G-Quadruplex/Hemin DNAzyme-Functionalized Silver Nanoclusters with Synergistic Antibacterial and Wound Healing Capabilities for Infected Wound Management.

Systematic management of infected wounds requires simultaneous antiinfection and wound healing, which has become the current treatment dilemma. Recently, a multifunctional silver nanoclusters (AgNCs)-based hydrogel dressing to meet these demands is developed. Here a diblock DNA with a cytosine-rich fragment (as AgNCs template) and a guanine-rich fragment (to form G-quadruplex/hemin DNAzyme, termed G4/hemin) is designed, for G4/hemin functionalization of AgNCs. Inside bacteria, G4/hemin can not only accelerate the oxidative release of Ag+ from AgNCs but also generate reactive oxygen species (ROS) via catalase- and peroxidase-mimic activities, which enhance the antibacterial effect. On the other hand, the AgNCs exhibit robust anti-inflammatory and antioxidative activities to switch M1 macrophages into M2 phenotype, which promotes wound healing. Moreover, the hemin is released to upregulate the heme oxygenase-1, an intracellular enzyme that can relieve oxidative stress, which significantly alleviates the cytotoxicity of silver. As a result, such silver-based dressing achieves potent therapeutic efficacy on infected wounds with excellent biosafety.

Freezing Functional Nucleic Acids: From Molecular Reactions to Surface Immobilization.

Biochemical reactions are typically slowed down by decreasing temperature. However, accelerated reaction kinetics have been observed for a long time. More recent examples have highlighted the unique role of freezing in fabricating supermaterials, degrading environmental contaminants, and accelerating bioreactions. Functional nucleic acids are DNA or RNA oligonucleotides with versatile properties, including target recognition, catalysis, and molecular co4mputing. In this review, we discuss the current observations and understanding of freezing-facilitated reactions involving functional nucleic acids. Molecular reactions such as ligation/conjugation, cleavage, and hybridization are discussed. Moreover, freezing-induced DNA-nanoparticle conjugations are introduced. Then, we describe our effect in immobilizing DNA on bulk surfaces. Finally, we address some critical questions and research opportunities in the field.

Highly Uniform DNA Monolayers Generated by Freezing-Directed Assembly on Gold Surfaces Enable Robust Electrochemical Sensing in Whole Blood.

Assembling DNA on solid surfaces is fundamental to surface-based DNA technology. However, precise control over DNA conformation and organization at solid-liquid interfaces remains a challenge, resulting in limited stability and sensitivity in biosensing applications. We herein communicate a simple and robust method for creating highly uniform DNA monolayers on gold surfaces by a freeze-thawing process. Using Raman spectroscopy, fluorescent imaging, and square wave voltammetry, we demonstrate that thiolated DNA is concentrated and immobilized on gold surfaces with an upright conformation. Moreover, our results reveal that the freezing-induced DNA surfaces are more uniform, leading to improved DNA stability and target recognition. Lastly, we demonstrate the successful detection of a model drug in undiluted whole blood while mitigating the effects of biofouling. Our work not only provides a simple approach to tailor the DNA-gold surface for biosensors but also sheds light on the unique behavior of DNA oligonucleotides upon freezing on the liquid-solid interface.

Understanding Carbon Nanotube-Based Ionic Diodes: Design and Mechanism.

The rectification of ion transport through biological ion channels has attracted much attention and inspired the thriving invention and applications of ionic diodes. However, the development of high-performance ionic diodes is still challenging, and the working mechanisms of ionic diodes constructed by 1D ionic nanochannels have not been fully understood. This work reports the systematic investigation of the design and mechanism of a new type of ionic diode constructed from horizontally aligned multi-walled carbon nanotubes (MWCNTs) with oppositely charged polyelectrolytes decorated at their two entrances. The major design and working parameters of the MWCNT-based ionic diode, including the ion channel size, the driven voltage, the properties of working fluids, and the quantity and length of charge modification, are extensively investigated through numerical simulations and/or experiments. An optimized ionic current rectification (ICR) ratio of 1481.5 is experimentally achieved on the MWCNT-based ionic diode. These results promise potential applications of the MWCNT-based ionic diode in biosensing and biocomputing. As a proof-of-concept, DNA detection and HIV-1 diagnosis is demonstrated on the ionic diode. This work provides a comprehensive understanding of the working principle of the MWCNT-based ionic diodes and will allow rational device design and optimization.

From general base to general acid catalysis in a sodium-specific DNAzyme by a guanine-to-adenine mutation.

Recently, a few Na+-specific RNA-cleaving DNAzymes were reported, where nucleobases are likely to play critical roles in catalysis. The NaA43 and NaH1 DNAzymes share the same 16-nt Na+-binding motif, but differ in one or two nucleotides in a small catalytic loop. Nevertheless, they display an opposite pH-dependency, implicating distinct catalytic mechanisms. In this work, rational mutation studies locate a catalytic adenine residue, A22, in NaH1, while previous studies found a guanine (G23) to be important for the catalysis of NaA43. Mutation with pKa-perturbed analogs, such as 2-aminopurine (∼3.8), 2,6-diaminopurine (∼5.6) and hypoxanthine (∼8.7) affected the overall reaction rate. Therefore, we propose that the N1 position of G23 (pKa ∼6.6) in NaA43 functions as a general base, while that of A22 (pKa ∼6.3) in NaH1 as a general acid. Further experiments with base analogs and a phosphorothioate-modified substrate suggest that the exocyclic amine in A22 and both of the non-bridging oxygens at the scissile phosphate are important for catalysis for NaH1. This is an interesting example where single point mutations can change the mechanism of cleavage from general base to general acid, and it can also explain this Na+-dependent DNAzyme scaffold being sensitive to a broad range of metal ions and molecules.

DNA Triplex and Quadruplex Assembled Nanosensors for Correlating K+ and pH in Lysosomes.

It remains an unanswered question whether the flux of K+ and H+ in lysosomes are correlated due to difficulties in simultaneously imaging these two ions. This question is of great value for understanding lysosomal acidification. Herein, we designed DNA quadruplex and triplex based luminescent nanosensors that can, respectively monitor K+ and pH in lysosomal lumen. Each sensor contained an upconversion nanoparticle luminophore and a gold nanoparticle quencher, producing green and blue luminescence signals for K+ and H+ , respectively. The sensors were tested in buffers showing dynamic ranges of 5 to 200 mM K+ and pH 5.0 to 8.2. Co-imaging using these two sensors in cells indicated that the influx of H+ was accompanied with the efflux of K+ , solving this long-standing question of the lysosomal biochemistry.

Heating Drives DNA to Hydrophobic Regions While Freezing Drives DNA to Hydrophilic Regions of Graphene Oxide for Highly Robust Biosensors.

Bioconjugation is often performed at ambient temperatures, while freezing and heating may allow different interfacial and inter-/intramolecular interactions. Herein, we report that both freezing and heating allowed more stable DNA adsorption on graphene oxide. Freezing stretched DNA oligonucleotides and drove them to the more oxidized hydrophilic regions on graphene oxide. Heating enhanced hydrophobic interactions and drove DNA to the carbon-rich regions. With a mixture of low-affinity T15 DNA and high-affinity C15 DNA, heating drove the high-affinity DNA to high-affinity regions, achieving ultrahigh adsorption stability, leaving the low-affinity DNA to the remaining low-affinity regions. Using a diblock DNA containing a high-affinity polycytosine block and heating, the nanoflare type of sensor achieved highly sensitive DNA detection in serum with 100-fold improved signal to background ratio, solving a longstanding biosensing problem for robust detection using physisorbed DNA probes.

Promoting DNA Adsorption by Acids and Polyvalent Cations: Beyond Charge Screening.

Adsorbing DNA oligonucleotides onto nanoparticles is the first step in developing DNA-based biosensors, drug delivery systems, and smart materials. Since DNA is a polyanion, it is repelled by negatively charged nanoparticles, which constitute the majority of commonly used nanomaterials. Adding salt such as NaCl to screen charge repulsion is a standard method of promoting DNA adsorption. However, Na+ does not supply additional attractive forces. In addition, adding a high concentration of NaCl can cause the aggregation of nanomaterials. In this feature article, we mainly summarize the methods developed in our laboratory to promote DNA adsorption by lowering the pH and by adding polyvalent metal ions, especially transition-metal ions. Various materials including noble metals (gold, silver, and platinum), 2D materials (graphene oxide, MoS2, WS2, and MXene), polydopamine, and several metal oxides are discussed. In general, low pH can protonate DNA bases and nanoparticle surfaces, reducing charge repulsion and even leading to attraction, although DNA folding at low pH can sometimes be detrimental to adsorption. Polyvalent metal ions can bridge additional interactions to achieve otherwise impossible adsorption. On the basis of the current understanding, a few future research directions are proposed to further improve DNA adsorption.

Stronger Adsorption of Phosphorothioate DNA Oligonucleotides on Graphene Oxide by van der Waals Forces.

Finding DNA sequences that can adsorb strongly on nanomaterials is critical for bioconjugate and biointerface chemistry. In most previous work, unmodified DNA with a phosphodiester backbone (PO DNA) were screened or selected for adsorption on inorganic surfaces. In this work, the adsorption of phosphorothioate (PS)-modified DNA (PS DNA) on graphene oxide (GO) is studied. By use of fluorescently labeled oligonucleotides as probes, all the tested PS DNA strands are adsorbed more strongly on GO compared to the PO DNA of the same sequence. The adsorption mechanism is probed by washing the adsorbed DNA with proteins, surfactants, and urea. Molecular dynamics simulations show that van der Waals forces are responsible for the tighter adsorption of PS DNA. Polycytosine (poly-C) DNA, in general, has a high affinity for the GO surface, and PS poly-C DNA can adsorb even stronger, making it an ideal anchoring sequence on GO. With this knowledge, noncovalent functionalization of GO with a diblock DNA is demonstrated, where a PS poly-C block is used to anchor on the surface. This conjugate achieves better hybridization than the PO DNA of the same sequence for hybridization with the complementary DNA.

Yttrium Oxide as a Strongly Adsorbing but Nonquenching Surface for DNA Oligonucleotides.

A large number of nanomaterials can strongly adsorb DNA and quench fluorescence, such as graphene oxide, gold nanoparticles, and most metal oxides. On the other hand, noncationic nanomaterials that adsorb DNA but cannot quench fluorescence are less known. These materials are attractive for studying the mechanism of DNA-based surface reactions. Y2O3 was found to have this property. Herein, we used fluorescently labeled oligonucleotides as probes to study the mechanism of DNA adsorption. The fluorescence was quenched at low concentrations of Y2O3 and then recovered and even enhanced with higher Y2O3 concentrations. The reason was attributed to the intermolecular quenching by the DNA bases of the neighboring strands. The fluorescence enhancement was due to breaking of the intramolecular fluorophore/DNA interactions, and the most enhancement was observed with a Cy3-labeled DNA. DNA adsorption followed the Langmuir isotherm on Y2O3. Desorption experiments suggested that DNA was adsorbed through the phosphate backbone, with FAM-G15 and FAM-C15 adsorbed more strongly than the other two DNA homopolymers. With a high salt concentration, no fluorescence change was observed, suggesting that the DNA adsorbed in a folded state reducing intermolecular quenching. Overall, Y2O3 might be useful as a model surface for investigating DNA hybridization on a surface.

Interactions between gold, thiol and As(iii) for colorimetric sensing.

As(iii) or arsenite is extremely toxic, and various colorimetric sensors were reported for its on-site detection. A highly cited example was based on gold nanoparticles (AuNPs) modified with a few thiol-containing compounds including dithiothreitol (DTT), reduced glutathione (GSH), and cysteine (Cys). As(iii) was believed to crosslink these surface ligands to aggregate AuNPs and produce a red-to-blue color change. Since As(iii) can also adsorb on AuNPs, we herein carefully studied this effect on these ligand-capped AuNPs. For citrate-capped AuNPs, 10 mM free citrate resulted in a strong blue color in the presence of As(iii) attributable to the elevated ionic strength, while common divalent cations resulted in no color change due to the chelation effect of free citrate. For AuNPs capped with the three thiol compounds, more than 5 mM As(iii) was needed to produce a color change, which was very different from the previously reported color change with nanomolar concentration of As(iii). Our color change was attributed to the displacement of the surface ligands by As(iii) instead of crosslinking by it. This conclusion was made based on the irreversibility of the color change, kinetics of the reaction, and high As(iii) concentration needed. This work has revealed that any two species from AuNPs, thiol and As(iii) can react. It also calls for care in the interpretation of related colorimetric sensing mechanisms, and the need to consider the previously overlooked As(iii) adsorption onto AuNPs.

Opposite salt-dependent stability of i-motif and duplex reflected in a single DNA hairpin nanomachine.

We herein report a DNA hairpin structure containing a polycytosine loop region, and this hairpin can operate like a nanomachine allowing independently controlled stability of the i-motif loop and duplex stem region. This was made possible by the opposite salt-dependent stability of DNA duplex and hairpin, thus providing a new method for designing molecular devices or switches design. A singly-labeled fluorescent method was used to measure the stability of an i-motif DNA in the presence of various metal ions. Salt in general destabilizes the i-motif but stabilizes duplex DNA, allowing us to engineer an i-motif containing hairpin for modulating the stability of each secondary structure independently.

Freezing-Driven DNA Adsorption on Gold Nanoparticles: Tolerating Extremely Low Salt Concentration but Requiring High DNA Concentration.

Attaching thiolated DNA to gold nanoparticles (AuNPs) is a highly important and useful reaction for many applications. Various methods such as adding salts, acids, polymers, and surfactants have been developed to facilitate the reaction. Recently, it was reported that a very high DNA density can be achieved simply by freezing AuNPs with the DNA without any other reagents. DNA oligonucleotides are also known to stretch and align upon freezing. In this work, a set of experiments were performed with a fluorophore and thiol dual-labeled DNA, and the DNA loading density and colloidal stability of AuNPs were measured. The initial salt concentration was unimportant, and even 0.1 mM Na+ allowed around 100 DNA attached to each 13 nm AuNPs. On the other hand, a high DNA concentration of 3 µM was needed to achieve the high DNA density and good colloidal stability of AuNPs. When the thiolated DNA was forced in stable secondary structures, the attachment was low, and preadsorbed DNA also inhibited the DNA attachment by the freezing method. Overall, nonstructured thiolated DNA strands need to align by freezing and quickly attached through the ends of the DNA. This work illustrates practical experiment design conditions and offers fundamental surface science insights for the DNA attachment by freezing.

Mn2+-Assisted DNA Oligonucleotide Adsorption on Ti2C MXene Nanosheets.

As a new type of 2D nanomaterial, MXene (transition metal carbide/nitride) nanosheets are already widely used in catalysis, sensing, and energy research. DNA is a popular sensing molecule. Compared to other 2D materials such as graphene oxide, MoS2, and WS2, few fundamental studies were carried out on DNA adsorption by MXene. Due to its exfoliation and delamination process, the surface of MXene is abundant in -F, -OH, and -O- groups, rendering the surface negatively charged and repelling DNA. In previous studies, surface modification of MXene was performed to promote DNA adsorption. Herein, Mn2+ was discovered to promote DNA adsorption on unmodified Ti2C MXene. Different from Ca2+ and Mg2+, Mn2+ can inverse the ζ-potential of the Ti2C MXene to positive. DNA mainly uses its phosphate backbone for adsorption, while its bases contribute significantly less. In addition, delayed DNA desorption was observed through the addition of inorganic phosphate due to the formation of manganese phosphate to gradually extract Mn2+ from the DNA/MXene complex. Finally, DNA-induced DNA desorption from the Ti2C MXene can hardly distinguish the complementary DNA from a random DNA, which is very different from that for graphene oxide. This difference is likely due to the distinct surface chemistry between the MXene and graphene oxide.

Fluorescein-Stabilized i-Motif DNA and Its Unfolding Leading to a Stronger Adsorption Affinity.

Several previous studies have indicated that polydeoxycytidine (poly-C) DNA has an anomalously high affinity for different types of surfaces. It was hypothesized that the formation of an i-motif structure could be a factor responsible for this enhanced affinity, but this is against the notion that a folded molecule should have fewer interactions with a surface. Herein, the properties of poly-C DNA were examined in detail, focusing on the presence or absence of a FAM (carboxyfluorescein) label and its subsequent adsorption on graphene oxide. Fluorescence and CD spectroscopy studies indicated that FAM can stabilize an i-motif structure in C15 DNA. In particular, the fluorescence of FAM is drastically quenched when the DNA is folded. This structure is irreversibly unfolded upon heating. Furthermore, the unfolded structure has an even higher affinity for graphene oxide than the folded structure. Finally, a large portion of the folded C15 unfolds upon desorption from graphene oxide, and unfolding could happen upon adsorption or desorption of the DNA. This study provides a method to further enhance the adsorption stability of poly-C DNA and calls for care when investigating the potential effects of dye labels on DNA.

Adsorption of Arsenite on Gold Nanoparticles Studied with DNA Oligonucleotide Probes.

Gold nanoparticles (AuNPs) have been extensively used for detecting arsenite, As(III). Many methods rely on a DNA aptamer that claimed to bind specifically to inorganic arsenic. In these cases, the focus was on arsenic binding to the aptamer, while the potential interactions between As(III) and the AuNP surface were ignored. Herein, a set of spectroscopic and isothermal titration calorimetry (ITC) experiments were conducted to measure the adsorption of As(III) by AuNPs and its competition with DNA adsorption. With 10 mM As(III), 18% of adsorbed DNA was displaced from AuNPs, while preadsorption of only 20 µM As(III) inhibited DNA adsorption by around 50%. The affinity of As(III) on AuNPs is comparable to Br- and guanosine. ITC and Raman spectroscopy both indicated that only As(III) can be adsorbed, while As(V) had no measurable interactions with the AuNPs. Based on this understanding, a random DNA sequence was used and a similar colorimetric response in the presence of As(III) was observed. This study confirmed the affinity between As(III) and the gold surface. The As(III)/gold interaction is strong enough to affect DNA adsorption, and care should be taken to interpret the observations based on the color change of AuNPs for the detection of As(III).

Adsorption of DNA Oligonucleotides by Boronic Acid-Functionalized Hydrogel Nanoparticles.

Boronic acid-functionalized hydrogels were commonly used for covalent binding of cis-diol-contained molecules such as glucose, but noncovalent adsorption by boronic acids was less studied. DNA as an important polymer has been used to enhance the function of hydrogels including boronic acid-containing gels. In this work, noncovalent interactions between DNA oligonucleotides and boronic acid-containing hydrogel nanoparticles were studied in detail. The gels were composed of either poly(N-isopropylacrylamide) or with additional 5.6 mol % of 3-acrylamidophenylboronic acid (AAPBA). DNA adsorption on the AAPBA-containing gels was achieved with a high salt concentration, which can be explained by electrostatic repulsion between DNA and boronic acid. The critical role of boronic acid was confirmed by adding competing cis-diol-containing molecules such as glucose, fructose, and cytidine. Hydrogen bonding and hydrophobic interactions are important for DNA adsorption based on inhibited adsorption by urea and dimethyl sulfoxide. Polycytosine DNA showed a higher adsorption capacity compared to the other three types of DNA homopolymers. When T15 and T14-rU were compared, no covalent binding was detected for T14-rU, suggesting that a single terminal diol was insufficient to support covalent binding at the low concentration of DNA used. Finally, the boronic acid-containing gels were able to adsorb an aptamer and inhibit its binding function. Binding was rescued by adding glucose to block the boronic acids. This study demonstrates noncovalent boronic acid interactions with DNA, and this information could be useful for designing and optimization of related biosensors and materials.

Freezing-directed Stretching and Alignment of DNA Oligonucleotides.

Most single-stranded DNA oligonucleotides are random coils with a persistence length of below 1 nm. So far, no good methods are available to stretch oligonucleotides. Herein, it is shown that freezing can stretch DNA, as confirmed using fluorescence resonance energy transfer, thiazole-orange staining, and surface-enhanced Raman spectroscopy. Lateral inter-strand interactions are critical, and the stretched DNA oligonucleotides are aligned. This work also provides a set of methods for studying frozen oligonucleotides. Upon freezing, DNA oligonucleotides are readily adsorbed onto various nanomaterials, including gold nanoparticles, graphene oxide, iron oxide, and WS2 via the most thermodynamically stable conformation, leading to more stable conjugates.

Bromide as a Robust Backfiller on Gold for Precise Control of DNA Conformation and High Stability of Spherical Nucleic Acids.

Functionalization of a gold surface with DNA is often complicated by kinetic traps from unintended DNA base adsorption. Herein, we communicate that Br- serves as a robust backfilling agent displacing selected DNA bases on gold. Traditional thiol backfillers are too strong, while even 300 mM Br- is well tolerated. Conjugates prepared with Br- hybridize 10-fold faster and resist DNA release with better colloidal stability yielding highly sensitive probes. From colorimetric and Raman assays, adsorption affinity ranks as F- < T ≈ Cl- < C < G ≈ Br- < A < I-, allowing Br- to displace nonpoly-A sequences from gold. This well-controlled biointerface will impact biosensing, drug delivery, and directed assembly of nanomaterials.

DNA-Functionalized Nanoceria for Probing Oxidation of Phosphorus Compounds.

Chemical reactions without an obvious optical signal change, such as fluorescence or color, are difficult to monitor. Often, more advanced analytical techniques such as high-performance liquid chromatography and mass spectroscopy are needed. It would be useful to convert such reactions to those with changes in optical signals. In this work, we demonstrate that fluorescently labeled DNA oligonucleotides adsorbed on nanomaterials can probe such reactions, and oxidation of phosphorus-containing species was used as an example. Various metal oxides were tested, and CeO2 nanoparticles were found to be the most efficient for this purpose. Among phosphate, phosphite, and hypophosphite, only phosphate produced a large signal, indicating its strongest adsorption on CeO2 to displace the DNA. This was further used to screen oxidation agents to convert lower oxidation-state compounds to phosphate, and bleach was found to be able to oxidize phosphite. Canonical discriminant analysis was performed to discriminate various phosphorus species using a sensor array containing different metal oxides. On the basis of this, glyphosate was studied for its adsorption and oxidation. Although this method is not specific enough for selective biosensors, it is useful as a tool to produce sensitive optical signals to follow important chemical transformations.