuracy. Furthermore, the assay's stability is extended due to its reliance on AuNPs rather than reagents like antibodies and enzymes, reducing costs and ensuring long-term viability. Our cost-effective μPADs therefore provide a real-time analysis of both contaminants, making them suitable for assessing water quality in resource-limited settings.