Low-cost point-of-care biosensor device for clinical diagnosis in developing countries

Patricia Ramirez Priego

Prof. Laura M. Lechuga

Tuberculosis (TB) is an infectious disease caused by the microorganism Mycobacterium tuberculosis. It is estimated that 10 million people worldwide fell ill with TB in 2020 (approximately 90% in developing countries), and the numbers are expected to worsen in 2020 and 2021. TB can be prevented, treated and cured, but it is vital that it is diagnosed as early as possible to prevent the spread of the pandemic. Currently, TB diagnostic techniques exhibit several difficulties: they must be carried out in centralized laboratories, using bulky equipment, complex reagents and qualified personnel; all of which increases the costs and time to obtain the results. For this reason, for some years now, much effort has been devoted to developing analytical techniques and platforms for the diagnosis of TB that are fast, efficient and reliable. With this aim, in this thesis an innovative portable biosensor for the rapid diagnosis of TB directly in urine samples has been fully developed. The new photonic biosensor is based on a highly sensitive interferometric transducer incorporated into a disposable microfluidic cartridge. The elements necessary for light coupling and optical reading are integrated in a prototype, which allows real-time monitoring, data processing and control of fluid injections into the sensor cartridge. The results obtained in this thesis have demonstrated the application of the innovative portable and integrated biosensor for the detection of a lipopolysaccharide present in the bacterial cell wall, lipoarabinomannan (LAM), directly in urine samples and in less than 15 minutes. This detection has been done directly and without any labeling or amplification, which greatly simplifies the process of obtaining results. Furthermore, thanks to the samples provided by several hospitals in Tanzania, the biosensor platform has been validated with real patient samples. Specifically, it has been possible to discriminate between TB patients and healthy patients directly in the urine and in 15 minutes regardless of HIV/AIDS coinfection, overcoming one of the main limitations of the rapid tests currently available. Additionally, these samples were analyzed with the diagnostic methods recommended by the World Health Organization (WHO), achieving excellent correlation and demonstrating that the technology is reliable and competitive. These results, validated in larger studies, could have important clinical implications, considering the added advantages of the innovative biosensor compared to the fastest method recommended by the WHO. Furthermore, it becomes an exceptional candidate for on-site use in primary care clinics or hospitals in developing countries. Additionally, the design of the portable biosensor allows the simultaneous detection of a panel of six different biomarkers from a single sample, opening the door to the use of the innovative biosensor for the rapid detection of different biomarkers or diseases.

The results achieved in this Doctoral Thesis have served to demonstrate the application potential of the innovative biosensor platform in the detection of biomarkers related to M. tuberculosis (the microorganism that causes TB) in less than 15 minutes directly in urine samples. One of the main challenges posed by the project was to fill the gap between the tests current high-end, sensitive but expensive, and the most economical and affordable tests, but less sensitive, especially for developing countries. Specifically, in the thesis the detection of four proteins together with a ipopolysaccharide has been studied commonly present in the urine of patients with active TB. It was decided to do so carry out a direct and individualized detection of each of the biomarkers selected in urine samples, that is to say, without the use of fluorescent labels or chromophores Among all the analyzed biomarkers, the direct detection of lipopolysaccharide lipoarabinomannano (LAM) has been the strategy that has offered more successful results and without There is no doubt that it has great potential to be applied directly in the clinical field. Itsindividualized detection, using designed monoclonal antibodies exclusively for the project, has shown that limits can be reached detection of 475 pg/mL directly in urine samples. Also, thanks to the collaboration of LIONEX Diagnostics & Therapeutics GmbH, different real patient samples have been analyzed. Specifically, the POC biosensor has been capable of discriminating the samples of patients with active TB from those of healthy patients regardless of co-infection by HIV/AIDS directly in their urine patients and in 15 minutes, something that is not possible with the rapid tests available ommercially for the detection of LAM in urine. Likewise, these samples were analyzed with the diagnostic methods recommended by the WHO, reaching one excellent correlation In summary, the results obtained have shown that the combination of the POC biosensor together with biochemical analysis methodologies developed in the thesis provide a fast, effective and reliable test that can be used as a diagnostic tool. In addition, given the degree of integration and portability that with the POC biosensor, this methodology can be carried out without the need for large infrastructures or highly qualified personnel. All these advantages associated with us POC biosensor make it an excellent candidate for in-situ use in controls routine in developing countries. On the other hand, it should also be noted that thanks to the simple and quick detection (15 minutes) of patients with Active tuberculosis, treatments could be started almost immediately. this would favor a slowdown in the transmission and expansion of TB worldwide, and finally, it would mean an increase in the survival rate. In view of the promising results obtained for the individual detection of the selected biomarkers and in order to take advantage of the design of the POC biosensor, it has carried out a proof of concept to corroborate the potential of the technology in the simultaneous detection of a panel of different biomarkers from the same sample of urine For this, it was decided to deposit two different bioreceptors in an orderly manner on the surface of sensado using automated dispensing technologies olumes, which are able to distribute tiny amounts (nL) of solutions controlled way Although in this thesis only the multiple analysis with two has been worked on bioreceptors; in the future, thanks to the use of these technologies, it will be possible to distribute up to six different biomarkers along the sensor surface, implying an improvement significant in the sensitivity and specificity of current diagnostic techniques. In the same way, it is relevant to add that when dispensing the bioreceptors in a localized way on the surface of nanosensado, bioreceptors related to others could be immobilized infectious agents or other relevant biomarkers for other diseases, thus opening the doors of this platform to an infinity of applications. We also note that, from the design stage of the biosensor, the application was considered of the POC biosensor so that it was compatible with single-use cartridges they will include sensor surfaces previously functionalized with specific bioreceptors for each disease or clinical condition. This would mean that the centers where they would perform the tests they could have the portable diagnostic platform (the biosensor POC) and buy different cartridges aimed at different diseases.

Tuberculosis (TB); Infectious Disease; Mycobacterium tuberculosis; Global Health; Developing Countries; Prevention; Treatment; Cure; Early Diagnosis; Pandemic Spread; TB Diagnostic Techniques; Centralized Laboratories; Bulky Equipment; Complex Reagents; Qualified Personnel; Costs; Time to Results; Fast Analytical Techniques; Efficient Analytical Techniques; Reliable Analytical Techniques; Portable Biosensor; Rapid Diagnosis; Urine Samples; Photonic Biosensor; Highly Sensitive Interferometric Transducer; Disposable Microfluidic Cartridge; Light Coupling; Optical Reading; Prototype; Real-time Monitoring; Data Processing; Fluid Injections; Sensor Cartridge; Lipopolysaccharide; Bacterial Cell Wall; Lipoarabinomannan (LAM); Direct Detection; Labeling-Free; Amplification-Free; Process Simplification; Tanzania Hospitals; Real Patient Samples; TB Patients; Healthy Patients; HIV/AIDS Coinfection; Rapid Tests Limitations; World Health Organization (WHO) Recommended Methods; Excellent Correlation; Reliable Technology; Competitive Technology; Clinical Implications; On-Site Use; Primary Care Clinics; Hospitals; Simultaneous Detection; Panel of Six Different Biomarkers; Single Sample; Different Biomarkers; Different Diseases.