The Measurement Of Salmonella Using Electrochemistry

Salmonella is a rod-shaped bacteria with a length of around 2 to 5 µm and a width of around 0.7 to 1.5 µm. It is one of the most problematic foodborne pathogens in the world. For example, the WHO estimated that of the 420,000 worldwide deaths from foodborne illnesses in 2010, around 60,000 came from non-typhoidal Salmonella.

The different variations within a species of bacteria are known as serotypes (or serovars). The serotype Salmonella typhi causes typhoid fever, while of the non-typhoidal forms of Salmonella, the most common are Salmonella typhimurium and Salmonella enteritids. In the developed world non-typhoidal salmonellosis usually causes diarrhea and abdominal pains, and healthy individuals usually recover. However, in sub-Saharan Africa outbreaks of non-typhoidal Salmonella are much more severe, which is in part due to a large proportion of the population having some degree of immune suppression for reasons such as HIV, malaria, and malnutrition. For example, in 2012, the non-typhoidal Salmonella outbreaks in sub-Saharan Africa had a mortality rate estimated at 20 – 25 %.

Most people become infected with Salmonella through eating food contaminated with feces. This can come about when raw meat and poultry is contaminated during the butchering process, seafood sourced from contaminated water, infected chickens laying contaminated eggs, and fresh fruit and vegetables being washed with unclean water.

Since foods contaminated by Salmonella generally look and smell normal, the monitoring of food samples is very important. This monitoring needs to be highly sensitive because salmonellosis can come from an extremely small infective dose (as low as 10 cells). The traditional method of detection is to pre-enrich the culture and then plate onto agar for identification. However, this can take 3 – 4 days for presumptive results and then 5 – 7 days for confirmation. Identification using DNA-based methods, such as the polymerase chain reaction, are much faster but require expensive equipment and reagents. Also, the analysis of foods can be problematic due to the presence of chemicals which inhibit the polymerase enzyme.

Electrochemical measurement can often be used to detect analytes at low concentrations, using relatively cheap equipment. A common electrochemical measurement principle (known as voltammetry) is that the oxidation or reduction of a chemical at an electrode will cause a flow of current, which is proportional to the chemical’s concentration. Electrochemical experiments are normally performed in a 3-electrode cell, where the voltage is controlled between a reference electrode and the electrode where the reaction of interest is taking place (known as the working electrode). The current flow is measured between this working and a counter electrode that is usually an inert metal.  Combining the reference and counter into a single electrode is also possible, and is often used in commercial devices for simplicity of construction.  

One way to detect Salmonella by electrochemical means is through an immunoassay. When an organism’s immune system encounters a foreign molecule (denoted an antigen) it produces a Y-shaped molecule called an antibody, which binds to the antigen as a means of removing it. Because this antibody-antigen binding is highly specific, it can be used as a means of analytical recognition. However, a problem with performing electrochemical immunoassays of food samples is that the sample matrix can often contain species which inhibit measurement by adsorbing to the electrode and thus making the electrode reaction inefficient. These species can include high concentrations of fats and proteins (in meat and dairy products), and polysaccharides and polyphenols (fruits and vegetables).

One way to circumvent the problem is to use immunomagnetic separation. In this technique, micron-sized magnetic beads are coated with antibodies selective to the analyte. Having been exposed to the sample matrix to allow antigen binding, the beads can then be magnetically collected. This allows us to remove the problematic sample matrix and resuspend the beads in a solution more amenable to electrochemical measurement. Using a solution of smaller volume will also have the effect of preconcentrating the analyte.

To detect Salmonella Typhimurium we recently constructed bi-functional magnetic beads, by also coating the beads with an electroactive compound. The organic molecule methylene blue can be reduced easily at an electrode, and also has the property of binding very strongly to carbon nanotubes. This is because of interaction between the benzene ring structure of the methylene blue and of the nanotubes themselves. These coated nanotubes were then adsorbed onto magnetic beads with a diameter of around 1 µm. Antibodies selective to Salmonella Typhimurium were then coated onto the beads by simple adsorption. From characterization experiments, we estimated that each magnetic particle delivered approximately 2 × 10⁸ molecules of methylene blue. This represented a relatively high level of electrical charge when reduced at the working electrode. It allowed us to measure Salmonella Typhimurium in milk at a fairly low concentration (17 Colony Forming Units per mL).

These findings are described in the article entitled Electrochemical Immunoassay for Salmonella Typhimurium Based on an Immuno-Magnetic Redox Label, published in the journal Electroanalysis. This work was co-supervised by Mithran Somasundrum from National Center for Genetic Engineering and Biotechnology, a part of the National Science and Technology Development Agency of Thailand.