Improving molecularly imprinted nanogels by pH modulation

Molecularly imprinted polymers (MIPs) are polymerized in the presence of a template molecule. After removing the template, the polymer scaffold can selectively rebind the template. The imprinting factor (IF) refers to the rebinding ratio of imprinted and non-imprinted polymers. Generally, the IFs of most reported MIPs are quite low (e.g. below 3.0). This is partially attributable to strong non-specific interactions. In this study, imprinted nanogels are prepared using two common dyes as templates, sulforhodamine B (SRhB) and fluorescein. By varying the buffer pH, non-specific electronic interactions between the template and the gels are reduced, leading to improved IF for the SRhB-MIPs from 1.5 (at pH 7.2) to 7.4 (at pH 9.0). At the same time, the binding capacity of the MIP remained similar. On the other hand, while pH tuning also improved the IF of the fluorescein-imprinted nanogels, the binding capacity dropped significantly. Using isothermal titration calorimetry (ITC), the SRhB-imprinted nanogels display a much higher affinity (Ka = 2.9 × 104 M−1) than the non-imprinted (Ka = 0.031 × 104 M−1) when rebinding is conducted in high pH (pH 9.0). This difference is mainly driven by enthalpy. This study suggests that pH tuning can be used to further improve MIPs.Natural Sciences and Engineering Research Council || STPGP 447472-1

Molecular Imprinting with Functional DNA and Nanozymes: Affinity Improvement and Selective Catalysis

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

Self-healing metal coordinated hydrogels using nucleotide ligands

A supramolecular gel formed by coordination of Zn2+ with adenosine monophosphate (AMP) is reported. The adenine base, the monophosphate, and Zn2+ are all important for gel formation. Mechanically disrupted gels can re-form upon centrifugation; applications of this gel for guest-molecule entrapment are explored.Beijing Higher Education Young Elite Teacher Project || ETP0520 Fundamental Research Funds for the Central Universities || YS1407 China Scholarship Council || Natural Sciences and Engineering Research Council |

Molecularly Imprinted Polymers with DNA Aptamer Fragments as Macromonomers

This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Applied Interfaces, © 2016 American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see Zhang, Z., & Liu, J. (2016). Molecularly Imprinted Polymers with DNA Aptamer Fragments as Macromonomers. Acs Applied Materials & Interfaces, 8(10), 6371–6378. https://doi.org/10.1021/acsami.6b00461Molecularly imprinted polymers (MIPs) are produced in the presence of a template molecule. After removing the template, the cavity can selectively rebind the template. MIPs are attractive functional materials with a low cost and high stability, but traditional MIPs often suffer from low binding affinity. This study employs DNA aptamer fragments as macromonomers to improve MIPs. The DNA aptamer for adenosine was first split into two halves, fluorescently labeled, and copolymerized into MIPs. With a fluorescence quenching assay, the importance of imprinting was confirmed. Further studies were carried out using isothermal titration calorimetry (ITC). 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 one of the aptamer fragments, and the DNA length was pushed to as short as six nucleotides, yielding MIPs with a dissociation constant of 27 mu M adenosine. This study provides a new method 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.Natural Sciences and Engineering Research Council of Canada (NSERC) [STPGP 447472-13

How Students\u27 Attitudes of Career Design Affect their Academic Achievements

Muroran Institute of Technology has been conducting questionnaire to ask undergraduates about their attitudes of career design. In this paper, in order to visualize how students\u27 attitudes of career design affect their academic achievements, a new method which combined modi ed principal component analysis and support vector machines has been proposed.ROMBUNNO.SS10-

Histocompatibility and Long-Term Results of the Follicular Unit-Like Wigs after Xenogeneic Hair Transplantation: An Experimental Study in Rabbits

Objective. This study was designed to observe the histocompatibility and long-term results of wigs after xenogeneic hair transplantation and to explore the possibility of industrial products in clinical application. Methods. The human hair and melted medical polypropylene were preceded into the follicular unit-like wigs according to the natural follicular unit by extrusion molding. 12 New Zealand rabbits were used as experimental animals for wigs transplantation. The histocompatibility of polypropylene and human hair was observed by H&E staining and scanning electron microscope. The loss rate of wigs was calculated to evaluate the long-term result after transplantation. Results. Mild infiltration by inflammatory cells around the polypropylene and human hair were seen during the early period after transplantation, accompanied with local epithelial cell proliferation. The inflammatory cells were decreased after 30 days with increased collagen fibers around the polypropylene and human hair. The follicular unit-like wigs maintained a good histocompatibility in one year. The degradation of hair was not significant. The loss rate of wigs was 4.1 ± 4.0% in one year. The appearance of hair was satisfactory. Conclusions. We successfully developed a follicular unit-like wigs, which were made of xenogeneic human hair with medical polypropylene, showing a good histocompatibility, a low loss rate, and satisfactory appearance in a year after transplantation. The follicular unit-like wigs may have prospective industrial products in clinical application

A Deep Instance Generative Framework for MILP Solvers Under Limited Data Availability

In the past few years, there has been an explosive surge in the use of machine learning (ML) techniques to address combinatorial optimization (CO) problems, especially mixed-integer linear programs (MILPs). Despite the achievements, the limited availability of real-world instances often leads to sub-optimal decisions and biased solver assessments, which motivates a suite of synthetic MILP instance generation techniques. However, existing methods either rely heavily on expert-designed formulations or struggle to capture the rich features of real-world instances. To tackle this problem, we propose G2MILP, the first deep generative framework for MILP instances. Specifically, G2MILP represents MILP instances as bipartite graphs, and applies a masked variational autoencoder to iteratively corrupt and replace parts of the original graphs to generate new ones. The appealing feature of G2MILP is that it can learn to generate novel and realistic MILP instances without prior expert-designed formulations, while preserving the structures and computational hardness of real-world datasets, simultaneously. Thus the generated instances can facilitate downstream tasks for enhancing MILP solvers under limited data availability. We design a suite of benchmarks to evaluate the quality of the generated MILP instances. Experiments demonstrate that our method can produce instances that closely resemble real-world datasets in terms of both structures and computational hardness. The deliverables are released at https://miralab-ustc.github.io/L2O-G2MILP