Plasmonic SERS Biosensor for Rapid and Accurate Bacterial Identification

In 2019, an estimated 7.7 million people died from bacterial infections, accounting for 13.6% of all global deaths. This means that more than 1 in 8 deaths were due to bacterial infections, making it the second leading cause of death worldwide. Deaths from pathogenic bacterial infections are predicted to reach 10 million per year by 2050 due to the emergence of superbugs. Identifying the correct type of bacteria is critical for proper prevention and treatment.<br/>Bacteria are categorized into cocci, bacillus, vibrio, spirilla, and spirochaetes based on their shape. While most bacillus (rod-shaped) bacteria are harmless, some, like Bacillus anthracis (causing anthrax) and Bacillus cereus (causing food poisoning), are deadly to humans. Identifying Bacillus species accurately is challenging due to their structural and genetic similarities (99.2-99.6%). PCR analysis is the industry standard, but it is accurate yet time-consuming. Rapid and accurate identification is crucial for high-risk pathogens, prompting the need for a faster analysis method.<br/>Surface-enhanced Raman spectroscopy (SERS) is emerging as an alternative to PCR analysis for the detection of biomaterials. SERS is a rapid, highly sensitive, accurate, and selective identification of extremely trace amounts of analytes by significantly enhancing the Raman signal from plasmon resonance on metal nanosurfaces. However, because the SERS effect is strongest at the metal nanosurface and decreases with distance, SERS-based measurements of bacteria can only utilize information about limited outer regions such as the cell wall, cell membrane, and membrane proteins. Therefore, SERS is not known to be a suitable method for the analysis of relatively large biomaterials such as cells or bacteria.<br/>In this study, we propose a bacterial pretreatment process to apply the high-potential SERS technique to bacterial identification and analyze the obtained Raman spectra to discriminate between structurally and genetically similar bacteria. The proposed pretreatment process suitable for SERS is a hybrid lysis process consisting of a mechanical lysis process and a chemical lysis process.<br/>Bacillus, Gram-positive bacteria, have a relatively thick cell wall layer, so mechanical lysis is primarily used to reduce the size of the macromolecule bacteria while releasing intracellular components. Alkaline lysis is used to further reduce the size of the analyte, including the crushed cell wall and intracellular components, and in particular, to denature the released DNA into single strands, making it suitable for SERS analysis.<br/>In parallel, for Raman signal enhancement, we fabricated a 20 nm three-dimensional nanowire network using nanotransfer printing technology to fabricate nanodevices with ultra-high density hotspot regions where electromagnetic fields are enhanced. The presence of numerous factors in the pretreated bacterial samples, including small crushed cell walls and intracellular components, which can be located in the ultra-high density hotspot regions formed on the fabricated nanodevices, resulted in new signal peak shapes or enhanced signal results.<br/>Finally, principal component analysis, which is used to analyze high-dimensional data, was performed to verify that discrimination of five structurally and genetically similar Bacillus bacteria was possible, confirming its applicability as a next-generation biosensor.<br/>The proposed mechanical/chemical hybrid lysis process, which is an appropriate pretreatment process for SERS analysis of macromolecules, is applicable to large biological materials such as cells as well as bacteria. In addition, as a rapid analysis method with high accuracy, it can be applied beyond the public health industry to the high-tech bioindustry, and high-tech defense industry and is expected to be used in the medical field, such as early diagnosis of diseases.