# Technique Evolutions for Microorganism Detection in Complex Samples: A Review
## 3 Developments in Innovative Detection Methods
### Improvements and Developments in Analytical Methods Requiring Sampling
#### 3.2.2 Microfluidics  
Recent advances in the microfluidic field makes it the most promising technique and place it at the crossroads of sample-based and real-time techniques. The major limitation of microfluidics remains the low volumes processed, but due to other sorting techniques (acoustophoresis) or multiplexing, microfluidics can become a near real-time technique. It offers many possibilities adapted to our problem, which we can see in this section. Microfluidics is the science of fluid manipulation at the micrometer scale. This field of research is inspired by the nature that already mastered these techniques perfectly, such as blood capillaries or sap transport in plants. Microfluidics can also be considered as a technology for manufacturing \"lab-on-a-chip\" with innovative applications in the field of chemistry, biology [83], medicine [84], and the food industry [85]. Microfluidic systems include a set of miniaturized components that can replace bulky and very expensive instruments [86, 87]. This true \"microprocessor for biology\" represents a revolution for biology and chemistry similar to that brought by microprocessors to electronics and computers. Sorting by Acoustophoresis  
Combined with a classical detection method, microfluidics can be used as a first step to sort pathogens from the rest of the complex environment. This can greatly help the detection of the signal of pathogens that would be otherwise hidden by the strong signal of other components. Several microfluidic methods exist to separate bacteria from a sample [88, 89]; here we present the acoustophoresis method in more detail. By applying intense sound waves on micro channels, flowing particles are sorted according to their size because the acoustically induced velocity of a suspended object scales with the square of their radius. For example, small bacteria can be separated from the larger blood cells as  
Figure 3: Representation of paper sensors principle. In each microzone, DNAzyme sensors are printed. Then, biological samples are deposited into test zones. If a sample contains the target bacteria, the DNAzyme produces a fluorescent signal. This figure is a partial reproduction from reference [77], licensed under CC BY 4.0.  
depicted in Figure 4. Blood flows in the periphery of the channel and when the acoustic field is turned on, it applies a force on blood cells. They are thereby moved into the central stream, which then flows in a channel separated from the rest of the blood. On the contrary, small bacteria are much less affected by the acoustic field and stay in the peripheric streams. [PERSON] et al. successfully sorted a mixture of _E. coli_ and human blood cells using acoustophore-sis [80]. The resulting solution containing the bacteria showed a purity of more than 96% bacteria (less than 4% blood cells). This made the following electrochemical detection more sensitive as the non-specific signals due to blood cells were eliminated. As for most of microfluidic systems, their device has the advantages of being cost-effective, automated, and miniaturized. Those characteristics are well-suited for bacterial detection in industrial production. Similarly, [PERSON] et al. invented a device for acoustic separation of bacteria from blood combined with PCR detection for rapid sepsis diagnostics [90]. The acoustic separation of whole blood was performed as in Figure 4, then bacteria were trapped onto suspended polystyrene particles. Finally, a PCR was performed on the bacteria trapped in a polymer microchip containing dried PCR reagents. Tested with _Pseudomonas putida_, they showed a detection under two hours with an LOD of \(10^{3}\) bacteria/mL. Compared with blood culture on samples from septic patients, their system detected half of the septic cases, indicating the real need for further improvement to compete with the reference method. Consequently, they presented a new microfluidics design two years later [81] to increase the throughput of bacteria separation from whole blood.