Acoustofluidic one-step production of plasmonic Ag nanoparticles for portable paper-based ultrasensitive SERS detection of bactericides.

SERS measurements for monitoring bactericides in dairy products are highly desired for food safety problems. However, the complicated preparation process of SERS substrates greatly impedes the promotion of SERS. Here, we propose acoustofluidic one-step synthesis of Ag nanoparticles on paper substrates for SERS detection. Our method is economical, fast, simple, and eco-friendly. We adopted laser cutting to cut out appropriate paper shapes, and aldehydes were simultaneously produced at the cutting edge in the pyrolysis of cellulose by laser which were leveraged as the reducing reagent. In the synthesis, only 5 µL of Ag precursor was added to complete the reaction, and no reducing agent was used. Our recently developed acoustofluidic device was employed to intensely mix Ag+ ions and aldehydes and spread the reduced Ag nanoparticles over the substrate. The SERS substrate was fabricated in 1 step and 3 min. The standard R6G solution measurement demonstrated the excellent signal and prominent uniformity of the fabricated SERS substrates. SERS detection of the safe concentration of three bactericides, including tetracycline hydrochloride, thiabendazole, and malachite green, from food samples can be achieved using fabricated substrates. We take the least cost, time, reagents, and steps to fabricate the SERS substrate with satisfying performance. Our work has an extraodinary meaning for the green preparation and large-scale application of SERS.

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.

Quencher-free fluorescent assays by controlled DNA partitioning in the aqueous two-phase system with crowding-enhanced kinetics.

Fluorescent DNA assays are promising in disease diagnosis, environmental monitoring, and drug screening, encompassing both heterogeneous and homogeneous assay types. Nevertheless, heterogeneous assays suffer from tedious washing steps and slow reaction kinetics, whereas homogenous assays require well-designed fluorophore pairs to modulate signal off/on. Herein, we developed a cost-effective and efficient quencher-free fluorescent DNA assay using an aqueous two-phase system (ATPS). Using a strand-displacement reaction, we showed that similar sensing performance could be achieved at a much lower cost. Furthermore, the unique crowding environment in ATPS accelerated strand-displacement reactions by up to six-fold and reduced DNA amplification time from 120 min to 30 min. Our assay demonstrated robust sensing in serum environments and successful detection of miRNA extracted from cells. This innovative assay format has the potential for biosensor development with both heterogeneous readout and rapid reaction kinetics in various applications.

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.

A facile method for purifying DNA-modified small particles and soft materials using aqueous two-phase systems.

We herein report a convenient and effective method for the purification of DNA-conjugated materials with a benchtop minicentrifuge. We demonstrate the fast isolation of DNA-modified small gold nanoparticles (5 nm), liposomes, and DNA nanostructures using fluorescent methods and gel electrophoresis. Our method is cost-effective and efficient and would accelerate the development of DNA nanotechnology.

A rapid fluorescent aptasensor for point-of-care detection of C-reactive protein.

In this paper, we report on a novel fluorescent aptasensor based on aptamers modified by both nitrogen-doped graphene quantum dots (N-GQDs) and gold nanoparticles (AuNPs) for the detection of C-reactive protein (CRP). FRET effect is utilized in our aptasensor by the change of aptamers conformation when binding with the target. An obvious fluorescence quench of the N-GQDs can be observed when CRP appears in the assay due to electron transfer between the donor and accepter. A detection limit of 0.2 ng/mL can be achieved by our sensor in PBS buffer which is much lower than the physiological CRP level in human serum. Also, CRP levels in different patients' serum are tested with our assay. Since our aptasensor is rapid (detection time less than 40 min), one-step and very simple to operate, we believe it has great potential to apply for point-of-care testing (POCT).

Construction of a Mesoporous Ceria Hollow Sphere/Enzyme Nanoreactor for Enhanced Cascade Catalytic Antibacterial Therapy.

Nanozyme has been regarded as one of the antibacterial agents to kill bacteria via a Fenton-like reaction in the presence of H2O2. However, it still suffers drawbacks such as insufficient catalytic activity in near-neutral conditions and the requirement of high H2O2 levels, which would minimize the side effects to healthy tissues. Herein, a mesoporous ceria hollow sphere/enzyme nanoreactor is constructed by loading glucose oxidase in the mesoporous ceria hollow sphere nanozyme. Due to the mesoporous framework, large internal voids, and high specific surface area, the obtained nanoreactor can effectively convert the nontoxic glucose into highly toxic hydroxyl radicals via a cascade catalytic reaction. Moreover, the generated glucose acid can decrease the localized pH value, further boosting the peroxidase-like catalytic performance of mesoporous ceria. The generated hydroxyl radicals could damage severely the cell structure of the bacteria and prevent biofilm formation. Moreover, the in vivo experiments demonstrate that the nanoreactor can efficiently eliminate 99.9% of bacteria in the wound tissues and prevent persistent inflammation without damage to normal tissues in mice. This work provides a rational design of a nanoreactor with enhanced catalytic activity, which can covert glucose to hydroxyl radicals and exhibits potential applications in antibacterial therapy.

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.

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.

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.

Enhancing the peroxidase-like activity and stability of gold nanoparticles by coating a partial iron phosphate shell.

Using citrate-capped gold nanoparticles (AuNPs) as peroxidase-mimicking enzymes to design biosensors is hindered by their low catalytic activity and poor colloidal stability, resulting in limited sensitivity and large variations. Herein, the growth of a partial iron phosphate (FeP) shell with Fe2+ ions on citrate-capped AuNPs boosted the activity of the AuNPs by up to 20-fold. The FeP-enhanced activity was demonstrated on AuNPs of different sizes, and gold nanostars. When the FeP layer is thick enough to block the access to the Au/FeP interface, the activity was inhibited. Capping the remaining Au surface by thiol also inhibited the activity, suggesting that faster reactions occurred at the interfaces of Au/FeP. Moreover, a FeP shell can stabilize AuNPs against freezing and a high NaCl concentration of 1 M. Sensitive detection of Fe2+ was achieved with a detection limit of 0.41 µM, while no other tested transition metal phosphates enhanced the peroxidase-like activity of AuNPs.

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.

Conjugation of antibodies and aptamers on nanozymes for developing biosensors.

Nanozymes are engineered nanomaterials with enzyme-like activities. Over the past decade, impressive progresses on nanozymes in biosensing have been made due to their unique advantages of high stability, low cost, and easy modification compared to natural enzymes. For many biosensors, it is critical to conjugate nanozymes to affinity ligands such as antibodies and aptamers. Since different nanomaterials have different surface properties, conjugation methods need to be compatible with these properties. In addition, the effect of biomolecules on nanozyme activity needs to be considered. In this review, we first categorized nanozyme-based biosensors into four parts, respectively describing noncovalent and covalent modifications with antibodies and aptamers. Meanwhile, recent advances in antibody and aptamer labeled nanozyme biosensors are summarized, and the methods of their conjugation are further illustrated. Finally, conclusions and future perspectives for the development and application of nanozyme bioconjugates are discussed.

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.

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.

A high local DNA concentration for nucleating a DNA/Fe coordination shell on gold nanoparticles.

Preparing DNA/Fe coordination nanoparticles in solution requires a high concentration of DNA. Herein we grew a DNA/Fe shell on DNA-functionalized gold nanoparticles. Taking advantage of the high local DNA density, the required DNA concentration decreased 60-fold, and the size can be controlled. This hybrid material allowed drug loading and colorimetric sensing.

Nucleoside-based fluorescent carbon dots for discrimination of metal ions.

Carbon dots (Cdots) play an important role in many biological and chemical applications. To prepare strongly fluorescent Cdots, the starting material should contain nitrogen in addition to carbon. Nucleobases are nitrogen rich with interesting metal binding properties. In this work, we prepared a series of Cdots with citrate as the carbon source, and ethylenediamine, adenosine, cytidine, thymidine or guanosine as the respective nitrogen sources. The resulting Cdots were all fluorescent with the ethylenediamine sample being the most strongly emissive. These Cdots were then tested for their metal sensitivity and all tested metal ions can quench their fluorescence. The fluorescence of the G-Cdots prepared with guanosine was quenched most efficiently by Cu2+, while the Cdots prepared with ethylenediamine were more sensitive to Hg2+. With the differential quenching by different metal ions, we prepared a sensor array to discriminate multiple metal ions, and quantified Cu2+ and Hg2+ at the same time. Our work has expanded the range of starting materials for preparing Cdots and showed that by tuning the precursor composition, Cdots with different optical and metal binding properties can be obtained, which is useful in constructing a sensing platform for a large number of metal ions.

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.

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.