(4as) Development of Nanozyme-Linked Immunosorbent Assays

I studied biology and chemistry during my undergraduate studies at Seoul Women’s University (SWU), and I earned a master’s degree at Seoul National University (SNU) in chemical and biological engineering. After this I did research at The Garden of Naturalsolution (GNS), a cosmetics ingredient company. Currently, I am a Ph.D. student in the Chemical Engineering department at Washington State University (WSU). After my doctoral studies, I desire to continue to develop effective detection methods for target analytes to improve of the welfare of humanity.

My doctoral research will lead to new methods for easily and inexpensively detecting two different analytes—small molecule pesticides and macromolecule tissue-engineered protein collagen type II– to improve the health and welfare of humanity and promote healthy ecosystems. Research has generally conducted the development of fast and effective new nanozyme-linked immunoassays for target analytes to avoid the need for the less stable enzymes commonly used. These two target analytes offer excellent opportunities for developing these techniques. I am also developing comprehensive review articles on multiplex lateral flow assays used to detect foodborne pathogens and nanozyme-amplified biosensors. My research is expected to provide potential strategies for various target analytes and in various fields such as bioengineering and diagnostic analysis.

Introduction

Pesticides, such as herbicides and insecticides, are valuable in many agricultural fields; however, their toxicity can cause acute poisoning, increase the risk of certain cancers [1], and negatively affect reproduction [2]. Therefore, various agencies, including EPA, had established the limit of detections (LODs) for pesticides to regulate their usage. Therefore, the development of pesticide quantification is essential.

Collagens are the main structural protein in the extracellular matrix of various tissues, including cartilage [3]. Collagens are categorized into 28 subtypes, including types I, II, and II [3]. Articular cartilage, which lessens the pressure between articulating joints, consists of 85 – 90% collagen type II [4]. Osteoarthritis, a progressive joint disease, causes the breakdown of articular cartilage [5]; as osteoarthritis worsens, the enzyme collagenase continuously breaks down collagen type II [6]. Since the underlying treatment for articular cartilage is limited [7], some researchers have been studied the regeneration of AC using approaches such as scaffold-based techniques, which applied collagen type II [7]. Therefore, the need for quantification of collagen type II and collagen type II for medical use is rising.

Nanozymes are defined as nanoparticles with enzyme-like catalytic activities. Mesoporous core-shell palladium@platinum (Pd@Pt) nanoparticles have peroxidase-like catalytic activity [8]. Pd@Pt nanoparticles can be simply synthesized by the self-assembly method using surfactants. Since Pd@Pt NPs have a mesoporous structure with a high surface area, they decrease mass transfer limitations, allowing them to have outstanding peroxidase-like catalytic activity. These nanoparticles also have both high pH and thermal stability [9] and the capability to be bound directly to the primary antibody, thereby eliminating the use of additional binding steps, such as an enzyme-labeled secondary antibody [10]. These characteristics led me to develop immunoassays for target analytes by replacing a traditional enzyme, horseradish peroxidase (HRP), which has commonly been used as a signal detecting agent.

Intellectual Merit

Since Pd@Pt nanoparticles are attractive because of their cost-effectiveness and high stability and sensitivity [8, 9], these nanozymes are utilized as alternative signal detecting agents to others such as enzymes, which have limited stability [10]. My research will present the first application of Pd@Pt nanoparticles in the enzyme-linked immunosorbent assay (ELISA)-like format with a competitive assay for small molecules, herbicide atrazine [10]. This research is also expanding to detect pesticides, atrazine and acetochlor, in real samples (fruits and vegetables) and is expected for developing additional pesticide detection methods. Furthermore, nanozyme-linked immunoassays are expanded by developing a sandwich assay to detect larger molecular analytes. Therefore, this research is expected that nanozyme-linked immunoassays can be applied in various areas by representing the quantification of two different types of analytes.

A comprehensive review paper about the development of lateral flow assays for pathogens is in preparation. The review will be expected to provide current advances and potential strategies for detecting three pathogens, Escherichia coli, Salmonella, and Listeria. Another review paper is also in the preparation of the nanozyme and its amplified biosenors. This review will be helpful to understand the trend of nanozymes and their amplified biosensors, thereby providing ideas to develop more sophisticated biosensors.

Teaching Interests

Currently, I have been volunteering as a peer mentor in the international program at WSU, a role I have had since April 2019. I assist new international graduate students during their transition to life at WSU and in the US. I contact weekly or biweekly with these students; provide useful information; and encourage and support them. From last year to the present, I have been assisting through constant contact with peer mentees who are experiencing a feeling of isolation and aloneness because of COVID-19 pandemic. Being a peer mentor provides great opportunities to learn and understand different countries and cultures.

During the Summer of 2019, I worked with an undergraduate student in an NSF/WSU Research Experience for Undergraduates program. I mentored this student to teach the principle of ELISA and the processes of nanozyme-linked immunoassay for detecting pesticides. During Fall 2019 and 2020, I presented my research to undergraduate students as a guest lecturer in the WSU CHE 476 Biomedical Engineering Principles course. I introduced my research and graduate studies to them for increasing their interest in STEM research. These opportunities allowed me to practice presenting technical principles and scientific logics to others in an official setting.

Conclusion

My PhD research will cover the development of nanozyme-amplified immunoassay detection methods for target analytes and include literature reviews about multiplex lateral flow assays for detecting foodborne pathogens and nanozymes with their amplified biosensors. These preliminary nanozyme-linked immunoassays are expected to broaden for detecting additional pesticides and proteins with other nanozymes. Results will provide evidence that these nanozyme-linked immunoassays attractive to be widely applied for various target analytes in multiple fields such as bioanalysis, food safety, and diagnostic analysis. I aspire to a long and dedicated career as a researcher and student mentor with my research to improve the well-being of humanity across environmental and medical diagnostic analysis.

References

1. Kim, K.-H., E. Kabir, and S.A. Jahan, Exposure to pesticides and the associated human health effects. Science of The Total Environment, 2017. 575: p. 525-535.
2. (WHO), W.H.O. Pesticides residues in food. 2018 Feburary 19, 2018 [cited 2021 January 25]; Available from: https://www.who.int/news-room/fact-sheets/detail/pesticide-residues-in-food.
3. Bielajew, B.J., J.C. Hu, and K.A. Athanasiou, Collagen: quantification, biomechanics and role of minor subtypes in cartilage. Nature Reviews Materials, 2020. 5(10): p. 730-747.
4. Takahashi, T., et al., Development of a novel immunoassay for the measurement of type II collagen neoepitope generated by collagenase cleavage. Clinica Chimica Acta, 2012. 413(19): p. 1591-1599.
5. Wu, W., et al., Sites of collagenase cleavage and denaturation of type II collagen in aging and osteoarthritic articular cartilage and their relationship to the distribution of matrix metalloproteinase 1 and matrix metalloproteinase 13. Arthritis & Rheumatism, 2002. 46(8): p. 2087-2094.
6. Bay-Jensen, A.-C., et al., Enzyme-linked immunosorbent assay (ELISAs) for metalloproteinase derived type II collagen neoepitope, CIIM—Increased serum CIIM in subjects with severe radiographic osteoarthritis. Clinical Biochemistry, 2011. 44(5): p. 423-429.
7. Makris, E.A., et al., Repair and tissue engineering techniques for articular cartilage. Nature Reviews Rheumatology, 2015. 11(1): p. 21.
8. Cheng, N., et al., Nanozyme-mediated dual immunoassay integrated with smartphone for use in simultaneous detection of pathogens. ACS Applied Materials & Interfaces, 2017. 9(46): p. 40671-40680.
9. Jiang, T., et al., Detection of p53 protein based on mesoporous Pt–Pd nanoparticles with enhanced peroxidase-like catalysis. ACS Sensors, 2016. 1(6): p. 717-724.
10. Kwon, E.Y., et al., Mesoporous Pd@Pt nanoparticle-linked immunosorbent assay for detection of atrazine. Analytica Chimica Acta, 2020. 1116: p. 36-44.