Freezing Directed Construction of Bio/Nano Interfaces: Reagentless Conjugation, Denser Spherical Nucleic Acids, and Better Nanoflares

While nanoparticle solutions cannot freeze in general, they may remain stable in the presence of polymer stabilizers. We herein communicate that gold nanoparticles (AuNPs) are stable in the presence of thiolated DNA after a freeze–thaw cycle. The DNA is conjugated to AuNPs during freezing without additional reagents and the conjugation can be completed in a few minutes. More importantly, the DNA density is 20–30% higher than that prepared by the typical salt-aging method. By lowering temperature, DNA hybridization is also promoted, allowing the construction of better nanoflares with doubled probe density and signaling sensitivity. This freezing method works for AuNPs from 5 to 100 nm and all tested DNA sequences. The mechanism was studied by separating the effect of temperature, freezing and thawing, where the exclusion of salt and AuNPs by the growing ice crystals is deemed critical. In addition to developing a simple method, this study articulates unique physical processes during freezing with important fundamental surface science implications, and it could be extended to other systems

Parallel Polyadenine Duplex Formation at Low pH Facilitates DNA Conjugation onto Gold Nanoparticles

DNA-functionalized gold nanoparticles (AuNPs) have been extensively used in sensing, drug delivery, and materials science. A key step is to attach DNA to AuNPs, forming a stable and functional conjugate. Although the traditional salt-aging method takes a full day or longer, a recent low-pH method allows DNA conjugation in a few minutes. The effect of low pH was attributed to the protonation of adenine (A) and cytosine (C), resulting in an overall lower negative charge density on DNA. In this work, the effect of DNA conformation at low pH is studied. Using circular dichroism (CD) spectroscopy, the parallel poly-A duplex (A-motif) is detected when a poly-A segment is linked to a random DNA, a design typically used for DNA conjugation. A DNA staining dye, thiazole orange, is identified for detecting such A-motifs. The A-motif structure is ideal for DNA conjugation because it exposes the thiol group to directly react with the gold surface while minimizing nonspecific DNA base adsorption. For nonthiolated DNA, the optimal procedure is to incubate DNA and AuNPs followed by lowering the pH. The i-motif formed by poly-C DNA at low pH is less favorable to the conjugation reaction because of its unique way of folding. The stability of poly-A and poly-G DNA at low pH is examined. An excellent stability of poly-A DNA is confirmed, but poly-G has lower stability. This study provides new fundamental insights into a practically useful technique of conjugating DNA to AuNPs

Cation-Size-Dependent DNA Adsorption Kinetics and Packing Density on Gold Nanoparticles: An Opposite Trend

The property of DNA is strongly influenced by counterions. Packing a dense layer of DNA onto a gold nanoparticle (AuNP) generates an interesting colloidal system with many novel physical properties such as a sharp melting transition, protection of DNA against nucleases, and enhanced complementary DNA binding affinity. In this work, the effect of monovalent cation size is studied. First, for free AuNPs without DNA, larger group 1A cations are more efficient in inducing their aggregation. The same trend is observed with group 2A metals using AuNPs capped by various self-assembled monolayers. After establishing the salt range to maintain AuNP stability, the DNA adsorption kinetics is also found to be faster with the larger Cs⁺ compared to the smaller Li⁺. This is attributed to the easier dehydration of Cs⁺, and dehydrated Cs⁺ might condense on the AuNP surface to reduce the electrostatic repulsion effectively. However, after a long incubation time with a high salt concentration, Li⁺ allows ∼30% more DNA packing compared to Cs⁺. Therefore, Li⁺ is more effective in reducing the charge repulsion among DNA, and Cs⁺ is more effective in screening the AuNP surface charge. This work suggests that physicochemical information at the bio/nanointerface can be obtained by using counterions as probes

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

Magnetic Iron Oxide Nanoparticle Seeded Growth of Nucleotide Coordinated Polymers

The introduction of functional molecules to the surface of magnetic iron oxide nanoparticles (NPs) is of critical importance. Most previously reported methods were focused on surface ligand attachment either by physisorption or covalent conjugation, resulting in limited ligand loading capacity. In this work, we report the seeded growth of a nucleotide coordinated polymer shell, which can be considered as a special form of adsorption by forming a complete shell. Among all of the tested metal ions, Fe³⁺ is the most efficient for this seeded growth. A diverse range of guest molecules, including small organic dyes, proteins, DNA, and gold NPs, can be encapsulated in the shell. All of these molecules were loaded at a much higher capacity compared to that on the naked iron oxide NP core, confirming the advantage of the coordination polymer (CP) shell. In addition, the CP shell provides better guest protein stability compared to that of simple physisorption while retaining guest activity as confirmed by the entrapped glucose oxidase assay. Use of this system as a peroxidase nanozyme and glucose biosensor was demonstrated, detecting glucose as low as 1.4 μM with excellent stability. This work describes a new way to functionalize inorganic materials with a biocompatible shell

Dissecting Colloidal Stabilization Factors in Crowded Polymer Solutions by Forming Self-Assembled Monolayers on Gold Nanoparticles

An ideal colloidal system should be highly stable in a diverse range of buffer conditions while still retaining its surface accessibility. We recently reported that dispersing citrate-capped gold nanoparticles (AuNPs) in polymers, such as polyethylene glycol (PEG), can achieve such a goal because of contributions from depletion stabilization. Because AuNPs can weakly adsorb PEG to exert steric stabilization and the remaining citrate can impart charge stabilization, the extent of the contribution of depletion stabilization is unclear. In this work, we aim to dissect the contribution of each stabilizing factor. This is achieved by coating AuNPs with a layer of thiolated compound, which inhibits the adsorption of PEG and also allows for the control of surface charge. We found that depletion stabilization alone was insufficient to stabilize AuNPs at room temperature. However, when working together with other stabilization mechanisms, ultrahigh stability can be achieved. The size of both AuNPs and PEG was systematically varied, and the trends were compared to theoretical calculations. Finally, we report the importance of the surface chemistry of commercial AuNPs

Rationally Designed Nucleobase and Nucleotide Coordinated Nanoparticles for Selective DNA Adsorption and Detection

Nanomaterials for DNA adsorption are useful for sequence-specific DNA detection. Current materials for DNA adsorption employ electrostatic attraction, hydrophobic interaction, or π–π stacking, none of which can achieve sequence specificity. Specificity might be improved by involving hydrogen bonding and metal coordination. In this work, a diverse range of nucleobase/nucleotide (adenine, adenosine, adenosine 5′-triphosphate (ATP), adenosine 5′-monophosphate (AMP), and guanosine 5′-triphosphate (GTP)) coordinated materials containing various metal ions (Au­(III), Ag­(I), Ce­(III), Gd­(III), and Tb­(III)) are prepared. In most cases, nanoparticles are formed. These materials have different surface charges, and positively charged particles only show nonspecific DNA adsorption. Negatively charged materials give different adsorption kinetics for different DNA sequences, where complementary DNA homopolymers are adsorbed faster than other sequences. Therefore, the bases in the coordinated materials can still form base pairs with the DNA. The adsorption strength is mainly controlled by the metal ions, where Au shows the strongest adsorption while lanthanides are weaker. These materials can be used as sensors for DNA detection and can also deliver DNA into cells with no detectable toxicity. By tuning the nanoparticle formulation, enhanced detection can be achieved. This study is an important step toward rational design of materials to achieve specific interactions between biomolecules and synthetic nanoparticle surfaces

Hydrogen Peroxide Displacing DNA from Nanoceria: Mechanism and Detection of Glucose in Serum

Hydrogen peroxide (H₂O₂) is a key molecule in biology. As a byproduct of many enzymatic reactions, H₂O₂ is also a popular biosensor target. Recently, interfacing H₂O₂ with inorganic nanoparticles has produced a number of nanozymes showing peroxidase or catalase activities. CeO₂ nanoparticle (nanoceria) is a classical nanozyme. Herein, a fluorescently labeled DNA is used as a probe, and H₂O₂ can readily displace adsorbed DNA from nanoceria, resulting in over 20-fold fluorescence enhancement. The displacement mechanism instead of oxidative DNA cleavage is confirmed by denaturing gel electrophoresis and surface group p<i>K</i>_a measurement. This system can sensitively detect H₂O₂ down to 130 nM (4.4 parts-per-billion). When coupled with glucose oxidase, glucose is detected down to 8.9 μM in buffer. Detection in serum is also achieved with results comparable with that from a commercial glucose meter. With an understanding of the ligand role of H₂O₂, new applications in rational materials design, sensor development, and drug delivery can be further exploited

Instantaneous Attachment of an Ultrahigh Density of Nonthiolated DNA to Gold Nanoparticles and Its Applications

The last 16 years have witnessed the landmark development of polyvalent thiolated DNA-functionalized gold nanoparticles (AuNP's) possessing striking properties within the emerging field of nanobiotechnology. Many novel properties of this hybrid nanomaterial are attributed to the dense DNA shell. However, the question of whether nonthiolated polyvalent DNA–AuNP could be fabricated with a high DNA density and properties similar to those of its thiolated counterpart has not been explored in detail. Herein, we report that by simply tuning the pH of the DNA–AuNP mixture an ultrahigh capacity of nonthiolated DNA can be conjugated to AuNP's in a few minutes, resulting in polyvalent DNA–AuNP conjugates with cooperative melting behavior, a typical property of polyvalent thiolated DNA-functionalized AuNP's. With this method, large AuNP's (e.g., 50 nm) can be functionalized to achieve the colorimetric detection of sub-nanometer DNA. Furthermore, this fast, stable DNA loading was employed to separate AuNP's of different sizes. We propose that a large fraction of the attached DNAs are adsorbed via one or a few terminal bases to afford the high loading capacity and the ability to hybridize with the complementary DNA. This discovery not only offers a time- and cost-effective way to functionalize AuNP's with a high density of nonthiolated DNA but also provides new insights into the fundamental understanding of how DNA strands with different sequences interact with AuNP's

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