Clinical diagnosis and pathogen detection with a novel multiplexed nanophotonic point-of-care biosensor

Núria Fabri Faja

Alonso Chamarro, Julian Estevez Alberola, Mari Carmen Prof. Laura M. Lechuga

Sepsis is a clinical condition in which the immune system reacts aberrantly when a systemic infection occurs. Every year, 31 million cases of sepsis occur worldwide with an estimated mortality of 35%. The diagnosis of sepsis is criticized because every hour of delay in starting treatment leads to a significant reduction in the survival rate. In addition, the techniques used today (cell culture) for diagnosis usually take between 24 and 72 h and require specialized laboratories and trained personnel. That is why the main objective of this thesis is the development and application of a portable biosensor device for the diagnosis in less than one hour of sepsis through the direct and simultaneous detection of several inflammatory biomarkers and bacteria (and other microorganisms) in human fluids. This thesis is based on a European project called RAIS (HORIZON2020) and arises mainly motivated by the lack of analytical techniques and platforms for clinical diagnosis that are fast, efficient and reliable. The results obtained in this thesis have demonstrated the potential application of a new portable and integrated biosensor for the detection of two bacteria (Escherichia coli and Pseudomonas aeruginosa) in just 10 microliters (µL) of blood plasma. This detection has been done directly and without any type of labeling or amplification, which greatly simplifies the measurement process. In addition, thanks to the participation of a renowned public hospital, the Vall D'Hebron hospital, it has been possible to validate the biosensor with real patient samples. Specifically, it has been possible to discriminate between samples from healthy patients and samples from patients with bacterial infection (with sepsis) in less than an hour (30 minutes of incubation and 1 minute of measurement with the biosensor). These samples were analyzed with classic analysis techniques and a good correlation was obtained, which demonstrates that the technology is reliable and competitive. By applying this technology, the presence of infection in the blood can be detected quickly and easily in less than an hour, which will allow treatment with antibiotics to be started almost immediately. This will increase patient survival, shorten hospitalization times and, ultimately, represent significant savings for the healthcare system. It should be noted that the design of the biosensor will allow in the future to expand simultaneous detection to many more biomarkers such as other microorganisms or biomolecules.

The aim of the project was the search for the multiple and simultaneous detection of several biomarkers related to sepsis. Specifically, the thesis has studied the detection and quantification of three proteins of different sizes together with two bacteria commonly found in cases of sepsis. In addition, this detection had to be carried out directly, that is to say, without the use of fluorescent or chromophoric labels. For this, it was decided to use the microarray format, which consists of the ordered and controlled distribution of several bioreceptors on the sensing surface. For the arrangement of these bioreceptors on the sensor surfaces, it was necessary to use and handle volume dispensing technologies which are capable of distributing tiny amounts (nL) of solutions in a controlled manner. Thanks to this, although in this thesis we have worked with three bioreceptors of proteins and two of bacteria, in the future, with the use of these technologies, it will be possible to place up to 1,600 different bioreceptors distributed along the sensor surface, which will allow us to reach one of the highest label-free multiplexes. Of the two types of biomarkers analyzed, the direct detection of bacteria has been the most successful strategy given that it has offered better results and without a doubt has great potential to be applied directly in the clinical setting. The two bacteria that have been worked with have been Escherichia coli and Pseudomonas aeruginosa and, their individualized detection, has shown that detection limits of 100 and 146 bacteria/mL can be reached, which extrapolated to the measurement volume (which is 10 microliters of blood plasma) results in the possibility of detecting 1 and 2 bacterial cells directly. In addition, thanks to the collaboration of the public hospital Vall D'Hebron, different real patient samples have been analyzed. Specifically, the POC biosensor has been able to discriminate samples from patients with sepsis from those from healthy patients. All this using only the mentioned 10 uL of plasma from the patients and in less than an hour (30 minutes of incubation plus 1 minute of reading by the biosensor). Likewise, these samples were also analyzed with classic diagnostic techniques and a good correlation was achieved. In summary, all this has shown that the combination of the POC biosensor together with the 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 the POC biosensor has, this test can be performed without the need for large infrastructures such as specialized microbiological laboratories or for highly qualified personnel. Through the application of this technology, the presence of infection can be detected quickly, easily and in less than an hour, which will allow antibiotic treatments to be started almost immediately. This will increase the survival rate, shorten the hospital stay and, finally, lead to significant savings for the public health systems. Likewise, it is relevant to add that, as previously mentioned, the bioreceptors are dispensed and located in a controlled manner on the nanosensing surface so that, although in this thesis the POC biosensor device has been applied for the detection of sepsis-causing bacteria, in the future it will be possible to immobilize bioreceptors related to other infectious agents or other relevant biomarkers for other diseases. We also note that in the biosensor design stage carried out for the European project, from the beginning the application of the POC biosensor was considered so that it would be compatible with single-use cartridges that would contain the sensor surface with the specific bioreceptors for each disease or clinical condition. This would mean that hospitals could have the portable diagnostic platform (the POC biosensor) and buy different cartridges aimed at different diseases. Finally, I would like to add that, based on the results obtained, the research group where the thesis was carried out has begun to negotiate possible commercial agreements with companies interested in having POC-type systems for the rapid analysis of infections and the possibility of extending it to the study of the antibiotic resistance of the detected microorganism, in order to provide more effectively the most suitable therapy for each patient.

Sepsis; Clinical Condition; Immune System; Systemic Infection; Mortality; Diagnosis Delay; Survival Rate; Cell Culture; Specialized Laboratories; Trained Personnel; Portable Biosensor Device; Rapid Diagnosis; Inflammatory Biomarkers; Bacteria Detection; Microorganisms; Human Fluids; European Project; RAIS (HORIZON2020); Analytical Techniques; Clinical Diagnosis; Fast; Efficient; Reliable; Potential Application; Escherichia coli; Pseudomonas aeruginosa; Blood Plasma; Direct Detection; Labeling-Free; Amplification-Free; Measurement Process Simplification; Vall D'Hebron Hospital; Real Patient Samples; Healthy Patients; Bacterial Infection; Incubation Time; Measurement Time; Classic Analysis Techniques; Good Correlation; Reliable Technology; Competitive Technology; Antibiotic Treatment; Patient Survival; Hospitalization Times; Healthcare System Savings; Simultaneous Detection Expansion; Other Microorganisms; Biomolecules.