# Intelligent Packaging for Real-Time Monitoring of Food-Quality: Current and Future Developments
## 3 Methods for Real-Time Food Monitoring
### Biosensors for Bacteria Detection  
Among others, bacteria are one of the primary food contaminants associated with disease and death [152]. The growth of microorganisms in food may affect product quality in two different ways. On the one hand, non-pathogenic microorganisms normally present in the food supply chain can induce food spoilage and determine a quality issue. On the other hand, pathogens deriving from contamination at a certain point of the supply chain represent a serious health risk [5; 152; 153]. With these premises, it is not surprising that bacteria are an extremely important target in food safety monitoring. However, commonly employed approaches make use of expensive, time-consuming, and sophisticated analytical techniques that are not suitable for large-scale applications [153; 154; 155; 156]. Accessibility problems have been solved via devices that do not require significant user intervention and that are not destructive [157]. For instance, the presence of microorganisms in various food packages has been detected via lateral-flow strip (LFTS) assays [158; 159; 160], in which gold colloidal nanoparticles labeled with antibodies directed against specific microorganisms are deposited on a chromatographic test strip as schematically shown in Figure 5a. A sample containing the bacterium is then inserted at one side of the strip via a proper solution. The solution runs along the strip until it encounters the functionalized particles with the specific antibodies that interact with the bacteria. In the test zone, a detection line bringing receptors for the bacterium-gold complex interacts with the latter and induces particle accumulation that gives rise to a colored line. At the same time, the essay also has a control line aimed at assessing the chromatographic process. This line brings a receptor that interacts with a second group of gold nanoparticles that are opportunely functionalized. Another type of bacteria sensor has been fabricated by using portable microfluidic devices containing specific antibodies, which are able to respond to different bacteria and can be integrated with sensor chips [161; 162; 163; 164]. For example, Figure 5b schematizes the working principle of a completely automated microfluidic-based electrochemical biosensor  
Figure 4: (**a**) Critical temperature sensing system based on an enzymatic reaction and capable of indicating if a product has been compromised. (**b**) Critical time–temperature sensing system that indicates how long a breach of the threshold temperature has occurred. Adapted with permission from [7]. Copyright © 2019 American Chemical Society. able to detect _E. coli_ bacteria with high sensitivity (i.e., detection limit of 50 cfu/mL) and specificity [164]. Specifically, such a biosensor relies on the enzymatic reaction between horseradish peroxidase (HRP) labeled _E. coli_ antibodies and 3,3\({}^{\prime}\),5,5\({}^{\prime}\)-tetramethylbenzidine (TMB). Additionally, the detection of bacteria for intelligent food packaging applications via graphene flexible sheets operating as electrodes [165], via the aggregation of paramagnetic silica beads [166], or via laser speckle decorrelation (Figure 5c) [167, 168] has been demonstrated. Despite these sensor systems allowing for the real-time monitoring of microorganisms in packages and the fact that they may be suitable for large-scale usage, it is noteworthy that they still require sophisticated instruments. To tackle the issues related to complex detection techniques or lack of on-site monitoring possibilities, photonic crystal-based sensors have started to assume a major role in microorganism detection for food packaging [170, 171]. Figure 6a,b reports a photograph and a schematic of a silica-titania multi-layered photonic crystal covered by a plasmonic Ag film. The system can selectively interact with the _E. coli_ bacterium modifying its transmittance spectrum (Figure 6c) [172, 173].