[Surface-Enhanced Raman Scattering to Improve the Sensitivity of the MTT Assay].

Abstract

Currently, empirical therapy regimens are often used in the treatment of infectious diseases that are not based on data on pathogen resistance. One of the main reasons for the unjustified prescription of antibacterial drugs is the lack of rapid and at the same time universal methods of determining the antibiotic resistance of the pathogen. The most widely used culture techniques, such as the microdilution method, require a long time to generate the necessary number of bacterial cells. Less time-consuming methods of resistance assessment (genomic or proteomic) are based on the determination of specific markers (resistance genes, overexpression of certain proteins, etc.); in this case, the specific protocol is most often applicable to a narrow number of both microorganism strains and antibiotics. Previously, we demonstrated the possibility of using Raman spectroscopy (RS) technology for quantitative determination of the product of bacterial cell activity in the MTT assay, formazan, directly in the cell suspension. The absence of the formazan isolation step simplifies the assay and increases its accuracy. The analysis time did not exceed 2 h while maintaining the versatility of the MTT assay itself. Limitations of the developed protocol for RS detection of the MTT assay results include a high sensitivity threshold of 10^(7) CFU/mL for the bacterial cell concentration, so a preliminary stage of cultivation is necessary for samples with a low bacterial content. Here, we propose a method to increase the sensitivity of formazan determination by utilizing the effect of surface-enhanced Raman scattering (SERS) on gold nanoparticles. As a result of this study, the optimal conditions for SERS analysis of formazan in both solution and suspension of Escherichia coli cells are selected. Formazan signal amplification due to the use of SERS on gold nanoparticles instead of RS allowed us to reduce the sensitivity threshold for the number of bacterial cells in the sample by at least 30 times, up to 3 x 10^(5) CFU/mL. This sensitivity is not the limit of the SERS technology capabilities because the introduction of other types of nanoparticles (more optimal in shape, size, concentration, etc.) into the experiment will allow one to achieve even higher signal amplification.