Polydiacetylene for the Detection of α-Hemolysin in Milk toward the Diagnosis of Bovine Mastitis

Early diagnosis of mastitis is essential to reduce the expenses it imparts on the dairy industry. Existing techniques fail to provide rapid and pathogen-specific information to direct the prescription of antibiotics. Polydiacetylene (PDA)-based colorimetric sensors have emerged as promising bacterial screening tools that show potential for application in the diagnosis of bacterial infections. However, there are challenges in operating PDA sensors in complex samples, as identified in our recent work. To overcome this challenge, herein, we design and synthesize a custom diacetylene monomer via a copper-catalyzed cross-coupling reaction to yield 6,8-tricosadiynoic acid. Photopolymerization of 6,8-tricosadiynoic acid monomers yields blue-phase PDA with superior chromatic stability to milk constituents and reduced chromatic response to temperature fluctuations, which diminish the likelihood of false positives. We then form functionalized PDA vesicles by insertion of 1,2-dimyristoyl-sn-glycero-3-phosphocholine phospholipid and cholesterol in the PDA membrane. This allows the detection of α-hemolysin, a hemolytic toxin excreted by Staphylococcus aureus, a predominant causative pathogen of bovine mastitis. We demonstrate the detection of α-hemolysin in phosphate-buffered saline (limit of detection (LOD) = 3.62 μg/mL) and milk samples (LOD = 6.62 μg/mL) by a blue to red color change visible to the naked eye. The color change is studied by absorption spectroscopy and digital colorimetric analysis of photographs. We attribute the color change to pore formation in the PDA membrane and lysis of the vesicles due to the action of the S. aureus α-hemolysin. This is evidenced by vesicle membrane destruction in transmission electron microscopy micrographs of PDA vesicles before and after incubation with the toxin. The specificity of the sensor is demonstrated by discrimination between α-hemolysin and other toxins and biomarkers for mastitis. This is the first custom-designed PDA for point-of-care application in bovine milk samples and the first documented detection of α-hemolysin using a PDA-based sensor.