The enzyme-linked immunospot (ELISpot) is a highly sensitive immunoassay that measures the frequency of cytokine-secreting cells at the single-cell level. The secreted molecules are detected by using a detection antibody system similar to that used in the enzyme-linked immunosorbent assay (ELISA). The ELISpot assay is carried out in a 96-well plate and an automated ELISpot reader is used for analysis. The assay is easy to perform, robust and allows rapid analysis of a large number of samples and is not limited to measurement of cytokines; it is suitable for almost any secreted protein where single-cell analysis is of interest.

The ability to detect, identify and quantify bacteria is crucial in clinical diagnostics, environmental testing, food security settings and in microbiology research. Recently, the threat of multidrug-resistant bacterial pathogens pushed the global scientific community to develop fast, reliable, specific and affordable methods to detect bacterial species. The use of synthetically modified enzyme substrates is a convenient approach to detect bacteria in a specific, economic and rapid manner. The method is based on the use of specific enzyme substrates for a given bacterial marker enzyme, conjugated to a signalogenic moiety. Following enzymatic reaction, the signalophor is released from the synthetic substrate, generating a specific and measurable signal. Several types of signalophors have been described and are defined by the type of signal they generate, such as chromogenic, fluorogenic, luminogenic, electrogenic and redox. Signalophors are further subdivided into groups based on their solubility in water, which is key in defining their application on solid or liquid media for bacterial culturing. This comprehensive review describes synthetic enzyme substrates and their applications for bacterial detection, showing their mechanism of action and their synthetic routes.

The rapid detection of Listeria monocytogenes contamination in food is essential to prevent food-borne illness in humans. The aim of this study was to differentiate non-contaminated milk from milk contaminated with L. monocytogenes using enzyme substrates coupled with the analysis of volatile organic compounds (VOCs). The method is based on the activity of β-glucosidase and hippuricase enzymes and the detection of a specific VOC i.e. 2-nitrophenol and 3-fluoroaniline, respectively. VOCs were extracted, separated and detected by headspace-solid phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME GC-MS). This approach required the inclusion of the selective agent\'s cycloheximide, nalidixic acid and acriflavine HCl in the growth medium to inhibit interfering bacteria. The VOCs were liberated by L. monocytogenes provided that samples contained at least 1-1.5×10(2) CFU ml(-1) of milk prior to overnight incubation. This approach shows potential for future development as a rapid method for the detection of L. monocytogenes contaminated milk.