|dc.description.abstract||Molecular imprinting refers to polymerization of functional monomers in the presence of a template molecule. It is a general method to prepare stable and cost-effective artificial ligands as antibody mimics (also known as plastic antibodies), and the resulting materials are called molecularly imprinted polymers (MIP). Many molecules have been used as templates for imprinting ranging from metal ions, small molecules, peptides and proteins, nucleic acids, to whole cells with a wide range of applications including chromatography, solid-phase extraction, biosensors, therapeutics, organic synthesis and catalysis. MIP however suffer from low affinity and limited signaling mechanisms for binding. DNA oligonucleotides possess many functions such as specific molecular recognition (aptamers) and catalytic activities (DNAzymes). In addition, DNA is stable and easily modified. Combining MIP with DNA has several advantages. First, DNA aptamers can further improve the affinity of MIPs. At the same time, they may enable signaling of MIP binding. Second, some DNAzymes such as those with peroxidase-like activities (G-quadruplex DNAzymes), have low substrate selectivity, and MIP could solve this problem by introducing specific substrate binding sites on the DNAzymes. The approach can also extend to other type enzyme mimics such as nanozymes. Finally, the imprinted polymer shell can also protect enzymes from degradation and facilitate intracellular uptake. In this thesis, molecular imprinting with functional DNA and enzyme mimics were systematically studied. The main aims of the thesis include improving the binding affinity of MIPs and achieving selective catalysis of enzyme mimics. The mechanism of MIP for improved catalysis was also explored.
In Chapter 1, the introduction, relevant background knowledge about molecular imprinting, DNA and enzyme mimics was introduced. A state-of-the-art research progress of the fields was also reviewed. The research goals and outline of the thesis were described in the end of the chapter.
In Chapter 2, DNA aptamer fragments were used in the MIPs for affinity improvement and signalling. While previous research all used full-length aptamers, aptamer fragments with lower cost and higher stability have not been studied. In this work, DNA aptamer for adenosine was used as a model aptamer. It was first split into two halves, fluorescently labeled, and copolymerized into MIPs. With a fluorescence quenching assay, we found that the affinity of MIPs was improved with the aptamer fragments incorporated. Compared to the mixture of the free aptamer fragments, their MIPs doubled the binding affinity. Each free aptamer fragment alone cannot bind adenosine, whereas MIPs containing each fragment are effective binders. We further shortened the aptamer fragment, and the DNA length was pushed to as short as six nucleotides, yielding MIPs still having a high binding affinity (Kd ~27 μM). The study provides a new strategy for preparing functional MIP materials by combining high-affinity biopolymer fragments with low-cost synthetic monomers, allowing higher binding affinity and providing a method for signaling binding based on DNA chemistry.
In Chapter 3, molecularly imprinted nanogels were synthesized around a peroxidase-mimicking DNAzyme (G-quadruplex DNAzyme) to solve the problem of poor specificity of enzyme mimics. The polymer shell was demonstrated that improved the stability and activity of the DNAzymes by 2-fold. When the MIP was prepared with the DNAzyme and its substrate, the catalytic efficiency, kcat/Km, was enhanced by 6-fold for the imprinted substrate over the non-imprinted, true for both TMB and ABTS as substrates, indicating that selectivity can be achieved via imprinting. Within MIPs, the DNAzyme was also stable against high temperature and allowed for repeated use. This study demonstrated that molecular imprinting provided a general and practical method to form hybrid materials and introduce substrate recognition to enzyme mimics.
In Chapter 4, following the work in the Chapter 3, a molecularly imprinted DNAzyme nanogel was prepared using Amplex red as the template. The MIP nanogels selectively oxidized Amplex red in the presence of H2O2 to form a fluorescent product resorufin, while the oxidations for other substrates (TMB, ABTS and dopamine) were inhibited. The MIP nanogel exhibited more than 1.6-fold higher activity than the free DNAzyme. At the same time, the gel matrix protected the DNAzyme from degradation by DNase Ⅰ. The nanogel was then internalized by HeLa cells and an intracellular oxidation was achieved. This work provided an integrated solution for biocatalysis inside cells and it might be an interesting solution for intracellular therapeutic applications.
In Chapter 5, molecularly imprinted nanogels were grown on nanozymes to create substrate binding pockets. Fe3O4 NPs with peroxidase-mimicking activity were chosen as a model nanozyme. Electron microscopy confirmed a shell of nanogel encapsulating the nanozyme core. By imprinting with an adsorbed substrate, moderate specificity was achieved with neutral monomers (around 2.4-fold). Further introducing charged monomers led to nearly 100-fold specificity for the imprinted substrate over the non-imprinted compared to that of bare Fe3O4. Selective substrate binding was further confirmed by ITC tests. Besides Fe3O4, the same method was also successfully applied for imprinting on gold nanoparticles (a peroxidase mimic) and nanoceria (an oxidase mimic). In this work, molecular imprinting advanced the functional enzyme mimicking aspect of nanozymes, and such hybrid materials will find applications in biosensor development, separation, environmental remediation, and drug delivery.
In Chapter 6, following the work in Chapters 4 and 5, the catalytic mechanism of molecular imprinted enzyme mimics was systematically studied. A surface science approach was taken by dissecting the catalysis into three steps: adsorption of substrates, reaction, and product release. Each step was individually studied using reaction kinetics measurement, dynamic light scattering, UV-vis spectrometry. Through imprinting, the local substrate concentration around enzyme mimics was enriched by around 8-fold, which contributed to the increased activity. Diffusion of the substrate across the imprinted gel layer was studied by a pre-incubation experiment, demonstrating the improved molecular transportation in the imprinted gel layers. The activation energy (Ea) was measured and a substrate imprinted sample had the lowest activation energy of 13.8 kJ mol−1. Product release was also improved after imprinting as indicated by ITC binding tests using samples respectively imprinted with the substrate and the product. This study has rationalized improved activity and specificity of molecularly imprinted enzyme mimics and guided further rational design of such functional materials.
Overall, molecular imprinting with DNA aptamer fragments improved the affinity and enabled binding signalling. Imprinting on the enzyme mimics including both DNAzyme and nanozymes effectively solved the problem of low substrate specificity. The catalytic activity was also improved due to the enriched local concentration of substrate and lowered activation energy. The thesis provides a new strategy for preparing functional materials by combining MIP with functional DNA and nanomaterials to advance the molecular recognition and selective catalysis field.||en