Optical methods for ultrafast screening of microorganisms

Resumo

Infectious diseases are currently responsible for over eight million deaths per year. Efficient treatments require accurate recognition of pathogens at low concentrations, which in the case of blood infection (septicemia) can go as low as one per milliliter. However, detecting and identifying bacteria in such low concentrations is challenging and typically requires costly techniques and lengthy cultures of large samples of blood (~1 milliliter) extending over 24-72 hours. This delay seriously compromises the health of patients and is largely responsible for the death toll of bacterial infections. Recent advances in nanoscience, spectroscopy, plasmonics, and microfluidics allow for the development of optical devices capable of monitoring minute amounts of analytes in liquid samples. Therefore, this doctoral thesis intended to develop and optimize a method for multiplex detection and quantification of the most common microorganisms causing bacterial infections. This detection approach envisions to directly use the biological matrix of the microorganisms avoiding pre-treatments of the samples with an unprecedented speed, low cost, and sensitivity. To demonstrate this concept, four different bacterial strains with high incidence rates in hospitals were selected. Specifically, two gram positive strains that are Streptococcus agalactiae and Staphylococcus aureus and other two gram negative strains, Escherichia coli and Pseudomonas aureginosa. The design of the system is based on variations in the SERS intensity. This is accomplished using encoded plasmonic nanoparticles functionalized with bio-recognition elements. Consequently, when a sample (biological fluid) containing the biological target to be identified interacts with the recognition elements attached to the nanoparticle, will induce an accumulation of them at the surface of the targeted microorganism. Therefore, each type of bacteria is driven to gather silver nanoparticles labeled with specific Raman-active molecules. This particle aggregation on the bacteria membranes renders a dense array of inter-particle gaps in which the Raman signal is amplified by several orders of magnitude relative to the dispersed particles, enabling a multiplexed deterministic identification of the microorganisms through the molecule-specific spectral fingerprints. Quantification is achieved by passing the sample through a microfluidic device with a collection window where a laser interrogates and classifies each of the induced bacteria–nanoparticle aggregates in real time. Moreover, to study the viability of the system in other biological fluids rather than blood, tree extra different real human fluids were used: urine, pleural fluid, and ascites fluid. To perform this study, Staphylococcus aureus was chosen because is a common cause of serious infections. Additionally, an extensive comparison between two of the most common bio-recognition elements (antibodies and aptamers) was performed. Determining that use of aptamers has several advantages. This new pathogen detection system opens exciting prospects for fast inexpensive diagnosis of bacterial infections.