# Technique Evolutions for Microorganism Detection in Complex Samples: A Review
## 3 Developments in Innovative Detection Methods
### Ligands for Classical Detection Techniques Improvement
#### 3.1.1 Broad-Spectrum Ligand  
The choice of the appropriate ligand is an important issue in detection methods [40]. A multitude of ligands seems potentially useful for the detection of bacteria, such as phage, aptamers, anti-microbial peptides, or antibodies (Figure 1). Ligands can be used for both detection and identification. For the subject of this review, the goal is to detect the presence or absence of any kind of bacteria. This is enough in some situations, such as monitoring the sterility of a therapeutic product or an industrial ATMP bioreactor. A ligand is usually very specific to a bacterial species. As a result, general detection requires numerous ligands at the risk of increasing the analysis time and cost. A ligand that can bind to several bacterial species or genera is thus more relevant for detection and appears as an objective to achieve. A search for ligands with a broader recognition spectrum was therefore undertaken. Their role would be to interact with several potential microorganisms responsible for the contamination of samples and to allow the detection of any bacterial species. This is however challenging because there is a trade-off between the affinity of the ligand, and its ability to bind to a broad spectrum of bacteria. Antibodies are the more widely used ligands and can be good candidates. Antibodies form immune complexes with antigens. The long-standing use of antibodies provides an extended literature on their engineering and use for bacterial detection. Moreover, a large variety of antibodies recognizing a wide range of antigens is commercially available. However, the specificity of the antibodies makes it difficult to detect a large number of different bacterial species simultaneously. Although some antibodies are capable of recognizing conserved epitopes in many bacteria, in practice, due to bacterial proteolytic capabilities, these antibodies have not proven conclusive for broad-spectrum detection [40]. Bacteriophages, or phages, are also interesting ligands. Phages are viruses that specifically infect bacteria. They are abundant on Earth and are involved in the regulation of  
Figure 1: Schematic representation of the binding of bacteria to a surface that can be coated (or functionalized) with different molecules acting as ligands such as antibodies, bacteriophages, antimicrobial peptides (AMPs), or DNAzyme. bacterial species. Using a phage as a ligand makes it possible to detect, fix, and eliminate bacteria. The recognition of bacteria by bacteriophages is performed via binding proteins with strong affinities to structures on the surface of the bacterial host. Modifications of these proteins can allow biotechnological adaptation to specific detection requirements [40; 41]. Whole bacteriophages or fragments of phage proteins can be grafted onto biosensing surfaces. In addition, the size of whole bacteriophages limits the sensitivity of some detection methods. While there is a growing interest in phages as ligands [22; 42], it is mainly used for identification application as the binding is very specific to the species of the bacteria. Enzymes are another example of molecules used for recognition. They are catalytic proteins that facilitate certain chemical reactions. They can be used for detection, taking advantage of their highly specific affinity for their substrates. In the case of pathogens, enzymes directed to specific proteins have been used for detection [43]. Upon recognition of the target, enzyme activity can be detected by optical (fluorescence, colorimetry) or electrochemical means. Redox enzymes have been widely used in biosensors as their redox activity can be monitored relatively easily with electrodes [44]. The specificity panel of the different enzymes makes them suitable for detection of broad-spectrum detection (esterases) or of single species of bacteria (bacterial enzyme considered as one of their virulent factor), and in specific cases for identification.