2021 Virtual Undergraduate Research Symposium
2021 Virtual Undergraduate Research Symposium
In Vitro Characterization of FNP Oxygen Nanosensors
In Vitro Characterization of FNP Oxygen Nanosensors
PROJECT NUMBER: 18 | AUTHOR: Tony Tien and Pilar Martin, Chemical and Biological Engineering
MENTOR: Kevin Cash, Chemical and Biological Engineering
ABSTRACT
Flash nanoprecipitation (FNP) is a rapid mixing technique that can be used to make nanoparticles of desired size and character given appropriate stream turbulence and composition. In collaboration with the Prud’homme group at Princeton University, the Cash Lab is working on the best methods to characterize FNP nanosensors designed to measure oxygen concentration. Ten oxygen nanosensor samples were formulated by the Prud’homme group using varying compositions of pegylated polystyrene particles (PS-PEG), 4-(4-Dihexadecylaminostyryl)-N-methylpyridinium iodide) (DiA), 5,10,15,20-(tetraphenyl)porphyrin (PtTPP), vitamin E or vitamin E acetate, and other inert core components. Fluorescence data were used to characterize the nanosensors using a combination of glucose/glucose oxidase tests (G+GOx) compared to ambient oxygen. In analyzing the fluorescence data, sample #5 was determined to have the best oxygenated to deoxygenated fluorescence ratio as well as the highest overall fluorescence for subsequent analysis. Additional characterization was performed with sample #5 to quantify oxygen response using direct gas bubbling with air and nitrogen; however, the nanosensors exhibited variations in the reference dye peak (DiA) compared to the oxygen-responsive peak (PtTPP) during bubbling, leading to inconclusive results regarding reliable oxygen detection. Future work aims to further refine fluorescence techniques and re-evaluate reference dyes for FNP oxygen detection.
PRESENTATION
AUTHOR BIOGRAPHY
Tony is a Chemical Engineering junior performing research within the department in the lab of Dr. Kevin Cash. His work in the Cash Lab has involved the development of analyte nanosensors to be applied to biological systems in vivo, including nanosensors for the detection of sodium, sulfate, and oxygen. He has also worked on a layer-by-layer biofilm incorporating Pseudomonas aeruginosa, designed to replicate a natural P. aeruginosa biofilm for improved diagnosis of biofilms in clinical settings. His current work involves researching methods of characterizing oxygen-sensitive nanosensors developed by the Prud’homme group at Princeton University for the determination of optimal formulations for desired use cases. In the future, he intends to go to grad school to continue research in fields related to biotechnology and is considering working towards a Ph.D.
Pilar Martin is a junior in the Chemical and Biological Engineering department. She has been conducting research within the department under Dr. Kevin Cash since Fall 2019. Her past projects involved analyzing and developing nitrate-sensitive and sulfate-sensitive nanosensors. She is currently working on testing oxygen-sensitive nanosensors in collaboration with the Robert Prud’homme research group. In the future, she hopes to test her nanosensors in more complex samples such as bacterial biofilms and microbial consortia.
Hi-Nice work Tony and Pilar. I have a couple of questions for you two.
1. Do you have any hypotheses to explain why the absorbance of the DiA reference compound appears to change upon exposure to O2?
2. How stable are the nanoparticles? Could exposure to O2 cause oxidation of the PtTPP compound and/or change the oxidation state of the Pt center? This could stop the complexation reaction with O2. I encountered this problem working with Fe-based porphyrins for reversibly binding O2, ultimately we couldn’t make them stable enough.
Hi Professor Way,
While we did not expect DiA’s signal to fluctuate with oxygen, we are currently exploring whether photodegradation or photobleaching of the dye is occurring over extended read times, which would result in a signal decrease. Another factor that we are analyzing is that our bubbling system used to change the oxygen concentration causes a small amount of the sensor solution to evaporate over time, which may be affecting DiA’s signal. However, our lab also uses a related dye, DiO, in other nanosensor applications, and since we have observed similar behavior with DiO, the signal fluctuation of the dye with extended exposure may be characteristic of this family of dyes.
DiA’s signal fluctuation will require additional investigation as these extended oxygen tests seem to cause signal fluctuation whereas endpoint tests of deoxygenated/oxygenated environments at a single point in time result in little (if any) fluctuation in DiA’s signal. Minimal fluctuation of DiA in these tests aligned more with our expectations of its use as a reference dye. We do not currently have any absorbance data for DiA’s response to oxygen over time, though that may be a consideration for improving the nanosensor formulation or fluorescence measurement techniques in the future.
In terms of stability, our biweekly lifetime tests in deoxygenated and oxygenated environments show that the sensors provide a relatively consistent response at least 60 days after production. We have not yet seen any issues with the response of PtTPP over time with extended oxygen exposure, though our data in Figure 7 on our poster (“Reversibility of Oxygen Sensing”) seems to suggest that the effect of deoxygenating and oxygenating the sensors three times in a row affects the signal minimally, even when considered with respect to DiA’s fluctuation.
Thank you so much for your questions and for stopping by our poster!
Sincerely,
Tony