# Technique Evolutions for Microorganism Detection in Complex Samples: A Review
## 3 Developments in Innovative Detection Methods
### Development of Physical and Computer Analysis Methods  
#### 3.3.1 Raman Spectroscopy  
Among instrumental analytical methods, vibrational spectroscopy techniques have long been established; they allow the identification and quantification of the chemical composition of samples in a non-destructive manner. Raman spectroscopy exploits the physical phenomenon in which molecular vibrations in a sample are excited by incoming monochromatic light. This energy transfer results in a small shift in the frequency of the subsequent scattered light. Consequently, measuring the spectrum of the Raman scattered light provides molecular information about the sample. The preparation time is zero or almost zero and samples can be of different types: solid, liquid, or gaseous. Raman spectroscopy offers the possibility of in situ measurements. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Specifications** & **IC 3D System** & **Blood Culture** \\ \hline
**Specimen types** & Diluted blood & Blood \\
**Sample volume** & Microliters to milliliters & Milliliters \\
**Culture enrichment** & No & Yes \\
**Time to results** & \textless{}90 min, yes or no & 10 h–20 h \\
**Limit of detection (CFU mL\({}^{-1}\))** & 1–10 & \textless{}100 \\
**Selective** & Yes & No \\
**Quantitative** & Yes & No \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Major specifications of the IC 3D test in comparison with blood culture for bacteria detection. Compilation adapted from [58], licensed under CC BY 4.0. Figure 5: Schematic description of the IC 3D technology. This figure is a reproduction from reference [58], licensed under CC BY 4.0. (**a**) DNAzyme and blood samples are mixed and then encapsulated in microdroplets. DNAzyme sensors produce a fluorescent signal in the droplets with bacteria. (**b**) The 3D particle counter scans several milliliters worth of droplets within minutes and detects precisely single-fluorescent droplets. In the study of bacteria, Raman spectroscopy is particularly well-suited for identification. Multiple bacteria can be identified simultaneously from the Raman spectrum of the analyzed sample by comparison with reference spectra of library [96] (see Figure 6). This point is similar to what exists for infrared spectroscopy. For example, [PERSON] et al. showed that 16 different species of _Staphylococcus_ can be distinguished using Raman spectroscopy [97]. Although many studies deal with the identification of bacteria, we focused here on their detection, in accordance with the scope of this review. However, one can keep in mind that Raman spectroscopy offers the possibility to perform both at the same time. Combined with microscopy, Raman spectroscopy allows the detection of a single cell [98]. This avoids the time-consuming step of incubation, and thus increases greatly the rapidity of the analysis. This is similar to solid phase cytometry, but the Raman spectrum allows for a more specific detection. In particular, auto-fluorescent particles can no longer be confused with pathogens. One drawback of the high resolution of microscopy is the time that is required to scan the sample looking for bacteria. [PERSON] et al. [99] overcame this issue by using lens-free imaging instead of a classical microscope, providing a wide field of view. Moreover, lens-free imaging is cheaper and allows for more compact systems. With such a setup, they could localize and detect single bacteria under 1 min, though the concentration of bacteria in the solution, after drying, was high (\(10^{8}\) CFU/mL). For bacterial detection applications, other approaches used Surface-Enhanced Raman Spectroscopy (SERS) to enhance the spontaneous Raman signal, which is normally of low intensity [101]. For example, [PERSON] et al. combined an aptamer, a DNA walker on magnetic nanoparticles, and SERS detection to reach an LOD as low as 4 CFU/mL for _S. typhimurium_[102]. This limit was made possible both by SERS and the enzymatic augmentation performed by the DNA walker. However, the method did not seem specific at this concentration.