Understanding the interactions between nanozymes and aptamers to rationally design aptasensors for the detection of ampicillin in milk samples
Creators: name: Balapitiya Liyanage, Piyumi
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310607
subject: Nanobiotechnology
classificationCode: 340108
subject: Sensor technology (incl. chemical aspects)
classificationCode: 401807
subject: Nanomaterials

description: Over two decades ago, the discovery of nanomaterials that can mimic the catalytic activity of natural enzymes has opened new avenues, bridging nanomaterials and enzymes. These enzyme mimicking nanomaterials commonly referred to as “nanozymes” have been shown to exhibit catalytic activities that resemble four of the six EC classes of natural enzymes. While nanozymes have been used in several applications, the field of sensing has largely benefited as nanozymes can be used as a viable alternative instead of natural enzymes for the detection of targets. Although nanozymes lack the ability to recognize a specific target, the incorporation of molecular recognition elements (MREs) has enabled nanozyme sensors to attain high target specificity. Aptamers are a new class of MREs that are notable for having high affinity to its target (similar to the workhorse of analytical research - antibodies). Recent studies have combined the strength of nanozymes and aptamers to create ‘nanozyme aptasensors’. In such sensors, the aptamers are bound to the surface of the nanozyme where the binding determines the underlying mechanism of the sensor operation. To date, two strategies have been described for the binding of nanozymes to aptamers viz. strong or weak interactions. In the case of strong interactions, the aptamers are bound to the surface of the nanozyme in a manner where the sensing principle does not require the aptamers do dissociate from the surface of the nanozyme. In contrast, the working principle of nanozyme aptasensors based on weak interactions require the aptamers to dissociate from the surface of the nanozyme specifically in the presence of the aptamer specific target. Specifically, the interaction of the aptamers with the nanozyme and the interaction of the aptamers with the target when the aptamer is bound to the nanozyme are the key for effective working of the sensor. Understanding the nature and the thermodynamics of the interactions between the aptamer-nanozyme and aptamer-target (when the aptamer is bound to the surface of the nanozyme) would enable to rationally design sensing platforms that would minimise the effect of the complex matrices where the sensors need to operate. The current work is an attempt to understand some key parameters in nanozymes and the aptamers that can influence such interactions. 

While several aspects of nanozymes (viz. composition, size, shape, surface) can influence its interaction with aptamers, understanding the influence of the composition of the nanozyme is one of the fundamental aspects. Therefore, Chapter 2 is focused on studying how monometallic nanozymes based on noble metals can influence its interaction with ampicillin aptamer. The steady-state fluorescence quenching principles allowed probing the interaction that showed Au nanozymes have favourable interaction capabilities. Chapter 3 then explores the influence of the aptamer composition, the length, and the folding energy on its interaction with Au nanozymes. Correlation analysis provided in depth fundamental understanding of which parameters of the interactions are key in allowing the aptamers to associate/dissociate from the surface of the Au nanozyme. Chapter 4 combines the strength of two monometallic nanozymes to create AuPd bimetallic nanozyme that shows favourable interactions with ampicillin aptamer. The use of the bimetallic nanozyme enhances the fluorescence response of the aptasensor. The fundamental knowledge gained from the chapters is the first step towards rationally designing nanozyme aptasensors.