Periodic Reporting for period 1 - COFsensor (Novel COF-based sensors for detecting organic agents in water)
Berichtszeitraum: 2021-04-01 bis 2023-03-31
Detection and elimination of disease-causing agents, and early detection of disease markers are two major strategies employed in protecting the human health and wellbeing. In the current project, we addressed both challenges from the materials science point of view. We resorted to fairly recent classes of materials called covalent organic polymers (COPs) and covalent organic frameworks (COFs) and prepared new types of materials whose structures enabled them to function as sensors for bacteria, or as detectors of low-oxygen environment in human cells.
Rapid detection of biological organic matter is essential in prevention of diseases caused by biological agents such as viruses, bacteria and fungi. The primary infections of drug-resistant bacteria alone cause approximately five million deaths annually, making such infections the third leading cause of mortality globally. Moreover, secondary bacterial infections that follow various viral infections, such as Covid-19, are also lethal. Therefore, developing efficient bacteria detection methods is of great scientific, medical, forensic, biodefense, and food safety interest. Conventional methods of bacterial detection involve classical culturing techniques that require several handling steps, or advanced scientific equipment including polymerase chain reaction (PCR) to detect nucleic acids and enzyme-linked immunosorbent assay (ELISA) to monitor antigen-antibody interactions. These methods are highly accurate and specific, but they also face several drawbacks. They are laborious, time-consuming, expensive, require trained operators, and fail to detect microorganisms in real-time or outside the laboratory. Thus, there is a pressing need for inexpensive, easily operable, rapid, label-free and portable detection methods that give a quantitative readout. The objective of our project was, therefore, to develop a polymeric material that is stable in the physiological environment where bacteria are found and is responsive to their presence. Furthermore, we aimed to build a setup based on this material that would enable fast detection and would be easy to operate. Rather than developing a sensor for a particular species of bacterium, we were interested in detecting bacterial cells in aqueous media in general.
When diseases are not caused by a particular agent that could be detected prior to entering the human body, such as a bacterium, detecting disease markers can be of major importance in slowing down the progress of the disease. Hypoxia is a physiological condition defined by inadequate oxygen supply to body tissues. This condition can be seen as a disease marker as it, for instance, allows for early-stage cancer diagnostics while other medical imaging tools require comparably larger sizes of tumors for diagnostics. Furthermore, detecting hypoxia is also relevant in the pathogenesis of the chronic renal disease, progression of rheumatoid arthritis, and blood vessel plaque vulnerability. Current detection of hypoxia relies on expensive imaging tools such as magnetic resonance imaging (MRI). Within the scope of this project, we aimed to develop a fluorescence-based material that could aid in detecting hypoxic conditions in human cells.
Rapid detection of biological organic matter is essential in prevention of diseases caused by biological agents such as viruses, bacteria and fungi. The primary infections of drug-resistant bacteria alone cause approximately five million deaths annually, making such infections the third leading cause of mortality globally. Moreover, secondary bacterial infections that follow various viral infections, such as Covid-19, are also lethal. Therefore, developing efficient bacteria detection methods is of great scientific, medical, forensic, biodefense, and food safety interest. Conventional methods of bacterial detection involve classical culturing techniques that require several handling steps, or advanced scientific equipment including polymerase chain reaction (PCR) to detect nucleic acids and enzyme-linked immunosorbent assay (ELISA) to monitor antigen-antibody interactions. These methods are highly accurate and specific, but they also face several drawbacks. They are laborious, time-consuming, expensive, require trained operators, and fail to detect microorganisms in real-time or outside the laboratory. Thus, there is a pressing need for inexpensive, easily operable, rapid, label-free and portable detection methods that give a quantitative readout. The objective of our project was, therefore, to develop a polymeric material that is stable in the physiological environment where bacteria are found and is responsive to their presence. Furthermore, we aimed to build a setup based on this material that would enable fast detection and would be easy to operate. Rather than developing a sensor for a particular species of bacterium, we were interested in detecting bacterial cells in aqueous media in general.
When diseases are not caused by a particular agent that could be detected prior to entering the human body, such as a bacterium, detecting disease markers can be of major importance in slowing down the progress of the disease. Hypoxia is a physiological condition defined by inadequate oxygen supply to body tissues. This condition can be seen as a disease marker as it, for instance, allows for early-stage cancer diagnostics while other medical imaging tools require comparably larger sizes of tumors for diagnostics. Furthermore, detecting hypoxia is also relevant in the pathogenesis of the chronic renal disease, progression of rheumatoid arthritis, and blood vessel plaque vulnerability. Current detection of hypoxia relies on expensive imaging tools such as magnetic resonance imaging (MRI). Within the scope of this project, we aimed to develop a fluorescence-based material that could aid in detecting hypoxic conditions in human cells.
In the realm of bacterial cell detection, we prepared and fully characterized a novel positively-charged polymeric material called CATN. Positive surface charge of the material was a crucial structural feature responsible for driving the detection of bacterial cells because these cells typically exhibit negative surface charge. Following the synthesis and purification of CATN, the material was fully characterized by standard molecular-level and microscopic materials characterization techniques. Interestingly, we noted that the prepared material exhibits spherical morphology under electron microscopy. Since the material was insoluble in common solvents, some form of deposition method had to be developed in order to build a sensor setup. Electrophoresis was used for this purpose, whereby a custom-made electrophoretic cell was built and CATN was deposited onto an interdigitated electrode array (IDEA). Finally, E. coli cells were used as a model organism in demonstrating the ability of CATN-coated IDEA to respond to the presence of bacterial cells. We observed a positive linear relationship between the concentration of bacteria and the electrochemical response of the sensor. On account of its sensitivity and common application to biosensors, electrochemical impedance spectroscopy (EIS) was used as the detection method. This work was published in ACS Sensors (ACS Sens. 2022, 7, 9, 2743–2749). It was also presented at two conferences, namely Macro 2022 – World Polymer Congress (Winnipeg, Canada, 17. – 21.7.2022) and the Slovene Chemical Days 2022 (Portorose, Slovenia, 21. – 23.9.2022) and at the Bimonthly Organic Solid-State Symposium (BOSS; New York University, New York, USA, 1.12.2022).
The project on detecting hypoxia has been completed experimentally, but the results have not yet been published. Hence, due to the confidentiality of these findings, further details cannot be provided at this stage.
The project on detecting hypoxia has been completed experimentally, but the results have not yet been published. Hence, due to the confidentiality of these findings, further details cannot be provided at this stage.
Both research projects within the scope of the action have surpassed the previous state-of-the-art. In determining the efficiency of an electrochemical sensor, one of the crucial parameters is its limit of detection (LOD), i.e. the smallest amount of the analyte that the sensor is able to respond to. CATN in our setup exhibited one of the lowest LOD values for E. coli cells reported in the literature. Furthermore, most sensors in the literature incorporate antibodies specific to a particular species of bacteria, so these systems are only able to respond to that particular bacterium. Our system was based on electrostatic interactions between the positively-charged material and the negatively-charged bacterial surface, so it can be implemented for detecting bacteria even when their exact identity is unknown. In the long run, systems such as ours could be adapted to suit particular real-life applications such as estimating the quality of drinking water without the need for advanced scientific equipment and trained personnel.
The research on hypoxia is a rare example of using a COF for detecting a particular condition inside cells by means of fluorescence imaging. Only occasionally have COFs been explored for bioimaging, and the reports primarily demonstrate the internalization process. We have shown that these materials can be tuned to respond differently to cells grown under low-oxygen conditions than to cells cultured under normal atmosphere. We hope that these reports will stimulate further developments of COPs and COFs for biosensor applications, as these classes of materials have only recently become important players in the biosensor arena.
The research on hypoxia is a rare example of using a COF for detecting a particular condition inside cells by means of fluorescence imaging. Only occasionally have COFs been explored for bioimaging, and the reports primarily demonstrate the internalization process. We have shown that these materials can be tuned to respond differently to cells grown under low-oxygen conditions than to cells cultured under normal atmosphere. We hope that these reports will stimulate further developments of COPs and COFs for biosensor applications, as these classes of materials have only recently become important players in the biosensor arena.