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Evaluating enzymatic reactions, microbial growth and small molecule detection with a new thermal principle: the heat-transfer method (HTM)

Betlem, Kai (2019) Evaluating enzymatic reactions, microbial growth and small molecule detection with a new thermal principle: the heat-transfer method (HTM). Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

The Heat Transfer Method (HTM), invented in 2012 by van Grinsven et al. 1, is a thermal analysis technique that can monitor DNA denaturation using only two thermocouples and a heat source. The advantage of this thermal sensing is its simplicity, low-cost and use as a portable set up that does not require a lab environment. This work will explore novel applications of the HTM including studying of enzyme catalysis, monitoring of microbial growth, and detection of biomolecules using novel Molecularly Imprinted Polymer-based sensor platforms. The first objective of this work was to determine whether HTM could be used to for the study of enzyme activity, this is yet unexplored in the HTM. Currently applied techniques for the study of enzyme activities are labour intensive or require expensive equipment whilst not all reactions can be studied. The HTM is not limited by these factors and with the ability to sense changes in DNA length a proof of concept is designed using the EcoR1 restriction enzyme, for which an microbial sample is available as next step. Therefore, it was necessary to functionalise DNA on gold electrodes. To this end, self-assembled monolayer (SAM) on gold electrodes was formed followed by conventional 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling to DNA strands bearing an amine end. This protocol is adapted from previous work by van Grinsven et al. 1, but fluorescence microscopy revealed limited attachment of the DNA to the surface. The short comings in the protocol are discussed and alternatives are suggested to promote DNA binding. Due to difficulties in studying of DNA with HTM, the focus switched towards real-time monitoring of microbial growth, which is important an important factor in many fields, such as study of quality control in food industry, antimicrobial resistance and waste water management, whilst is also an unexplored area for the HTM. Adaptations were made to the existing flow-cell design in order to facilitate longitudinal measurements. First measurements were performed with baking yeast, Saccharomyces cerevisiae, and suspensions of the microorganisms were applied to a flow cell containing a plain gold electrode. After it was confirmed that it was possible to study the kinetics of yeast growth, including factors (pH, presence of toxic compounds, temperature) that impact on microbial growth, Staphylococcus aureus was studied. Measurements conducted in buffered solutions showed that the growth of Staphylococcus is temperature dependent, and the optimum growth temperature is in accordance with literature. Following on, the growth of S. aureus in a complex digestate sample is studied that is composed of several colonies of microorganisms. It is shown that the developed thermal sensor platform is capable of determining the overall microbial load, corresponding to both the amount of S. aureus in the system and microorganisms present in the digestate sample. While this could have useful applications in the wastewater and food industry, gold electrodes used as recognition elements are not selective towards particular bacterial strains. Therefore, Molecularly Imprinted Polymers were developed and integrated onto electrode materials. MIPs are synthetic mimics of antibodies that possess high affinity for their target molecule but are low-cost, can be produced in large quantities, and possess superior thermal and chemical stability compared to their natural counterparts. A novel functionalisation procedure is proposed that directly incorporates the polymer particles into screen-printing ink. The main advantage of using MIP-modified Screen-Printed Electrodes (SPEs) is the simplicity of this sensor platform, low-cost, and high reproducibility due to the use of SPEs. Furthermore, the electrodes can be printed onto paper which provides a sustainable alternative compared to polyester as traditional support material. Measurements with MIP-modified SPEs were conducted with a range of neurotransmitters and caffeine. Caffeine serves as an anthropogenic marker of water quality and therefore it was of high relevance to study its presence in digestate samples. It is proven that the detection of caffeine with HTM is temperature dependent, and the measurement temperature can be used to fine tune the detection limit of the sensor platform. Sensitivity of the MIP-based platform can be improved by the use of nanoMIPs, nanoparticles that are produced via a solid-phase approach. These particles possess superior affinity (into the sub nano molar range) and are water-soluble due to their small size, which enables the simple functionalisation of thermocouples using dip-coating. These functionalised thermocouples are positioned into flow cells of the HTM set up and it is proven that the temperature recorded by the thermocouple is dependent on the biomolecule concentration. These measurements are conducted for a range of compounds, highlighting the versatility of this system and the possibility to measure from small molecules (vancomycin) to larger macromolecules (protein EGFR). The limit of detection compared to the use of traditional MIP bulk micro particles improved with an order of three, indicating the superior affinity of the nanoMIPs. Therefore, it is shown that the HTM is a highly versatile diagnostic tool that can has the potential to offer a quick and easy analysis for a variety of analyses, ranging from high precise medical diagnostics to quality control in for industry and monitoring of environmental changes. While the monitoring of enzyme catalysis was not successful, it is possible to monitor microbial loads in complex samples using simple gold electrodes. Furthermore, by the use of MIPs, it is possible to determine trace amounts of organic molecules and micro-pollutants in water samples.

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