Covalent Attachment of Horseradish Peroxidase to Single-Walled Carbon Nanotubes for Hydrogen Peroxide Detection.

Single-walled carbon nanotubes (SWCNTs) are desirable nanoparticles for sensing biological analytes due to their photostability and intrinsic near-infrared fluorescence. Previous strategies for generating SWCNT nanosensors have leveraged nonspecific adsorption of sensing modalities to the hydrophobic SWCNT surface that often require engineering new molecular recognition elements. An attractive alternate strategy is to leverage pre-existing molecular recognition of proteins for analyte specificity, yet attaching proteins to SWCNT for nanosensor generation remains challenging. Towards this end, we introduce a generalizable platform to generate protein-SWCNT-based optical sensors and use this strategy to synthesize a hydrogen peroxide (H 2 O 2 ) nanosensor by covalently attaching horseradish peroxidase (HRP) to the SWCNT surface. We demonstrate a concentration-dependent response to H 2 O 2 , confirm the nanosensor can image H 2 O 2 in real-time, and assess the nanosensor's selectivity for H 2 O 2 against a panel of biologically relevant analytes. Taken together, these results demonstrate successful covalent attachment of enzymes to SWCNTs while preserving both intrinsic SWCNT fluorescence and enzyme function. We anticipate this platform can be adapted to covalently attach other proteins of interest including other enzymes for sensing or antibodies for targeted imaging and cargo delivery.

Transition-Metal Dichalcogenide Artificial Antibodies with Multivalent Polymeric Recognition Phases for Rapid Detection and Inactivation of Pathogens.

Antibodies are recognition molecules that can bind to diverse targets ranging from pathogens to small analytes with high binding affinity and specificity, making them widely employed for sensing and therapy. However, antibodies have limitations of low stability, long production time, short shelf life, and high cost. Here, we report a facile approach for the design of luminescent artificial antibodies with nonbiological polymeric recognition phases for the sensitive detection, rapid identification, and effective inactivation of pathogenic bacteria. Transition-metal dichalcogenide (TMD) nanosheets with a neutral dextran phase at the interfaces selectively recognized S. aureus, whereas the nanosheets bearing a carboxymethylated dextran phase selectively recognized E. coli O157:H7 with high binding affinity. The bacterial binding sites recognized by the artificial antibodies were thoroughly identified by experiments and molecular dynamics simulations, revealing the significance of their multivalent interactions with the bacterial membrane components for selective recognition. The luminescent WS2 artificial antibodies could rapidly detect the bacteria at a single copy from human serum without any purification and amplification. Moreover, the MoSe2 artificial antibodies selectively killed the pathogenic bacteria in the wounds of infected mice under light irradiation, leading to effective wound healing. This work demonstrates the potential of TMD artificial antibodies as an alternative to antibodies for sensing and therapy.

Sustainable Nanosheet Antioxidants for Sepsis Therapy via Scavenging Intracellular Reactive Oxygen and Nitrogen Species.

Sepsis is an aberrant systemic inflammatory response mediated by excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Developing an efficient antioxidant therapy for sepsis via scavenging ROS and RNS remains a big challenge owing to the insufficient activity and sustainability of conventional antioxidants. Herein, biocompatible transition-metal dichalcogenide antioxidants with excellent scavenging activity and sustainability for H2O2, O2•-, OH•, and nitric oxide are developed for effective sepsis treatment. WS2, MoSe2, and WSe2 nanosheets exfoliated and functionalized with a biocompatible polymer effectively scavenge mitochondrial and intracellular ROS and RNS in inflammatory cells. Among the nanosheets, WS2 most efficiently suppresses the excessive secretion of inflammatory cytokines along with scavenging ROS and RNS without affecting the expression levels of the anti-inflammatory cytokine and ROS-producing enzymes. The WS2 nanosheets significantly improve the survival rate up to 90% for severely septic mice by reducing systemic inflammation. The pharmacokinetics suggests that the WS2 nanosheets can be excreted from mice 3 days after intravenous injection. This work demonstrates the potential of therapeutic nanosheet antioxidants for effective treatment of ROS and RNS-related diseases.

Modulation of oligonucleotide-binding dynamics on WS2 nanosheet interfaces for detection of Alzheimer's disease biomarkers.

Non-covalent adsorption and desorption of oligonucleotides on two-dimensional nanosheets are widely employed to design nanobiosensors for the rapid optical detection of targets. A precise control over the weak interactions between nanosheet interfaces and oligonucleotides is crucial for a high-sensing performance. Herein, the interface of ultrathin WS2 nanosheets used as a fluorescence quencher was engineered by four different dextran polymers in an aqueous solution to control the adsorption kinetics and thermodynamics of the DNA probe. The WS2 nanosheets, functionalized by the carboxyl group-bearing dextran (CM-dex-WS2) or the trimethylammonium-modified dextran (TMA-dex-WS2), exhibited 3.6-fold faster adsorption rates of the fluorescein-labeled DNA probe (FAM-DNA), which led to the effective fluorescence quenching of FAM, compared to the nanosheets functionalized with pristine dextran (dex-WS2) or the hydrophobic phenoxy groups-bearing dextran (PhO-dex-WS2). Isothermal titration calorimetry measurements showed that the adsorption strength of FAM-DNA for CM-dex-WS2 was one order of magnitude greater than its hybridization energy for a target microRNA (miR-29a) that is well-known as an Alzheimer's disease (AD) biomarker, leading to the unfavorable desorption of the DNA probe from the surface. In contrast, TMA-dex-WS2 exhibited the proper adsorption strength of FAM-DNA, which was lower than its hybridization energy for miR-29a, leading to its favorable desorption from the nanosheet surface along with the noticeable restoration of the quenched fluorescence after its hybridization with miR-29a. Finally, the interface modulation of WS2 nanosheets allowed the selective and sensitive recognition of miR-29a against non-complementary RNA and single base-mismatched RNA in human serum via increases in target-specific fluorescence.

Ultrathin WO3 Nanosheets Converted from Metallic WS2 Sheets by Spontaneous Formation and Deposition of PdO Nanoclusters for Visible Light-Driven C-C Coupling Reactions.

It is not facile to obtain ultrathin two-dimensional (2D) WO3 nanosheets through the exfoliation of their bulk counterpart in solution due to strong covalent interaction between interlayers. In addition, they require additional functionalization with cocatalysts to expand their applicability in photocatalytic organic reactions owing to their insufficient conduction band edge position. Here, we report a chemical approach for the simultaneous production and functionalization of ultrathin 2D WO3 nanosheets through the direct conversion of metallic WS2 nanosheets, accomplished by the spontaneous formation and deposition of PdO nanoclusters on the nanosheet surface in H2O. When chemically exfoliated metallic WS2 nanosheets were simply mixed with K2PdCl4 in H2O under mild conditions (50 °C, 1 h), they were converted to semiconducting WO3 nanosheets on which PdO nanoclusters of a uniform size (∼3 nm) were spontaneously formed, leading to the production of PdO-functionalized ultrathin WO3 (PdO@WO3) nanohybrids. The conversion yield of WO3 nanosheets from metallic WS2 nanosheets increased with increasing coverage of PdO nanoclusters on the nanosheet surface. In addition, the conversion of WO3 nanosheets induced by PdO nanocluster formation was effective only in H2O but not in organic solvents, such as N-methylpyrrolidone and acetonitrile. A mechanical study suggests that the chemisorption of hydrated Pd precursors on the chalcogens of metallic WS2 nanosheets leads to their facile oxidation by water molecules, producing WO3 nanosheets covered with PdO nanoclusters. The as-prepared PdO@WO3 nanosheets exhibited excellent photocatalytic activity and recyclability in Suzuki cross-coupling reactions of various aryl halides under visible light irradiation.

Effective Suppression of Oxidative Stress on Living Cells in Hydrogel Particles Containing a Physically Immobilized WS2 Radical Scavenger.

We report a tungsten disulfide (WS2) nanosheet-immobilized hydrogel system that can inhibit oxidative stress on living cells. First, we fabricated a highly stable suspension of WS2 nanosheets as a radical scavenger by enveloping them with the amphiphilic poly(ε-caprolactone)- b-poly(ethylene oxide) copolymer (PCL- b-PEO) during in situ liquid exfoliation in aqueous medium. After the PCL- b-PEO-enveloped WS2 nanosheets were embedded in three types of hydrogel systems, including carrageenan gum/locust bean gum bulk hydrogels, physically cross-linked alginate microparticles, and covalently cross-linked PEG hydrogel microparticles, they retained their characteristic optical properties. Intriguingly, the WS2 nanosheet-immobilized hydrogel particles exhibited sustainable radical scavenging performance without any deterioration in the original activity of the WS2 nanosheets, even after repeated use. This implies that the hydrogen atoms dissociated from the chalcogen of the WS2 nanosheets effectively scavenged free radicals through the hydrogel mesh. Because of this unique behavior, the coexistence of the WS2 nanosheets with living cells in the hydrogel matrix improved cell viability up to 40%, which demonstrates that the WS2 nanosheets can suppress oxidative stress on living cells.

Reaction Kinetics-Mediated Control over Silver Nanogap Shells as Surface-Enhanced Raman Scattering Nanoprobes for Detection of Alzheimer's Disease Biomarkers.

It is very challenging to accurately quantify the amounts of amyloid peptides Aß40 and Aß42, which are Alzheimer's disease (AD) biomarkers, in blood owing to their low levels. This has driven the development of sensitive and noninvasive sensing methods for the early diagnosis of AD. Here, an approach for the synthesis of Ag nanogap shells (AgNGSs) is reported as surface-enhanced Raman scattering (SERS) colloidal nanoprobes for the sensitive, selective, and multiplexed detection of Aß40 and Aß42 in blood. Raman label chemicals used for SERS signal generation modulate the reaction rate for AgNGSs production through the formation of an Ag-thiolate lamella structure, enabling the control of nanogaps at one nanometer resolution. The AgNGSs embedded with the Raman label chemicals emit their unique SERS signals with a huge intensity enhancement of up to 107 and long-term stability. The AgNGS nanoprobes, conjugated with an antibody specific to Aß40 or Aß42, are able to detect these AD biomarkers in a multiplexed manner in human serum based on the AgNGS SERS signals. Detection is possible for amounts as low as 0.25 pg mL-1 . The AgNGS nanoprobe-based sandwich assay has a detection dynamic range two orders of magnitude wider than that of a conventional enzyme-linked immunosorbent assay.

Fluorescent 2D WS2 Nanosheets Bearing Chemical Affinity Elements for the Recognition of Glycated Hemoglobin.

It is required to exfoliate and functionalize 2D transition metal dichalcogenides (TMDs) in an aqueous solution for biological and medical applications. Herein, the approach for the simultaneous exfoliation and functionalization of 2D WS2 nanosheets using boronic acid-modified poly(vinyl alcohol) (B-PVA) in an aqueous solution is reported, and the B-PVA-functionalized WS2 nanosheets (B-PVA-WS2 ) are exploited as a fluorescent biosensor for the detection of glycated hemoglobin, HbA1c. The synthetic B-PVA polymer facilitates the exfoliation and functionalization of WS2 nanosheets from the bulk counterpart in the aqueous solution via a pulsed sonication process, resulting in fluorescent B-PVA-WS2 nanohybrids with a specific recognition of HbA1c. The fluorescence of the B-PVA-WS2 is quenched in the presence of HbA1c, whereas PVA-functionalized WS2 (PVA-WS2 ), not bearing boronic acid as a recognition moiety, shows no fluorescence changes upon the addition of the target. The B-PVA-WS2 is able to selectively detect HbA1c at the concentration as low as 3.3 × 10-8 m based on its specific fluorescence quenching.

ß-Lactoglobulin Peptide Fragments Conjugated with Caffeic Acid Displaying Dual Activities for Tyrosinase Inhibition and Antioxidant Effect.

The regulation of tyrosinase activity and reactive oxygen species is of great importance for the prevention of dermatological disorders in the fields of medicine and cosmetics. Herein, we report a strategy based on solid-phase peptide chemistry for the synthesis of ß-lactoglobulin peptide fragment/caffeic acid (CA) conjugates (CA-Peps) with dual activities of tyrosinase inhibition and antioxidation. The purity of the prepared conjugates, CA-MHIR, CA-HIRL, and CA-HIR, significantly increased to 99%, as acetonide-protected CA was employed in solid-phase coupling reactions on Rink amide resins. The tyrosinase inhibitory activities of all CA-Pep derivatives were higher than the activity of kojic acid, and CA-MHIR exhibited the highest tyrosinase inhibition activity (IC50 = 47.9 µM). Moreover, CA-Pep derivatives displayed significantly enhanced antioxidant activities in the peroxidation of linoleic acid as compared to the pristine peptide fragments. All CA-Pep derivatives showed no cytotoxicity against B16-F1 melanoma cells.

Adjustable Intermolecular Interactions Allowing 2D Transition Metal Dichalcogenides with Prolonged Scavenging Activity for Reactive Oxygen Species.

There is an increasing demand for control over the dimensions and functions of transition metal dichalcogenides (TMDs) in aqueous solution toward biological and medical applications. Herein, an approach for the exfoliation and functionalization of TMDs in water via modulation of the hydrophobic interaction between poly(ε-caprolactone)-b-poly(ethylene glycol) (PCL-b-PEG) and the basal planes of TMDs is reported. Decreasing the hydrophobic PCL length of PCL-b-PEG from 5000 g mol-1 (PCL5000 ) to 460 g mol-1 (PCL460 ) significantly increases the exfoliation efficiency of TMD nanosheets because the polymer-TMD hydrophobic interaction becomes dominant over the polymer-polymer interaction. The TMD nanosheets exfoliated by PCL460 -b-PEG5000 (460-WS2 , 460-WSe2 , 460-MoS2 , and 460-MoSe2 ) show excellent and prolonged scavenging activity for reactive oxygen species (ROS), but each type of TMD displays a different scavenging tendency against hydroxyl, superoxide, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals. A mechanistic study based on electron paramagnetic resonance spectroscopy and density functional theory simulations suggests that radical-mediated oxidation of TMDs and hydrogen transfer from the oxidized TMDs to radicals are crucial steps for ROS scavenging by TMD nanosheets. As-prepared 460-TMDs are able to effectively scavenge ROS in HaCaT human keratinocytes, and also exhibit excellent biocompatibility.

Monodisperse Microshell Structured Gelatin Microparticles for Temporary Chemoembolization.

Embolization is a nonsurgical, minimally invasive procedure that deliberately blocks a blood vessel. Although several embolic particles have been commercialized, their much wider applications have been hampered owing mainly to particle size variation and uncontrollable degradation kinetics. Herein we introduce a microfluidic approach to fabricate highly monodisperse gelatin microparticles (GMPs) with a microshell structure. For this purpose, we fabricate uniform gelatin emulsion precursors using a microfluidic technique and consecutively cross-link them by inbound diffusion of glutaraldehyde from the oil continuous phase to the suspending gelatin precursor droplets. A model micromechanic study, carried out in an artificial blood vessel, demonstrates that the extraordinary degradation kinetics of the GMPs, which stems from the microshell structure, enables controlled rupturing while exhibiting drug release under temporary chemoembolic conditions.

Structuring Pd Nanoparticles on 2H-WS2 Nanosheets Induces Excellent Photocatalytic Activity for Cross-Coupling Reactions under Visible Light.

Effective photocatalysts and their surface engineering are essential for the efficient conversion of solar energy into chemical energy in photocatalyzed organic transformations. Herein, we report an effective approach for structuring Pd nanoparticles (NPs) on exfoliated 2H-WS2 nanosheets (WS2/PdNPs), resulting in hybrids with extraordinary photocatalytic activity in Suzuki reactions under visible light. Pd NPs of different sizes and densities, which can modulate the photocatalytic activity of the as-prepared WS2/PdNPs, were effectively structured on the basal plane of 2H-WS2 nanosheets via a sonic wave-assisted nucleation method without any reductants at room temperature. As the size of Pd NPs on WS2/PdNPs increased, their photocatalytic activity in Suzuki reactions at room temperature increased substantially. In addition, it was found that protic organic solvents play a crucial role in activating WS2/PdNPs catalysts in photocatalyzed Suzuki reactions, although these solvents are generally considered much less effective than polar aprotic ones in the conventional Suzuki reactions promoted by heterogeneous Pd catalysts. A mechanistic investigation suggested that photogenerated holes are transferred to protic organic solvents, whereas photogenerated electrons are transferred to Pd NPs. This transfer makes the Pd NPs electron-rich and accelerates the rate-determining step, i.e., the oxidative addition of aryl halides under visible light. WS2/PdNPs showed the highest turnover frequency (1244 h-1) for photocatalyzed Suzuki reactions among previously reported photocatalysts.

2H-WS2 Quantum Dots Produced by Modulating the Dimension and Phase of 1T-Nanosheets for Antibody-Free Optical Sensing of Neurotransmitters.

Modulating the dimensions and phases of transition metal dichalcogenides is of great interest to enhance their intrinsic properties or to create new physicochemical properties. Herein, we report an effective approach to synthesize 2H-WS2 quantum dots (QDs) via the dimension and phase engineering of 1T-WS2 nanosheets. The solvothermal reaction of chemically exfoliated 1T-WS2 nanosheets in N-methyl-2-pyrrolidone (NMP) under an N2 atmosphere induced their chopping and phase transition at lower temperature to produce 2H-WS2 QDs with a high quantum yield (5.5 ± 0.3%). Interestingly, this chopping and phase transition process showed strong dependency on solvent; WS2 QDs were not produced in other solvents such as 1,4-dioxane and dimethyl sulfoxide. Mechanistic investigations suggested that NMP radicals played a crucial role in the effective production of 2H-WS2 QDs from 1T-WS2 nanosheets. WS2 QDs were successfully applied for the selective, sensitive, and rapid detection of dopamine in human serum (4 min, as low as 23.8 nM). The intense fluorescence of WS2 QDs was selectively quenched upon the addition of dopamine and Au3+ ions due to fluorescence resonance energy transfer between WS2 QDs and the quickly formed Au nanoparticles. This new sensing principle enabled us to discriminate dopamine from dopamine-derivative neurotransmitters including epinephrine and norepinephrine, as well as other interference compounds.

Optical Detection of Enzymatic Activity and Inhibitors on Non-Covalently Functionalized Fluorescent Graphene Oxide.

It has been of great interest to measure the activity of acetylcholinesterase (AChE) and its inhibitor, as AChE is known to accelerate the aggregation of the amyloid beta peptides that underlie Alzheimer's disease. Herein, we report the development of graphene oxide (GO) fluorescence-based biosensors for the detection of AChE activity and AChE inhibitors. To this end, GO was non-covalently functionalized with phenoxy-modified dextran (PhO-dex-GO) through hydrophobic interaction; the resulting GO showed excellent colloidal stability and intense fluorescence in various aqueous solutions as compared to pristine GO and the GO covalently functionalized with dextran. The fluorescence of PhO-dex-GO remarkably increased as AChE catalyzed the hydrolysis of acetylthiocholine (ATCh) to give thiocholine and acetic acid. It was found that the turn-on fluorescence response of PhO-dex-GO to AChE activity was induced by protonation of carboxyl groups on it from the product of the enzymatic hydrolysis reaction, acetic acid. On the basis of its turn-on fluorescence response, PhO-dex-GO was able to report kinetic and thermodynamic parameters involving a maximum velocity, a Michaelis constant, and an inhibition dissociation constant for AChE activity and inhibition. These parameters enable us to determine the activity of AChE and the efficiency of the inhibitor.

Photoactive WS2 nanosheets bearing plasmonic nanoparticles for visible light-driven reduction of nitrophenol.

Semiconducting WS2 nanohybrids with different sizes of silver nanoparticles are designed via amine-assisted in situ reduction and growth of Ag(+) ions. These nanohybrids exhibit characteristic photocatalytic activity for the reduction of 4-nitrophenol as a function of their structure.

Orientation and density control of bispecific anti-HER2 antibody on functionalized carbon nanotubes for amplifying effective binding reactivity to cancer cells.

Nanomaterial bioconjugates have gained unabated interest in the field of sensing, imaging and therapy. As a conjugation process significantly affects the biological functions of proteins, it is crucial to attach them to nanomaterials with control over their orientation and the nanomaterial-to-protein ratio in order to amplify the binding efficiency of nanomaterial bioconjugates to targets. Here, we describe a targeting nanomaterial platform utilizing carbon nanotubes functionalized with a cotinine-modified dextran polymer and a bispecific anti-HER2 × cotinine tandem antibody. This new approach provides an effective control over antibody orientation and density on the surface of carbon nanotubes through site-specific binding between the anti-cotinine domain of the bispecific tandem antibody and the cotinine group of the functionalized carbon nanotubes. The developed synthetic carbon nanotube/bispecific tandem antibody conjugates (denoted as SNAs) show an effective binding affinity against HER2 that is three orders of magnitude higher than that of the carbon nanotubes bearing a randomly conjugated tandem antibody prepared by carbodiimide chemistry. As the density of a tandem antibody on SNAs increases, their effective binding affinity to HER2 increases as well. SNAs exhibit strong resonance Raman signals for signal transduction, and are successfully applied to the selective detection of HER2-overexpressing cancer cells.

Graphene oxide-encoded Ag nanoshells with single-particle detection sensitivity towards cancer cell imaging based on SERRS.

Developing ultrasensitive Raman nanoprobes is one of the emerging interests in the field of biosensing and bioimaging. Herein, we constructed a new type of surface-enhanced resonance Raman scattering nanoprobe composed of an Ag nanoshell as a surface-enhanced Raman scattering-active nanostructure, which was encapsulated with 4,7,10-trioxa-1,13-tridecanediamine-functionalized graphene oxide as an ultrasensitive Raman reporter exhibiting strong resonance Raman scattering including distinct D and G modes. The designed nanoprobe was able to produce much more intense and simpler Raman signals even at a single particle level than the Ag nanoshell bearing a well-known Raman reporter, which is beneficial for the sensitive detection of a target in a complex biological system. Finally, this ultrasensitive nanoprobe successfully demonstrated its potential for bioimaging of cancer cells using Raman spectroscopy.

Chemically-modulated photoluminescence of graphene oxide for selective detection of neurotransmitter by "turn-on" response.

Designing artificial nanomaterials capable of selectively detecting targets without the use of expensive and fragile antibodies is of great interest in the applications of nanomedicine. Here, we show that the photoluminescence (PL) of graphene oxide (GO) was chemically modulated for the selective detection of a neurotransmitter without the use of antibodies. GO was functionalized with nitrotriacetic acid (NTA) on which four different metal ions were chelated (M-NTA-GO), which led to its different PL responses to neurotransmitters. In particular, the Cu-NTA-GO hybrid was able to selectively detect norepinephrine at nanomolar concentrations in a simple manner via its "turn-on" PL. Moreover, it was successfully applied to the selective detection of norepinephrine secreted from living PC-12 cells.