Nanoparticle-enabled efficient spectroscopic techniques for biomolecules detection
Creators: name: WEERATHUNGE, Pabudi
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310607
subject: Nanobiotechnology
classificationCode: 340108
subject: Sensor technology (incl. chemical aspects)
classificationCode: 401807
subject: Nanomaterials

description: The last decade has seen a rise in the use of enzyme-mimicking nanoparticles, nanomaterials found to exhibit enzyme-like catalytic characteristics, as a viable alternative to natural enzymes. These artificial enzymes, more commonly known as "NanoZymes", are highly stable and low-cost alternatives to natural enzymes that have large implications in a wide range of applications including biosensing, pollutant removal, cell growth etc. A myriad of novel smart nanomaterials has been designed to show a wide range of enzyme-mimic activity that can be tailored by simply changing the nanoparticle composition, size, shape, surface chemistry. The application of NanoZymes in biosensors is particularly interesting as it offers a unique opportunity to visualise the sensing event (colour generation through the oxidation of enzymatic substrates), thereby offering the potential for on-site/ point-of-care detection by nontechnical users. Although interesting, NanoZyme-based biosensing platforms are still in their infancy. In addition to the generation of colour by the NanoZyme, target recognition is also the key for the detection of chemical and biological analytes. Although a myriad of recognition moieties has been used in sensor platforms, combining the strengths of NanoZymes with an MRE such as aptamers has not been studied. Aptamers are single stranded DNA or RNA molecules generated through a process of in vitro evolution called Systematic Evolution of Ligands by EXponential Enrichment (SELEX). Considering that aptamers can be raised against a myriad of targets including small molecules to whole cell, this work outlined herein is an attempt to explore the potential of using NanoZymes with aptamers and proteins to generate a colorimetric sensing technological platform that can be applied in the environment (Chapter 3), food (Chapter 4 and Chapter 5) and health (Chapter 6 and Chapter 7) sector. <br><br>

Chapter 3, Chapter 4 and Chapter 5 demonstrates a "turn-off/turn-on" strategy for the detection of environmentally important pesticide - acetamiprid, food contaminants - kanamycin and norovirus. In all cases, the sensor probe is fabricated by deactivating the NanoZyme activity of GNPs using target-specific aptamers and reactivating the inhibited activity in presence of the cognate target. Specifically, in chapter 3, target-specific S-18 aptamer was used to develop a quantitative method for acetamiprid detection. The fabricated sensor showed 0.1 ppm sensitivity within 10 minutes of assay time and a visual colour change resulted only in presence of acetamiprid. Chapter 4 was built upon this knowledge, where the sensor probe was created using Ky2 aptamer. This "turn-off/turn-on" biosensing approach allows detection of 1.49 nM concentrations of kanamycin which is at least 10 folds more sensitive than the conventional GNP-aptamer-based approach. In addition to the excellent sensitivity, the sensor also displayed excellent specificity, linear range, response time, accuracy and precision further demonstrating the suitability of this strategy as a universal method for detecting small molecules. Chapter 5 uses a similar strategy but replaces small molecules with whole cells. The colorimetric NanoZyme aptasensor allowed a rapid (10 min) and ultrasensitive (LoD of 3 viruses) detection of murine norovirus (MNV), a cultivable surrogate for NoV. In addition to testing the sensor performance in simulated conditions, the ability of the sensor to detect norovirus in complex biological matrix of shellfish lysate further shows the robustness of the NanoZyme aptasensor for real-time norovirus sensing. This new assay format can therefore be of significant importance as it allows ultrasensitive norovirus detection rapidly within minutes, while also offering the simplicity of use and need for non-specialized laboratory infrastructure.<br><br>

Considering the importance of identifying pathogenic strains and virulence factors in pathogens such as Staphylococcus aureus, one of the foremost causative agent resulting in minor to major healthcare infections, a new chemical/nose tongue approach was developed. In this case, multiple aptamers that target different regions of the S. aureus cell wall were employed. This resulted in a unique colorimetric response pattern from the sensing events. Discriminant analysis could reliably identify five strains of S. aureus, and improved the sensitivity of bacterial detection to 100 cfu/mL. Hierarchical cluster analysis could further group these bacteria based on the presence/ absence of virulence factors on bacterial surfaces. These virulence factors are extremely important in inducing pathogenicity during bacterial infections. Such detailed information about bacterial pathogenesis and disease progression from a simple assay will allow appropriate treatment strategies, and reinforces the importance of combining the proposed NanoZyme biosensing strategy with appropriate statistical algorithms. The final chapter describes the use of proteins as recognition moieties in combinations with NanoZymes. The overexpression of endogenous transferrin receptor in cancer cells serves as a ubiquitous biomarker for specific targeting of cancer cells. A distinct tonality in the colorimetric response was observed for cancer cells based on the interaction of the sensor probe with cancer vs. healthy cells. This not only allowed for the detection of cancer cells, but also in principle, allows profiling of different cell types based on the expression profile of cell surface receptors.