# Freshness Monitoring of Packaged Vegetables
## 4 Intelligent Packaging (IP)
### Commercial Biosensors  
Toxin Alert has produced a visual biosensor known as Toxin Guard(tm) [104; 105]. This biosensor immunoassay uses the concept of antigen/antigen-specific antibodies to detect pathogens in the form of antibodies sandwich with thickness of 100 \(\upmu\)m [106]. There are two types of antibodies used: the capture antibodies and the detector antibodies. Capture antibodies are immobilized and incorporated into a thin layer of polymer plastic films such as polyethylene and are patterned in the form of icons in a nutrient gel such as agarose gel [106]. The detector antibodies are labeled with colorimetric enzymes or luminescent chromophore dyes that will change color upon binding to antigens and are free to move in the nutrient gel layer. Then, a permeable protector gel coat is applied forming compartments that act as test sites and allow for specific pathogens to penetrate. Detector ligands will bind to pathogen antigens, and the nutritious gel will support the rapid growth of penetrated pathogens. The detector antibodies binding to the antigens of pathogens will then migrate toward the captured ligand due to affinity, leading to a distinctive pattern shapes where the chromophores are concentrated [106]. The response time is 30 min to show different patterns and up to 72 h to give a distinct dark color change [106]. The advantages of this biosensor lies in its ability to simultaneously detect and identify several bacterial types such as: _E. coli_, _E. coli O157:H7_, _Salmonella_, and _Listeria_, its stability and suitability for warm and cold environments, and its shelf life of one year [104; 105; 106; 107]. \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Transducer**} & \multirow{2}{*}{**Analytic**} & **Limit of Detection** & **Response** & \multirow{2}{*}{**Reference**} \\  & & **(CFU/mL)** & **Time** & \\ \hline \multirow{4}{*}{Amperometric} & \multirow{4}{*}{_E. coli O157:H7_} & 77 \(\pm\) 0.4 & 6 min & [80] \\  & & 7.8 \(\times 10^{1}\) & 10 min & [81] \\  & & 6 \(\times 10^{2}\) cells/mL & 2 h & [98] \\ \multirow{2}{*}{Potentiometric} & _E. coli_ & 10 cells/mL & 1.5 h & [99] \\  & _E. coli O157:H7_ & 7.9 \(\pm\) 0.3 \(\times 10^{1}\) & 10 min & [100] \\ \multirow{2}{*}{Conductometric} & _E. coli_ & 7.5 \(\pm\) 0.3 \(\times 10^{1}\) & 10 min & [100] \\  & _Salmonella_ spp. & 8.3 \(\pm\) 0.6 \(\times 10^{1}\) & 10 min & [100] \\ \multirow{2}{*}{Impedimetric} & _Bacillus cereus_ & 35–88 & 6 min & [101] \\  & _E. coli_ & 10\({}^{1}\)–10\({}^{7}\) & - & [102] \\ \multirow{2}{*}{Impedimetric} & _L. monocytogenes_ & 4.1 pg/mL & 30 min & [103] \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Electrochemical foodborne pathogen detection. However, small amounts of micro-organisms that may still cause diseases are not captured due to sensitivity limitations [104; 105; 107]. Figure 10 shows a picture of Toxin Guard biosensors. Recently, a new technology known as Janus emulsion droplets was developed for the detection of bacteria using _E. coli_ as the model system for testing [108; 109]. This technique, based on Janus particles, shows a promise to be part of vegetable packaging as pathogens detectors. Janus particles have been used for many sensing applications such as in biomedicine and highly selective sensors [87; 110]. These nanoparticles have different hemispheres with distinct physical and biochemical properties. The surface nature of these particles is like that of the living cells which enabled their use as _E. coli_ detector. The droplets are prepared with the use of surface-active agents that are made of carbohydrates and their optical properties (allow light to pass) change because of pathogen recognition through carbohydrate-lectin interactions.