Rapid Label-Free Detection of Pathogens by Local pH Modulation

Abstract

Pathogenic bacteria present major issues for human health across the world. One of the ways to mitigate the negative impacts from contaminated food and water sources is to decrease the time required to test potentially contaminated sources. This study examined a new method of label free detection using local pH modulation to quantitatively detect bacteria. By tagging antibodies with a pH-sensitive fluorescent dye it was possible to detect the presence of bacteria bound to antibodies. Local pH can be effected by the presence of charged molecules because they attract counter ions. By utilizing the negatively charged surface of bacteria to attract counter ions in the form of hydrogen ions the local pH can be lowered, thereby lowering the fluorescence of fluorescein. By measuring fluorescence with respect to bacterial cell concentration a relationship between bacteria concentration and fluorescence can be established. It is also advantageous to know if the pathogens detected are active and alive or dead. Adding a rapidly uptaken carbon source (glucose) allows for differences between live and dead cells to be detected. This approach was tested in microtiter plates and using immunomagnetic beads as the testing platforms. Using microtiter plates concentrations of ~10^6 E. coli cells could be detected although not to a statistically significant level. The addition of glucose showed that live cells could be distinguished from UV killed cells but cell numbers could not be established. Immunomagnetic beads displayed inconclusive results indicating the need for continued experiments.