Zijie Zhang

Molecular Imprinting on Inorganic Nanozymes for Hundred-fold Enzyme Specificity

Enzyme-mimicking nanomaterials (nanozymes) are more cost-effective and robust than protein enzymes, but they lack specificity. Herein, molecularly imprinted polymers were grown on Fe3O4 nanozymes with peroxidase-like activity to create substrate binding pockets. Electron microscopy confirmed a shell of nanogel. By imprinting with an adsorbed substrate, moderate specificity was achieved with neutral monomers. Further introducing charged monomers led to nearly 100-fold specificity for the imprinted substrate over the nonimprinted compared to that of bare Fe3O4. Selective substrate binding was further confirmed by isothermal titration calorimetry. The same method was also successfully applied for imprinting on gold nanoparticles (peroxidase mimics) and nanoceria (oxidase mimics). Molecular imprinting furthers the functional enzyme mimicking aspect of nanozymes, and such hybrid materials will find applications in biosensor development, separation, environmental remediation, and drug delivery.

Co-immobilization of multiple enzymes by metal coordinated nucleotide hydrogel nanofibers: improved stability and an enzyme cascade for glucose detection

Preserving enzyme activity and promoting synergistic activity via co-localization of multiple enzymes are key topics in bionanotechnology, materials science, and analytical chemistry. This study reports a facile method for co-immobilizing multiple enzymes in metal coordinated hydrogel nanofibers. Specifically, four types of protein enzymes, including glucose oxidase, Candida rugosa lipase, α-amylase, and horseradish peroxidase, were respectively encapsulated in a gel nanofiber made of Zn(2+) and adenosine monophosphate (AMP) with a simple mixing step. Most enzymes achieved quantitative loading and retained full activity. At the same time, the entrapped enzymes were more stable against temperature variation (by 7.5 °C), protease attack, extreme pH (by 2-fold), and organic solvents. After storing for 15 days, the entrapped enzyme still retained 70% activity while the free enzyme nearly completely lost its activity. Compared to nanoparticles formed with AMP and lanthanide ions, the nanofiber gels allowed much higher enzyme activity. Finally, a highly sensitive and selective biosensor for glucose was prepared using the gel nanofiber to co-immobilize glucose oxidase and horseradish peroxidase for an enzyme cascade system. A detection limit of 0.3 μM glucose with excellent selectivity was achieved. This work indicates that metal coordinated materials using nucleotides are highly useful for interfacing with biomolecules.

Multicopper Laccase Mimicking Nanozymes with Nucleotides as Ligands

Using nanomaterials to achieve functional enzyme mimics (nanozymes) is attractive for both applied and fundamental research. Laccases are multicopper oxidases highly important for biotechnology and environmental remediation. In this work, we report an exceptionally simple yet functional laccase mimic based on guanosine monophosphate (GMP) coordinated copper. It forms an amorphous metal-organic framework (MOF) material. The ratio of copper and GMP is 3:4 as determined by isothermal titration calorimetry. It has excellent laccase-like activity and converts a diverse range of phenol containing substrates such as hydroquinone, naphthol, catechol and epinephrine. Comparative work shows that the activity is originated from guanosine coordination instead of phosphate binding in GMP. Cu2+ is required and cannot be substituted by other metal ions. At the same mass concentration, the Cu/GMP nanozyme has a higher Vmax and similar Km compared to the protein laccase. To achieve the same catalytic efficiency, the cost of the Gu/GMP is ∼2400-fold lower than that of laccase. The Cu/GMP is much more stable at extreme pH, high salt, high temperature and for long-term storage. This is one of the first laccase-mimicking nanozymes, which will find important applications in analytical chemistry, environmental protection, and biotechnology.

Molecular Imprinting for Substrate Selectivity and Enhanced Activity of Enzyme Mimics

Using molecular imprinting, a peroxidase-mimicking DNAzyme has achieved substrate specificity with enhanced activity in a nanoscale gel for three different substrates.

New insights into a classic aptamer: binding sites, cooperativity and more sensitive adenosine detection

Abstract The DNA aptamer for adenosine (also for AMP and ATP) is a highly conserved sequence that has recurred in a few selections. It it a widely used model aptamer for biosensor development, and its nuclear magnetic resonance structure shows that each aptamer binds two AMP molecules. In this work, each binding site was individually removed by rational sequence design, while the remaining site still retained a similar binding affinity and specificity as confirmed by isothermal titration calorimetry. The thermodynamic parameters of binding are presented, and its biochemical implications are discussed. The number of binding sites can also be increased, and up to four sites are introduced in a single DNA sequence. Finally, the different sequences are made into fluorescent biosensors based on the structure-switching signaling aptamer design. The one-site aptamer has 3.8-fold higher sensitivity at lower adenosine concentration with a limit of detection of 9.1 μM adenosine, but weaker fluorescence signal at higher adenosine concentrations, consistent with a moderate cooperativity in the original aptamer. This work has offered insights into a classic aptamer for the relationship between the number of binding sites and sensitivity, and a shorter aptamer for improved biosensor design.

Kinetic Discrimination of Metal Ions Using DNA for Highly Sensitive and Selective Cr3+ Detection

Most metal sensors are designed for a strong binding affinity toward target metal ions, and the underlying principle relies on binding thermodynamics. The kinetic aspect of binding, however, was rarely explored for sensing. In this work, the binding kinetics of 19 common or toxic metal ions are compared based on a fluorescence quenching assay using DNA oligonucleotides as ligands. Among these metals, Cr3+ shows uniquely slow fluorescence quenching kinetics, and the quenched fluorescence cannot be recovered by EDTA or sulfide. Most other metals quenched fluorescence instantaneously and can be fully recovered by these metal chelators. Various factors such as DNA sequence and length, chelating agent, pH, and fluorophore type were studied to understand the binding mechanism, leading to a unique two-stage binding model for Cr3+. This system has a wide dynamic range of up to 50 μM Cr3+ and a low limit of detection of 80 nM. It is also useful for measuring Cr3+ in lake water. This work proposes a new metal sensor design by monitoring binding kinetics with Cr3+ being a primary example.

Intracellular delivery of a molecularly imprinted peroxidase mimicking DNAzyme for selective oxidation

Efficient delivery of protein enzymes and their selective catalysis in living cells is challenging due to their high cost, low stability and large protein size, and the complex intracellular environment. We aimed to develop artificial enzyme mimics that could replace proteins for this purpose. DNAzymes are a type of enzyme mimic made from highly stable DNA. In this study, a molecularly imprinted DNAzyme nanogel was prepared using Amplex Red as the template. The nanogel could selectively oxidize Amplex Red in the presence of H2O2 to form a fluorescent product, while the oxidation of other substrates was inhibited. The activity of this imprinted DNAzyme was even higher than that of the free DNAzyme. The nanogel was efficiently internalized by HeLa cells and intracellular oxidation was also achieved. Therefore, this material provides an integrated solution for biocatalysis inside cells and it might be an interesting solution for intracellular therapeutic applications.

Robust Hydrogels from Lanthanide Nucleotide Coordination with Evolving Nanostructures for a Highly Stable Protein Encapsulation

Metal coordination with organic ligands often produce crystalline metal-organic frameworks and sometimes amorphous nanoparticles. In this work, we explore a different type of material from the same chemistry: hydrogels. Lanthanides are chosen as the metal component because of their important technological applications and continuously tunable properties. Adenosine monophosphate (AMP) and lanthanides form two types of coordination materials: the lighter lanthanides from La3+ to Tb3+ form nanoparticles, whereas the rest heavier ones initially form nanoparticles but later spontaneously transform to hydrogels. This slow sol-to-gel transition is accompanied by heat release, as indicated by isothermal titration calorimetry. The transition is also accompanied by a morphology change from nanoparticles to nanofibers, as indicated by transmission electron microscopy. These gels are insensitive to ionic strength or temperature with excellent stability. Gelation is unique to AMP because other nucleotides or other adenine derivatives only yield nanoparticles or soluble products. Entrapment of guest molecules such as glucose oxidase is also explored, where the hydrogels allow a better enzyme activity and stability compared to nanoparticles. Further applications of lanthanide coordinated hydrogels might include biosensors, imaging agents, and drug delivery.

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.

Continuously Tunable Nucleotide/Lanthanide Coordination Nanoparticles for DNA Adsorption and Sensing

Metal–organic coordination polymers (CPs) have attracted great research interest because they are easy to prepare, porous, flexible in composition, and designable in structure. Their applications in biosensor development, drug delivery, and catalysis have been explored. Lanthanides and nucleotides can form interesting CPs, although most previous works have focused on a single type of metal ligand. In this work, we explored mixed nucleotides and studied their DNA adsorption properties using fluorescently labeled oligonucleotides. Adenosine monophosphate (AMP) and guanosine monophosphate (GMP) formed negatively charged CP nanoparticles with most lanthanides, and thus a salt was required to adsorb negatively charged DNA. DNA adsorption was faster and reached a higher capacity with lighter lanthanides. Desorption of pre-adsorbed DNA by inorganic phosphates, urea, proteins, surfactants, and competing DNA was successively carried out. The results suggested the importance of the DNA phosphate backbone, although hydrogen bonding and DNA bases also contributed to adsorption. The AMP CPs adsorbed DNA more strongly than the GMP ones, and using mixtures of AMP and GMP, continuous tuning of DNA adsorption affinity was achieved. Such CPs were also used as a sensor for DNA detection based on the different affinities of single- and double-stranded DNA, and a detection limit of 0.9 nM target DNA was achieved. Instead of tuning DNA adsorption by varying the length and sequence of DNA, the composition of CPs can also be controlled to achieve this goal.