# Technique Evolutions for Microorganism Detection in Complex Samples: A Review
## 3 Developments in Innovative Detection Methods
### Improvements and Developments in Real-Time and Online Analysis Techniques
#### 3.4.2 Surface Plasmon Resonance  
Surface Plasmon Resonance (SPR) has been extensively used in the past decade for optical biosensors [116; 117]. Its principle works as follows: when a beam of light illuminates an interface between two media, a portion of the incident light is reflected on the interface and the other portion of the light is refracted through the surface. If a thin layer of metal, rich in free electrons, is deposited at the interface, they come into resonance with the photons of the incident beam. This phenomenon is called Surface Plasmon Resonance. The resonance is visible through a loss of intensity in the reflected beam at a given angle. Any deposit on the surface can be detected because it modifies the angle at which the plasmon resonance occurs. In biosensors, this sensitive detection is used to detect the binding of species present in solutions to ligands grafted to the surface (Figure 9) [118; 119]. By grafting ligands specific to different types of pathogens on different positions on the surface, it is possible to perform a multiplex assay. This method is called SPR imaging (SPRi) as the surface is imaged to cover all ligand positions. The identification of the pathogens can be performed simultaneously to detection, depending on the specificity of the ligands used. The technology of an SPR biosensor has been successfully used for the detection of various analytes such as biomolecules [120] and proteins, bacterial toxins [121], or to monitor the formation of a bacterial biofilm and its degradation [122]. In the case of microorganisms, SPRi have been used for their detection in various media. [PERSON] el al. detected _E. coli_ in PBS and human urine in under 35 min for concentrations ranging from \(10^{3}\) to \(10^{9}\) CFU/mL and an LOD of \(10^{2}\) CFU/mL [123]. In food samples, [PERSON] et al. demonstrated the detection of _Salmonella_, _E. coli_ and _Listeria_ with a SPRi-based immunoassay. Their system had an LOD around \(10^{6}\) CFU/mL for direct measurement on a chicken carcass rinse or \(1\) CFU/mL with overnight enrichment [124]. [PERSON] et al. [125; 126; 127; 41; 128] proposed a method to make the detection of bacteria by SPRi as fast as possible where they no longer analyze a bacterial suspension after a growth step. The suspension to be tested is injected directly in contact with the microarray. The possible growth of the bacteria and their detection is thus performed at the same time (Figure 10). The detection limits claimed for standard SPRi in sample-based detection are  
Figure 9: Schematic representation of SPRi. analytes flowing in the channel bind to ligands grafted on a gold surface. This interaction is detected by a change in the intensity of the light reflected on the other side of the gold surface. often less relevant. Thus, by combining the growth of microorganisms and their detection, the time saving is real. This technique has similarities with blood cultures with comparable analysis times. The advantage here is real-time monitoring, whereas blood culture bottles provide a positive response without allowing quantification. In this method, the analysis time is governed by the growth rate of the microorganism; the faster the growth, the sooner the detection threshold of the sensor can be reached. (Figure 10) The method has been validated on various strains of _Salmonella_, _Listeria_, _Staphylococcus_, and _Escherichia_. Figure 10: Principle of the method “Culture-Capture-Measurement” for the detection of bacteria by SPRi. The bacteria initially present in small numbers in the sample multiply and end up interacting with the AMPs placed on the surface provided that some have an affinity for them. These interactions are visualized either on the differential images where a difference of color intensity with respect to the initial levels is carried out, or on the kinetics of variation in reflectivity (\(\Delta\)R) as a function of time. SPRi is suitable for bacteria detection in whole blood or cellular products.