Transient Metallo-Lipidoid Assemblies Amplify Covalent Catalysis of Aqueous and Non-Aqueous Reactions.

Dissipative supramolecular assemblies are hallmarks of living systems, contributing to their complex, dynamic structures and emerging functions. Living cells can spatiotemporally control diverse biochemical reactions in membrane compartments and condensates, regulating metabolite levels, signal transduction or remodeling of the cytoskeleton. Herein, we constructed membranous compartments using self-assembly of lipid-like amphiphiles (lipidoid) in aqueous medium. The new double-tailed lipidoid features Cu(II) coordinated with a tetravalent chelator that dictates the binding of two amphiphilic ligands in cis-orientation. Hydrophobic interactions between the lipidoids coupled with intermolecular hydrogen bonding led to a well-defined bilayer vesicle structure. Oil-soluble SNAr reaction is efficiently upregulated in the hydrophobic cavity, acting as a catalytic crucible. The modular system allows easy incorporation of exposed primary amine groups, which augments the catalysis of retro aldol and C-N bond formation reactions. Moreover, a higher-affinity chelator enables consumption of the Cu(II) template leveraging the differential thermodynamic stability, which allows a controllable lifetime of the vesicular assemblies. Concomitant temporal upregulation of the catalytic reactions could be tuned by the metal ion concentration. This work offers new possibilities for metal ion-mediated dynamic supramolecular systems, opening up a massive repertoire of functionally active dynamic "life-like" materials.

Photo-Controlled Gating of Selective Bacterial Membrane Interaction and Enhanced Antibacterial Activity for Wound Healing.

Reversible biointerfaces are essential for on-demand molecular recognition to regulate stimuli-responsive bioactivity such as specific interactions with cell membranes. The reversibility on a single platform allows the smart material to kill pathogens or attach/detach cells. Herein, we introduce a 2D-MoS2 functionalized with cationic azobenzene that interacts selectively with either Gram-positive or Gram-negative bacteria in a light-gated fashion. The trans conformation (trans-Azo-MoS2 ) selectively kills Gram-negative bacteria, whereas the cis form (cis-Azo-MoS2 ), under UV light, exhibits antibacterial activity against Gram-positive strains. The mechanistic investigation indicates that the cis-Azo-MoS2 exhibits higher affinity towards the membrane of Gram-positive bacteria compared to trans-Azo-MoS2 . In case of Gram-negative bacteria, trans-Azo-MoS2 internalizes more efficiently than cis-Azo-MoS2 and generates intracellular ROS to kill the bacteria. While the trans-Azo-MoS2 exhibits strong electrostatic interactions and internalizes faster into Gram-negative bacterial cells, cis-Azo-MoS2 primarily interacts with Gram-positive bacteria through hydrophobic and H-bonding interactions. The difference in molecular mechanism leads to photo-controlled Gram-selectivity and enhanced antibacterial activity. We found strain-specific and high bactericidal activity (minimal bactericidal concentration, 0.65 µg/ml) with low cytotoxicity, which we extended to wound healing applications. This methodology provides a single platform for efficiently switching between conformers to reversibly control the strain-selective bactericidal activity regulated by light.

Light-gated specific oxidase-like activity of a self-assembled Pt(II) nanozyme for environmental remediation.

Artificial enzyme equivalents, also known as nanozymes, are a practical tool for environmental remediation when compared to their natural counterparts due to their high operational stability, efficiency, and cost-effectiveness. Specific oxidase mimicking nanozymes are well suited to degrade toxic chemicals from industrial waste such as phenols and azo dyes. Therefore, photocatalytic nanozymes using visible/sunlight would provide a viable strategy for sustainable environmental remediation. Herein, we introduce an aggregation-induced emissive Pt(II) complex, which self-assembles in water providing NanoPtA nanotapes. These structures exhibit a specific oxidase-like nanozyme activity driven by light. The NanoPtA structure assists in the photogeneration of singlet oxygen in water via a triplet excited 3MMLCT state, leading to a specific oxidase-like activity instead of a peroxidase-like activity. The self-assembled nanozyme showed great stability under harsh environmental conditions and exhibited photo-induced specific oxidase-mimetic activity, which was considerably more efficient than the natural enzyme or other specific nanozymes. We demonstrated efficient NanoPtA-induced photocatalytic degradation of various phenolic compounds and azo dyes within 5-10 minutes of light irradiation. Notably, the system operates under sunlight and exhibits reusability over twenty cycles of catalytic reactions. Another fascinating aspect of NanoPtA is the unaltered catalytic performance for more than 75 days, providing a robust enzyme-equivalent for practical sustainable environmental remediation.

A transient vesicular glue for amplification and temporal regulation of biocatalytic reaction networks.

Regulation of enzyme activity and biocatalytic cascades on compartmentalized cellular components is key to the adaptation of cellular processes such as signal transduction and metabolism in response to varying external conditions. Synthetic molecular glues have enabled enzyme inhibition and regulation of protein-protein interactions. So far, all the molecular glue systems based on covalent interactions operated under steady-state conditions. To emulate dynamic biological processes under dissipative conditions, we introduce herein a transient supramolecular glue with a controllable lifetime. The transient system uses multivalent supramolecular interactions between guanidinium group-bearing surfactants and adenosine triphosphate (ATP), resulting in bilayer vesicle structures. Unlike the conventional chemical agents for dissipative assemblies, ATP here plays the dual role of providing a structural component for the assembly as well as presenting active functional groups to "glue" enzymes on the surface. While gluing of the enzymes on the vesicles achieves augmented catalysis, oscillation of ATP concentration allows temporal control of the catalytic activities similar to the dissipative cellular nanoreactors. We further demonstrate temporal upregulation and control of complex biocatalytic reaction networks on the vesicles. Altogether, the temporal activation of biocatalytic cascades on the dissipative vesicular glue presents an adaptable and dynamic system emulating heterogeneous cellular processes, opening up avenues for effective protocell construction and therapeutic interventions.

Sequestration of histidine kinases by non-cognate response regulators establishes a threshold level of stimulation for bacterial two-component signaling.

Bacterial two-component systems (TCSs) consist of a sensor histidine kinase (HK) that perceives a specific signal, and a cognate response regulator (RR) that modulates the expression of target genes. Positive autoregulation improves TCS sensitivity to stimuli, but may trigger disproportionately large responses to weak signals, compromising bacterial fitness. Here, we combine experiments and mathematical modelling to reveal a general design that prevents such disproportionate responses: phosphorylated HKs (HK~Ps) can be sequestered by non-cognate RRs. We study five TCSs of Mycobacterium tuberculosis and find, for all of them, non-cognate RRs that show higher affinity than cognate RRs for HK~Ps. Indeed, in vitro assays show that HK~Ps preferentially bind higher affinity non-cognate RRs and get sequestered. Mathematical modelling indicates that this sequestration would introduce a 'threshold' stimulus strength for eliciting responses, thereby preventing responses to weak signals. Finally, we construct tunable expression systems in Mycobacterium bovis BCG to show that higher affinity non-cognate RRs suppress responses in vivo.

Metal Coordination-Driven Supramolecular Nanozyme as an Effective Colorimetric Biosensor for Neurotransmitters and Organophosphorus Pesticides.

Analytical methods for detecting neurotransmitters (NTs) and organophosphorus (OP) pesticides with high sensitivity are vitally necessary for the rapid identification of physical, mental, and neurological illnesses, as well as to ensure food safety and safeguard ecosystems. In this work, we developed a supramolecular self-assembled system (SupraZyme) that exhibits multi-enzymatic activity. SupraZyme possesses the ability to show both oxidase and peroxidase-like activity, which has been employed for biosensing. The peroxidase-like activity was used for the detection of catecholamine NTs, epinephrine (EP), and norepinephrine (NE) with a detection limit of 6.3 µM and 1.8 µM, respectively, while the oxidase-like activity was utilized for the detection of organophosphate pesticides. The detection strategy for OP chemicals was based on the inhibition of acetylcholine esterase (AChE) activity: a key enzyme that is responsible for the hydrolysis of acetylthiocholine (ATCh). The corresponding limit of detection of paraoxon-methyl (POM) and methamidophos (MAP) was measured to be 0.48 ppb and 15.8 ppb, respectively. Overall, we report an efficient supramolecular system with multiple enzyme-like activities that provide a versatile toolbox for the construction of sensing platforms for the colorimetric point-of-care detection of both NTs and OP pesticides.

Ligand Exchange on MoS2 Nanosheets: Applications in Array-Based Sensing and Drug Delivery.

Two-dimensional MoS2 nanosheets (2D-MoS2) have been widely used in many biological applications due to their distinctive physicochemical properties. Further, the development of surface modification using thiolated ligands allows us to use them for many specific applications. But the effect of possible ligand exchange on 2D-MoS2 has never been explored, which can play an important role in diverse biological applications. In this study, we have observed the ligand-exchange phenomenon on 2D-MoS2 in the presence of different thiolated ligands. The initial study proceeded with boron-dipyrromethene (BODIPY) functionalized MoS2 with different concentrations of glutathione (GSH), which is the most abundant thiol species in the cytoplasm of various cancer cells. It was found that in the presence of GSH the fluorescence of BODIPY can be regenerated, which is time and concentration dependent. We have also examined this phenomenon with different thiol ligands and transition-metal dichalcogenides (TMDs). We observed a variable rate of ligand exchange in different solvents, surface functionality, and receptor environments that helped us to construct sensor arrays. Interestingly, a ligand-exchange process was not observed in the presence of dithiols. Further, this concept was applied to a cancerous cell line for in vitro delivery. We found that BODIPY-functionalized 2D-MoS2 undergoes thiol exchange by intracellular GSH and subsequently enhanced the fluorescence in the cytoplasm of cancer cells. This strategy can be applied to the development of 2D-TMD-based materials for various biological applications related to ligand exchange.

Temporally Controlled Multienzyme Catalysis Using a Dissipative Supramolecular Nanozyme.

The development of superior functional enzyme mimics (nanozymes) is essential for practical applications, including point-of-care diagnostics, biotechnological applications, biofuels, and environmental remediation. Nanozymes with the ability to control their catalytic activity in response to external fuels offer functionally valuable platforms mimicking nonequilibrium systems in nature. Herein, we fabricated a supramolecular coordination bonding-based dynamic vesicle that exhibits multienzymatic activity. The supramolecular nanozyme shows effective laccase-like catalytic activity with a KM value better than the native enzyme and higher stability in harsh conditions. Besides, the nanostructure demonstrates an efficient peroxidase-like activity with NADH peroxidase-like properties. Generation of luminescence from luminol and oxidation of dopamine are efficiently catalyzed by the nanozyme with high sensitivity, which is useful for point-of-care detections. Notably, the active nanozyme exhibits dynamic laccase-mimetic activity in response to pH variation, which has never been explored before. While a neutral/high pH leads to the self-assembly, a low pH disintegrates the assembled nanostructures and consequently turns off the nanozyme activity. Altogether, the self-assembled Cu2+-based vesicular nanostructure presents a pH-fueled dissipative system demonstrating effective temporally controlled multienzymatic activity.

High affinity protein surface binding through co-engineering of nanoparticles and proteins.

Control over supramolecular recognition between proteins and nanoparticles (NPs) is of fundamental importance in therapeutic applications and sensor development. Most NP-protein binding approaches use 'tags' such as biotin or His-tags to provide high affinity; protein surface recognition provides a versatile alternative strategy. Generating high affinity NP-protein interactions is challenging however, due to dielectric screening at physiological ionic strengths. We report here the co-engineering of nanoparticles and protein to provide high affinity binding. In this strategy, 'supercharged' proteins provide enhanced interfacial electrostatic interactions with complementarily charged nanoparticles, generating high affinity complexes. Significantly, the co-engineered protein-nanoparticle assemblies feature high binding affinity even at physiologically relevant ionic strength conditions. Computational studies identify both hydrophobic and electrostatic interactions as drivers for these high affinity NP-protein complexes.

A Robust Liposomal Platform for Direct Colorimetric Detection of Sphingomyelinase Enzyme and Inhibitors.

The enzyme sphingomyelinase (SMase) is an important biomarker for several diseases such as Niemann Pick's, atherosclerosis, multiple sclerosis, and HIV. We present a two-component colorimetric SMase activity assay that is more sensitive and much faster than currently available commercial assays. Herein, SMase-triggered release of cysteine from a sphingomyelin (SM)-based liposome formulation with 60 mol % cholesterol causes gold nanoparticle (AuNP) aggregation, enabling colorimetric detection of SMase activities as low as 0.02 mU/mL, corresponding to 1.4 pM concentration. While the lipid composition offers a stable, nonleaky liposome platform with minimal background signal, high specificity toward SMase avoids cross-reactivity of other similar phospholipases. Notably, use of an SM-based liposome formulation accurately mimics the natural in vivo substrate: the cell membrane. We studied the physical rearrangement process of the lipid membrane during SMase-mediated hydrolysis of SM to ceramide using small- and wide-angle X-ray scattering. A change in lipid phase from a liquid to gel state bilayer with increasing concentration of ceramide accounts for the observed increase in membrane permeability and consequent release of encapsulated cysteine. We further demonstrated the effectiveness of the sensor in colorimetric screening of small-molecule drug candidates, paving the way for the identification of novel SMase inhibitors in minutes. Taken together, the simplicity, speed, sensitivity, and naked-eye readout of this assay offer huge potential in point-of-care diagnostics and high-throughput drug screening.

Bloch surface wave enhanced biosensor for the direct detection of Angiopoietin-2 tumor biomarker in human plasma.

Quantitative detection of angiogenic biomarkers provides a powerful tool to diagnose cancers in early stages and to follow its progression during therapy. Conventional tests require trained personnel, dedicated laboratory equipment and are generally time-consuming. Herein, we propose our developed biosensing platform as a useful tool for a rapid determination of Angiopoietin-2 biomarker directly from patient plasma within 30 minutes, without any sample preparation or dilution. Bloch surface waves supported by one dimensional photonic crystal are exploited to enhance and redirect the fluorescence arising from a sandwich immunoassay that involves Angiopoietin-2. The sensing units consist of disposable and low-cost plastic biochips coated with the photonic crystal. The biosensing platform is demonstrated to detect Angiopoietin-2 in plasma samples at the clinically relevant concentration of 6 ng/mL, with an estimated limit of detection of approximately 1 ng/mL. This is the first Bloch surface wave based assay capable of detecting relevant concentrations of an angiogenic factor in plasma samples. The results obtained by the developed biosensing platform are in close agreement with enzyme-linked immunosorbent assays, demonstrating a good accuracy, and their repeatability showed acceptable relative variations.

Cancer Cell Discrimination Using Host-Guest "Doubled" Arrays.

We report a nanosensor that uses cell lysates to rapidly profile the tumorigenicity of cancer cells. This sensing platform uses host-guest interactions between cucurbit[7]uril and the cationic headgroup of a gold nanoparticle to non-covalently modify the binding of three fluorescent proteins of a multi-channel sensor in situ. This approach doubles the number of output channels to six, providing single-well identification of cell lysates with 100% accuracy. Significantly, this classification could be extended beyond the training set, determining the invasiveness of novel cell lines. The unique fingerprint of these cell lysates required minimal sample quantity (200 ng, ∼1000 cells), making the methodology compatible with microbiopsy technology.

Facet-Dependent Interactions of Islet Amyloid Polypeptide with Gold Nanoparticles: Implications for Fibril Formation and Peptide-Induced Lipid Membrane Disruption.

A comprehensive understanding of the mechanisms of interaction between proteins or peptides and nanomaterials is crucial for the development of nanomaterial-based diagnostics and therapeutics. In this work, we systematically explored the interactions between citrate-capped gold nanoparticles (AuNPs) and islet amyloid polypeptide (IAPP), a 37-amino acid peptide hormone co-secreted with insulin from the pancreatic islet. We utilized diffusion-ordered spectroscopy, isothermal titration calorimetry, localized surface plasmon resonance spectroscopy, gel electrophoresis, atomic force microscopy, transmission electron microscopy (TEM), and molecular dynamics (MD) simulations to systematically elucidate the underlying mechanism of the IAPP-AuNP interactions. Because of the presence of a metal-binding sequence motif in the hydrophilic peptide domain, IAPP strongly interacts with the Au surface in both the monomeric and fibrillar states. Circular dichroism showed that AuNPs triggered the IAPP conformational transition from random coil to ordered structures (α-helix and ß-sheet), and TEM imaging suggested the acceleration of IAPP fibrillation in the presence of AuNPs. MD simulations revealed that the IAPP-AuNP interactions were initiated by the N-terminal domain (IAPP residues 1-19), which subsequently induced a facet-dependent conformational change in IAPP. On a Au(111) surface, IAPP was unfolded and adsorbed directly onto the Au surface, while for the Au(100) surface, it interacted predominantly with the citrate adlayer and retained some helical conformation. The observed affinity of AuNPs for IAPP was further applied to reduce the level of peptide-induced lipid membrane disruption.

Ratiometric Array of Conjugated Polymers-Fluorescent Protein Provides a Robust Mammalian Cell Sensor.

Supramolecular complexes of a family of positively charged conjugated polymers (CPs) and green fluorescent protein (GFP) create a fluorescence resonance energy transfer (FRET)-based ratiometric biosensor array. Selective multivalent interactions of the CPs with mammalian cell surfaces caused differential change in FRET signals, providing a fingerprint signature for each cell type. The resulting fluorescence signatures allowed the identification of 16 different cell types and discrimination between healthy, cancerous, and metastatic cells, with the same genetic background. While the CP-GFP sensor array completely differentiated between the cell types, only partial classification was achieved for the CPs alone, validating the effectiveness of the ratiometric sensor. The utility of the biosensor was further demonstrated in the detection of blinded unknown samples, where 121 of 128 samples were correctly identified. Notably, this selectivity-based sensor stratified diverse cell types in minutes, using only 2000 cells, without requiring specific biomarkers or cell labeling.

A Multichannel Biosensor for Rapid Determination of Cell Surface Glycomic Signatures.

Cell surface glycosylation serves a fundamental role in dictating cell and tissue behavior. Cell surface glycomes differ significantly, presenting viable biomarkers for identifying cell types and their states. Glycoprofiling is a challenging task, however, due to the complexity of the constituent glycans. We report here a rapid and effective sensor for surface-based cell differentiation that uses a three-channel sensor produced by noncovalent conjugation of a functionalized gold nanoparticle (AuNP) and fluorescent proteins. Wild-type and glycomutant mammalian cells were effectively stratified using fluorescence signatures obtained from a single sensor element. Blinded unknowns generated from the tested cell types were identified with high accuracy (44 out of 48 samples), validating the robustness of the multichannel sensor. Notably, this selectivity-based high-throughput sensor differentiated between cells, employing a nondestructive protocol that required only a single well of a microplate for detection.

Colorimetric Detection of Small Molecules in Complex Matrixes via Target-Mediated Growth of Aptamer-Functionalized Gold Nanoparticles.

A versatile and sensitive colorimetric assay that allows the rapid detection of small-molecule targets using the naked eye is demonstrated. The working principle of the assay integrates aptamer-target recognition and the aptamer-controlled growth of gold nanoparticles (Au NPs). Aptamer-target interactions modulate the amount of aptamer strands adsorbed on the surface of aptamer-functionalized Au NPs via desorption of the aptamer strands when target molecules bind with the aptamer. Depending on the resulting aptamer coverage, Au NPs grow into morphologically varied nanostructures, which give rise to different colored solutions. Au NPs with low aptamer coverage grow into spherical NPs, which produce red-colored solutions, whereas Au NPs with high aptamer coverage grow into branched NPs, which produce blue-colored solutions. We achieved visible colorimetric response and nanomolar detection limits for the detection of ochratoxin A (1 nM) in red wine samples, as well as cocaine (1 nM) and 17ß-estradiol (0.2 nM) in spiked synthetic urine and saliva, respectively. The detection limits were well within clinically and physiologically relevant ranges, and below the maximum food safety limits. The assay is highly sensitive, specific, and able to detect an array of analytes rapidly without requiring sophisticated equipment, making it relevant for many applications, such as high-throughput drug and clinical screening, food sampling, and diagnostics. Furthermore, the assay is easily adapted as a chip-based platform for rapid and portable target detection.

A multichannel nanosensor for instantaneous readout of cancer drug mechanisms.

Screening methods that use traditional genomic, transcriptional, proteomic and metabonomic signatures to characterize drug mechanisms are known. However, they are time consuming and require specialized equipment. Here, we present a high-throughput multichannel sensor platform that can profile the mechanisms of various chemotherapeutic drugs in minutes. The sensor consists of a gold nanoparticle complexed with three different fluorescent proteins that can sense drug-induced physicochemical changes on cell surfaces. In the presence of cells, fluorescent proteins are rapidly displaced from the gold nanoparticle surface and fluorescence is restored. Fluorescence 'turn on' of the fluorescent proteins depends on the drug-induced cell surface changes, generating patterns that identify specific mechanisms of cell death induced by drugs. The nanosensor is generalizable to different cell types and does not require processing steps before analysis, offering an effective way to expedite research in drug discovery, toxicology and cell-based sensing.

Rapid identification of bacterial biofilms and biofilm wound models using a multichannel nanosensor.

Identification of infectious bacteria responsible for biofilm-associated infections is challenging due to the complex and heterogeneous biofilm matrix. To address this issue and minimize the impact of heterogeneity on biofilm identification, we developed a gold nanoparticle (AuNP)-based multichannel sensor to detect and identify biofilms based on their physicochemical properties. Our results showed that the sensor can discriminate six bacterial biofilms including two composed of uropathogenic bacteria. The capability of the sensor was further demonstrated through discrimination of biofilms in a mixed bacteria/mammalian cell in vitro wound model.

Tunable elastic modulus of nanoparticle monolayer films by host-guest chemistry.

The elastic modulus of an ultrathin nanoparticle (NP) monolayer film is tuned by modulating the binding strength between the NPs on a molecular level. NP monolayer films constructed by crosslinking NPs of different binding affinities are fabricated at oil/water interfaces. By inducing buckling patterns on these films, the correlation between the binding affinity of the NPs and the elastic modulus is investigated.

Optimizing the selective recognition of protein isoforms through tuning of nanoparticle hydrophobicity.

We demonstrate that ligand hydrophobicity can be used to increase affinity and selectivity of binding between monolayer-protected cationic gold nanoparticles and ß-lactoglobulin protein isoforms containing two amino acid mutations.