Development of a portable magnetic flow cytometer for the detection of hospital bacterial infections

Abstract

The rising incidence of infectious diseases, increased food and water safety concerns, and the new government initiatives and funding to detect and control antimicrobial-resistant species are demanding breakthrough advancements in bioanalytical tools for pathogen detection. Moreover, the clinical prevalence of Pseudomonas aeruginosa, and Klebsiel/a pneumoniae pathogens underscores the importance of rapid detection approaches to contain nosocomial infections and reduce the development of drug resistance. Current microbiological detection systems often lack precision, affordability, and portability. Furthermore, they require long detection times, sophisticated infrastructures, and trained personnel, which prevent their applicability at resource-limited settings.

In this thesis work, some of these challenges were addressed by developing a magnetic flow cytometer technology. Flow cytometers have become essential instruments in biomedical research and routine clinical tests for disease diagnosis , prognosis, and treatment monitoring. Magnetic flow cytometers ally the potential for cell detection and counting to the advantages of a lab-on-a-chip technology, pushing forward the progress in integrated and compact systems for On-site testing. In particul ar, magnetoresistive sensors (MR) and magnetic labelling processes were exploited, promoting high capture efficiencies of magnetically labelled targets and generating strong magnetic fields .

The MR chip was integrated with a microfluidic system with a small heigh dimension to improve the system sensitivity by promoting the labeled targets to be closer to the sensing units. Chip design optimizations regarding the maximization of the sensor sensitivity and reduction of electromagnetic interferences, as well as improving the processing signal analysis and boosting multiplex capabilities were perfo rmed .

Protocols for the functionalization of bacteriophages and antibodies onto the magnetic nanoparticles surfaces were optimized and validated for the magnetic labelling of bacterial cells (K pneumoniae and P aeruginosa) spiked in buffer solution, as well as, directly from clinical rectal swa bs. The results showed capture efficiencies over 70 % with specificity.

The whole system was integrated with an electronic acquisition setup for the sensors addressing and read-out in collaboration with INESC ID. A signal classification

method based on simulations was proposed to aid in the identification of the number of magnetically labelled bacteria associated with each detection event, as well as in the ability to distinguish signatures of free or clustered magnetic particles from magnetically labelled targets.

This platform was tested and validated for the detection of K/ebsie//a pneumonia in laboratory samples as well as a pilot study was conducted with clinical rectal swab samples collected from 45 patients admitted to the emergency department of Hospital Beatriz Angelo (Loures, Portugal).

Overall, this work mostly outlies relevant aspects concerning biosensor development, demonstrating how magnetic flow cytometry can be explored as an accurate, sensitive, and portable device that can be applied for bacteria detection and identification.