Biofunctional nanoparticles for magnetic immunoassaysapplication to the detection of pneumolysin for the rapid diagnosis of pneumococcal pneumonia.

Abstract

Humanity faces news problems as it evolves: overpopulation and aging, globalization, global warming, and pollution put administrations in check almost every day. Today, cancer is the disease that causes the most deaths, a virus spreads from one region of the world to its opposite in the blink of an eye antibiotic-resistant bacteria do not stop growing, and there is a high risk of contamination of our food chain by pharmaceutical or agricultural wastes. To address these problems quickly and effectively, detection tools that allow the obtention in situ of reliable results easily and in the shortest possible time are needed. These devices, called point- of-care, do not depend on sophisticated equipment or qualified personnel, being very useful, especially in remote areas and developing countries. Currently, lateral flow immunoassays are the most widely used point-of-care tests. The home pregnancy test or the rapid antigen tests used during the COVID-19 pandemic are the most relevant examples. However, some improvements such as increasing its sensitivity or the possibility of quantifying the analyte would allow a more extensive use. The use of magnetic nanoparticles as detection labels would allow both purposes. This thesis’ general objective is to study and characterize magnetic nanoparticles for their application in lateral flow immunoassays that allow the detection and quantification of biomolecules of interest, using an inductive sensor and optimizing their sensitivity. The biomedical applications of magnetic nanoparticles are multiple. The first chapter describes magnetite nanoparticles with a double layer of three different fatty acids: Their physicochemical, structural, and magnetic characterization, and performance in nuclear magnetic resonance imaging, magnetic hyperthermia, and biosensing tests are shown. The results showed that such nanoparticles (1) increase the contrast between the tissues, exceeding the commercial reference agent; (2) they have an excellent heating capacity; and (3) they could be used as labels in quantitative assays using the biotin-neutravidin affinity model. The second chapter describes the search for optimal properties of magnetite nanoparticles in inductive biosensing. Different samples of magnetite were synthesized by thermal decomposition and characterized structurally, physiochemically, and magnetically as well as in the inductive sensor. In this way, the initial magnetic susceptibility was determined as an essential parameter for good detection within the superparamagnetic threshold. The particles’ response in flow tests was also studied using the biotin-neutravidin model, verifying that both the initial aggregates or the ones produced during the bioconjugation process increase the magnetic mass per unit of the biomolecule. This aggregation amplifies the signal. The last two chapters describes how magnetic clusters with the optimal characteristics were used in two applications of real interest: Detecting antibodies generated by SARS-CoV-2 and quantifying pneumolysin. The latter is a protein that indicates pneumococcal pneumonia when detected in the urine. Pneumococcal pneumonia diagnosis currently involves taking challenging and invasive samples, which are not decisive, and often resolved with indiscriminate antibiotic prescriptions. The test developed is a helpful tool for detecting pneumolysin. It also takes advantage of the magnetic properties of nanoparticle clusters to pre-concentrate samples whose concentrations are outside the detection limit and increase the test signal thanks to magnetic relocation. Finally, SARS-CoV-2 antibodies were also detected at concentrations of clinical interest. Thus, they might monitor the population’s immune response during infection and after vaccination.