Bacterial sensor: pathogen detected with fluorescence

Pathogenic sense

A new biosensor platform for the detection of bacterial pathogens, specifically demonstrated with E coli, has been developed based on long-range surface plasmon-enhanced fluorescence spectroscopy (LRSP-FS).

Chun-Jen Huang, Jakub Dostalek, Angela Sessitsch and Wolfgang Knoll of the Health and Environment Department, at the Austrian Institute of Technology GmbH, in Vienna, explain how increasing awareness of food safety and the risks associated with various microbial pathogens carried in food and drinking water, in particular Escherichia coli O157:H7, mean identifying when such pathogens are present is becoming more and more important.

O157 is an enterohaemorrhagic strain of the bacterium and not only causes severe diarrhoea but can lead to potentially lethal kidney damage. Transmission is most commonly via the faecal-oral route and recent high-profile outbreaks have generally been traced back to contaminated beef products that have been undercooked or more rarely to exposure to farm animal faeces or to swimming in contaminated water. O157's Shiga-like toxins make it an acutely harmful and widespread food borne pathogen. Unfortunately ingestion of just 50 to 100 microbial cells is sufficient to cause infection. The US Centers for Disease Control (CDC) reports that outbreaks of E. coli O157:H7 lead to at least ten thousand cases of food poisoning each year with thousands of hospitalizations, and dozens of deaths in the USA alone. Similarly exposure and illness rates might be assumed among other developed nations.

Speeding up O157 detection

Detecting O157 usually requires a specialist laboratory with access to polymerase chain reaction (PCR) for confirmation of the pathogen in a sample by genetic identification. Alternatively, the powerful technique called ELISA (enzyme-linked immunosorbent assays) can be used. Again, ELISA needs careful handling by trained staff and laborious sample pre-treatment. Both techniques can take hours or even days to confirm that a food sample is contaminated, which means many more people can become infected following an initial outbreak than would be necessary with a faster test.

Knoll and colleagues have now turned to long-range surface plasmon-enhanced fluorescence spectroscopy (LRSP-FS) to help them develop a rapid and simple to carry out test for O157. Until now, however, this and related techniques were sensitive to the pathogen at levels much higher than the infective limit. Researchers have previously tried to make the technique more sensitive by using nanoparticle assays, better sample preparation and more accurate sensors to amplify the signal and reduce the detection threshold to closer to the 50-100 cell limit.

The Austrian team believes it has surmounted the various obstacles. They embedded a thin metallic film in dielectrics of similar refractive index, which allows more intense resonant excitation to take place when a fluorophore-labelled molecule is captured on the metallic sensor surface and exposed to the electromagnetic field of long-range surface plasmons, made available by the specific sensor architecture the team has developed. The binding of the target molecule is displayed by the high intensity fluorescence. The approach allows the team to probe the entire volume of a trapped bacterial cell due to the evanescent field penetrating up to several micrometres from the sensor surface.

Glowing reference for one microbe

"The potential of the developed sensor platform is demonstrated in an experiment in which the detection of E. coli O157:H7 was carried out using sandwich immunoassays," the team explains. The limit of detection was below 10 cells per millilitre and detection time was a mere 40 minutes. This detection limit is three to four orders of magnitude lower than equivalent surface plasmon resonance techniques. Moreover, it is highly specific to the pathogenic strain, the team showed, as it does not respond to the closely related but non- pathogenic E. coli K12 strain.

"At the moment, we are looking for industrial partner to implement this technology into a portable device," Dostalek told SpectroscopyNOW.