Rapid colorimetric detection of pathogenic bacteria by antibiotic-conjugated gold nanoparticles in a laser-printed µPAD

Abstract

With the global spread of resistant pathogenic bacteria, the lack of effective point-of-care diagnostics will increase selective pressure on existing antibiotics and prolong the treatment of resistant bacterial infections. Antibiotic-gold nanoparticle conjugates have been trialed previously to improve the potency of pharmaceuticals against resistant bacteria, but little has been done to evaluate antibiotic resistance infections at the point of care (POC). Here, we estimated the response of these conjugates to different ranges of bacterial concentration and tested them using a paper microfluidic device to solve this problem. Gold nanoparticles (AuNPs) were synthesized, conjugated to a panel of four antibiotics (imipenem [IMI], meropenem [MER], ciprofloxacin [CIP] and augmentin [AUG]), and characterized. The conjugates were first validated to detect gram-positive and gram-negative bacteria in colloidal suspensions. The microfluidic paper analytical device (µPAD) was fabricated using laser printing technology and heated to create a hydrophobic barrier. Antibiotic-decorated AuNPs were embedded into the detection zones of the laser-printed µPADs for the detection of cultured bacteria to assess the functionality of the antibiotic-AuNPs within a biosensor configuration. With limits of detection (LODs) of 1.86 × 10⁻³ CFU mL⁻¹ (AuNPs@AUG), 6.26 × 10⁻⁴ CFU mL⁻¹ (AuNPs@CIP), and 3.72 × 10⁻⁴ CFU mL⁻¹ (AuNPs@MER) in colloidal suspensions, the antibiotic conjugates effectively detected low concentrations of bacteria. Tests of the hydrophobic barrier demonstrated that fluid could flow within the hydrophilic channels without crossing the barrier, and detection of the bacteria occurred at the µPAD but with less sensitivity than in solution. Thus, antibiotics are an effective ligand in diagnostics, while antibiotic-decorated AuNPs could function in the µPAD to provide reliable point-of-care diagnostics for the early detection and real-time monitoring of pathogenic bacteria in water, food and biological samples.