Discrete LAT condensates encode antigen information from single pMHC:TCR binding events.

LAT assembly into a two-dimensional protein condensate is a prominent feature of antigen discrimination by T cells. Here, we use single-molecule imaging techniques to resolve the spatial position and temporal duration of each pMHC:TCR molecular binding event while simultaneously monitoring LAT condensation at the membrane. An individual binding event is sufficient to trigger a LAT condensate, which is self-limiting, and neither its size nor lifetime is correlated with the duration of the originating pMHC:TCR binding event. Only the probability of the LAT condensate forming is related to the pMHC:TCR binding dwell time. LAT condenses abruptly, but after an extended delay from the originating binding event. A LAT mutation that facilitates phosphorylation at the PLC-γ1 recruitment site shortens the delay time to LAT condensation and alters T cell antigen specificity. These results identify a function for the LAT protein condensation phase transition in setting antigen discrimination thresholds in T cells.

Height, but not binding epitope, affects the potency of synthetic TCR agonists.

Under physiological conditions, peptide-major histocompatibility complex (pMHC) molecules can trigger T cell receptors (TCRs) as monovalent ligands that are sparsely distributed on the plasma membrane of an antigen-presenting cell. TCRs can also be triggered by artificial clustering, such as with pMHC tetramers or antibodies; however, these strategies circumvent many of the natural ligand discrimination mechanisms of the T cell and can elicit nonphysiological signaling activity. We have recently introduced a synthetic TCR agonist composed of an anti-TCRß Fab' antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor. This Fab'-DNA ligand efficiently triggers TCR as a monomer when membrane associated and exhibits a potency and activation profile resembling agonist pMHC. In this report, we explore the geometric requirements for efficient TCR triggering and cellular activation by Fab'-DNA ligands. We find that T cells are insensitive to the ligand binding epitope on the TCR complex but that length of the DNA tether is important. Increasing, the intermembrane distance spanned by Fab'-DNA:TCR complexes decreases TCR triggering efficiency and T cell activation potency, consistent with the kinetic-segregation model of TCR triggering. These results establish design parameters for constructing synthetic TCR agonists that are able to activate polyclonal T cell populations, such as T cells from a human patient, in a similar manner as the native pMHC ligand.

Nanoparticle-based computing architecture for nanoparticle neural networks.

The lack of a scalable nanoparticle-based computing architecture severely limits the potential and use of nanoparticles for manipulating and processing information with molecular computing schemes. Inspired by the von Neumann architecture (VNA), in which multiple programs can be operated without restructuring the computer, we realized the nanoparticle-based VNA (NVNA) on a lipid chip for multiple executions of arbitrary molecular logic operations in the single chip without refabrication. In this system, nanoparticles on a lipid chip function as the hardware that features memory, processors, and output units, and DNA strands are used as the software to provide molecular instructions for the facile programming of logic circuits. NVNA enables a group of nanoparticles to form a feed-forward neural network, a perceptron, which implements functionally complete Boolean logic operations, and provides a programmable, resettable, scalable computing architecture and circuit board to form nanoparticle neural networks and make logical decisions.

A Lipid-Nanopillar-Array-Based Immunosorbent Assay.

Since infectious diseases, particularly viral infections, have threatened human health and caused huge economical losses globally, a rapid, sensitive, and selective virus detection platform is highly demanded. Enzyme-linked immunosorbent assay (ELISA) with flat solid substrates has been dominantly used in detecting whole viruses for its straightforwardness and simplicity in assay protocols, but it often suffers from limited sensitivity, poor quantification range, and a time-consuming assay procedure. Here, a lipid-nanopillar-array-based immunosorbent assay (LNAIA) is developed with a nanopillar-supported lipid bilayer substrate with fluorophore-modified antibodies for rapid, sensitive, and quantitative detection of viruses. 3D nanopillar array structures and fluid antibodies with fluorophores facilitate faster and efficient target binding and rapid fluorophore localization for quick, reliable analysis on binding events with a conventional fluorescence microscopy setup. LNAIA enables quantification of H1N1 virus that targets down to 150 virus particles with 5-orders-of-magnitude dynamic range within 25 min, which cannot be achieved with conventional ELISA platforms.

Plasmonic Nanoparticle-Interfaced Lipid Bilayer Membranes.

Plasmonic nanoparticles are widely exploited in diverse bioapplications ranging from therapeutics to biosensing and biocomputing because of their strong and tunable light-matter interactions, facile and versatile chemical/biological ligand modifications, and biocompatibility. With the rapid growth of nanobiotechnology, understanding dynamic interactions between nanoparticles and biological systems at the molecular or single-particle level is becoming increasingly important for interrogating biological systems with functional nanostructures and for developing nanoparticle-based biosensors and therapeutic agents. Therefore, significant efforts have been devoted to precisely design and create nano-bio interfaces by manipulating the nanoparticles' size, shape, and surface ligand interactions with complex biological systems to maximize their performance and avoid unwanted responses, such as their agglomeration and cytotoxicity. However, investigating physicochemical interactions at the nano-bio interfaces in a quantitative and controllable manner remains challenging, as the interfaces involve highly complex networks between nanoparticles, biomolecules, and cells across multiple scales, each with a myriad of different chemical and biological interactions. A lipid bilayer is a membrane made of two layers of lipid molecules that forms a barrier around cells and plays structural and functional roles in diverse biological processes because they incorporate and present functional molecules (such as membrane proteins) with lateral fluidity. Plasmonic nanoparticles conjugated on lipid membranes provide reliable analytical labels and functional moieties that allow for studying and manipulating interactions between nanoparticles and molecules with single-particle resolution; they also serve as efficient tools for applying optical, mechanical, and thermal stimuli to biological systems, which stem from plasmonic properties. Recently, new opportunities have emerged by interfacing nanoparticle-modified lipid bilayers (NLBs) with complex systems such as molecular circuits and living systems. In this Account, we briefly review how plasmonic properties can be beneficially harnessed on lipid bilayer membranes to investigate the structures and functions of cellular membranes and to develop new platforms for biomedical applications. In particular, we discuss the versatility of supported lipid bilayers (SLBs), which are planar lipid bilayers on hydrophilic substrates, as dynamic biomaterials that provide lateral fluidity and cell membrane-like environments. We then summarize our efforts to create a quantitative analytical platform utilizing nanoparticles as active building blocks and SLBs as integrative substrates. Through this bottom-up approach, various functionalized nanoparticles have been introduced onto lipid bilayers to render nanoparticle-nanoparticle, nanoparticle-lipid bilayer, and biomolecule-lipid bilayer interfaces programmable. Our system provides a new class of tools for studying thermodynamics and kinetics in complex networks of nanostructures and for realizing unique applications in biosensing and biocomputing.

Biocomputing with Nanostructures on Lipid Bilayers.

Biocomputation is the algorithmic manipulation of biomolecules. Nanostructures, most notably DNA nanostructures and nanoparticles, become active substrates for biocomputation when modified with stimuli-responsive, programmable biomolecular ligands. This approach-biocomputing with nanostructures ("nano-bio computing")-allows autonomous control of matter and information at the nanoscale; their dynamic assemblies and beneficial properties can be directed without human intervention. Recently, lipid bilayers interfaced with nanostructures have emerged as a new biocomputing platform. This new nano-bio interface, which exploits lipid bilayers as a chemical circuit board for information processing, offers a unique reaction space for realizing nanostructure-based computation at a previously unexplored dimension. In this Concept, recent advances in nano-bio computing are briefly reviewed and the newly emerging concept of biocomputing with nanostructures on lipid bilayers is introduced.

Monitoring Glutathione Dynamics and Heterogeneity in Living Stem Cells.

Glutathione (GSH) is a major antioxidant in cells, and plays vital roles in the cellular defense against oxidants and in the regulation of redox signals. In a previous report, we demonstrated that stem cell function is critically affected by heterogeneity and dynamic changes in cellular GSH concentration. Here, we present a detailed protocol for the monitoring of GSH concentration in living stem cells using FreSHtracer, a real-time GSH probe. We describe the steps involved in monitoring GSH concentration in single living stem cells using confocal microscopy and flow cytometry. These methods are simple, rapid, and quantitative, and able to demonstrate intracellular GSH concentration changes in real time. We also describe the application of FreSHtracer to the sorting of stem cells according to their GSH content using flow cytometry. Typically, microscopic or flow cytometric analyses of FreSHtracer and MitoFreSHtracer signals in living stem cells take ~2~3 h, and the fractionation of stem cells into subpopulations on the basis of cellular GSH levels takes 3~4.5 h. This method could be applied to almost every kind of mammalian cell with minor modifications to the protocol described here.

Nano-bio-computing lipid nanotablet.

Using nanoparticles as substrates for computation enables algorithmic and autonomous controls of their unique and beneficial properties. However, scalable architecture for nanoparticle-based computing systems is lacking. Here, we report a platform for constructing nanoparticle logic gates and circuits at the single-particle level on a supported lipid bilayer. Our "lipid nanotablet" platform, inspired by cellular membranes that are exploited to compartmentalize and control signaling networks, uses a lipid bilayer as a chemical circuit board and nanoparticles as computational units. On a lipid nanotablet, a single-nanoparticle logic gate senses molecules in solution as inputs and triggers particle assembly or disassembly as an output. We demonstrate a set of Boolean logic operations, fan-in/fan-out of logic gates, and a combinational logic circuit such as a multiplexer. We envisage that our approach to modularly implement nanoparticle circuits on a lipid bilayer will create new paradigms and opportunities in molecular computing, nanoparticle circuits, and systems nanoscience.

Nonnoble-Metal-Based Plasmonic Nanomaterials: Recent Advances and Future Perspectives.

The application scope of plasmonic nanostructures is rapidly expanding to keep pace with the ongoing development of various scientific findings and emerging technologies. However, most plasmonic nanostructures heavily depend on rare, expensive, and extensively studied noble metals such as Au and Ag, with the limited choice of elements hindering their broad and practical applications in a wide spectral range. Therefore, abundant and inexpensive nonnoble metals have attracted attention as new plasmonic nanomaterial components, allowing these nonnoble-metal-based materials to be used in areas such as photocatalysis, sensing, nanoantennas, metamaterials, and magnetoplasmonics with new compositions, structures, and properties. Furthermore, the use of nonnoble metal hybrids results in newly emerging or synergistic properties not observed from single-metal component systems. Here, the synthetic strategies and recent advances in nonnoble-metal-based plasmonic nanostructures comprising Cu, Al, Mg, In, Ga, Pb, Ni, Co, Fe, and related hybrids are highlighted, and a discussion and perspectives in their synthesis, properties, applications, and challenges are presented.

Sensitive, Quantitative Naked-Eye Biodetection with Polyhedral Cu Nanoshells.

One of the most heavily used methods in chemical and biological labeling, detection, and imaging is based on silver shell-based enhancement on Au nanoparticles (AuNPs) that is useful for amplifying Rayleigh scattering, colorimetric signal, surface-enhanced Raman scattering, and electrical signal, but poor structural controllability and nonspecific growth of silver shells have limited its applications, especially with respect to signal reproducibility and quantification. Here, a highly specific, well-defined Cu nanopolyhedral shell overgrowth chemistry is developed with the aid of polyethyleneimine (PEI) on AuNPs, and the use of this PEI-mediated Cu polyhedral nanoshell (CuP) chemistry is shown as a means of light-scattering signal enhancement for the development of naked-eye-based highly sensitive and quantitative detections of DNA and viruses. Remarkably, these CuPs are exclusively formed on AuNPs in a controllable manner, with no noticeable nonspecific CuP growth. The findings enable to acquire clearly visible signals without analytic instrumentation, detectable down to 8 × 10-15 m of DNA (anthrax sequence) and 2700 copies of viruses (noroviruses in clinical stool samples) with broad dynamic ranges on archetypal assay platforms. This new method provides a general platform in controlling Cu shell nanostructures and their optical signals, and opens up revenues for highly reliable, quantitative onsite naked-eye biodetection.

Optokinetically Encoded Nanoprobe-Based Multiplexing Strategy for MicroRNA Profiling.

Multiplexed real-time analysis on multiple interacting molecules and particles is needed to obtain information on binding patterns between multiple ligands and receptors, specificity of bond formations, and interacting pairs in a complex medium, often found in chemical and biological systems, and difference in binding affinity and kinetics for different binding pairs in one solution. In particular, multiplexed profiling of microRNA (miRNA) in a reliable, quantitative manner is of great demand for the use of miRNA in cell biology, biosensing, and clinical diagnostic applications, and accurate diagnosis of cancers with miRNA is not possible without detecting multiple miRNA sequences in a highly specific manner. Here, we report a multiplexed molecular detection strategy with optokinetically (OK) coded nanoprobes (NPs) that show high photostability, distinct optical signals, and dynamic behaviors on a supported lipid bilayer (SLB) (OK-NLB assay). Metal NPs with three distinct dark-field light scattering signals [red (R), green (G), and blue (B)] and three different target miRNA half-complements were tethered to a two dimensionally fluidic SLB with mobile (M) or immobile (I) state. In situ single-particle monitoring and normalized RGB analysis of the optokinetically combinatorial assemblies among three M-NPs and three I-NPs with dark-field microscopy (DFM) allow for differentiating and quantifying 9 different miRNA targets in one sample. The OK-NP-based assay enables simultaneous detection of multiple miRNA targets in a highly quantitative, specific manner within 1 h and can be potentially used for diagnosis of different cancer types. We validated the OK-NLB assay with single-base mismatched experiments and HeLa cell-extracted total RNA samples by comparing the assay results to the quantitative reverse transcription polymerase chain reaction (qRT-PCR) results.

Highly Controlled Synthesis and Super-Radiant Photoluminescence of Plasmonic Cube-in-Cube Nanoparticles.

The plasmonic properties of metal nanostructures have been heavily utilized for surface-enhanced Raman scattering (SERS) and metal-enhanced fluorescence (MEF), but the direct photoluminescence (PL) from plasmonic metal nanostructures, especially with plasmonic coupling, has not been widely used as much as SERS and MEF due to the lack of understanding of the PL mechanism, relatively weak signals, and the poor availability of the synthetic methods for the nanostructures with strong PL signals. The direct PL from metal nanostructures is beneficial if these issues can be addressed because it does not exhibit photoblinking or photobleaching, does not require dye-labeling, and can be employed as a highly reliable optical signal that directly depends on nanostructure morphology. Herein, we designed and synthesized plasmonic cube-in-cube (CiC) nanoparticles (NPs) with a controllable interior nanogap in a high yield from Au nanocubes (AuNCs). In synthesizing the CiC NPs, we developed a galvanic void formation (GVF) process, composed of replacement/reduction and void formation steps. We unraveled the super-radiant character of the plasmonic coupling-induced plasmon mode which can result in highly enhanced PL intensity and long-lasting PL, and the PL mechanisms of these structures were analyzed and matched with the plasmon hybridization model. Importantly, the PL intensity and quantum yield (QY) of CiC NPs are 31 times and 16 times higher than those of AuNCs, respectively, which have shown the highest PL intensity and QY reported for metallic nanostructures. Finally, we confirmed the long-term photostability of the PL signal, and the signal remained stable for at least 1 h under continuous illumination.

Plasmonically Engineered Nanoprobes for Biomedical Applications.

The localized surface plasmon resonance of metal nanoparticles is the collective oscillation of electrons on particle surface, induced by incident light, and is a particle composition-, morphology-, and coupling-dependent property. Plasmonic engineering deals with highly precise formation of the targeted nanostructures with targeted plasmonic properties (e.g., electromagnetic field distribution and enhancement) via controlled synthetic, assembling, and atomic/molecular tuning strategies. These plasmonically engineered nanoprobes (PENs) have a variety of unique and beneficial physical, chemical, and biological properties, including optical signal enhancement, catalytic, and local temperature-tuning photothermal properties. In particular, for biomedical applications, there are many useful properties from PENs including LSPR-based sensing, surface-enhanced Raman scattering, metal-enhanced fluorescence, dark-field light-scattering, metal-mediated fluorescence resonance energy transfer, photothermal effect, photodynamic effect, photoacoustic effect, and plasmon-induced circular dichroism. These properties can be utilized for the development of new biotechnologies and biosensing, bioimaging, therapeutic, and theranostic applications in medicine. This Perspective introduces the concept of plasmonic engineering in designing and synthesizing PENs for biomedical applications, gives recent examples of biomedically functional PENs, and discusses the issues and future prospects of PENs for practical applications in bioscience, biotechnology, and medicine.

Supported lipid bilayers as dynamic platforms for tethered particles.

Nanoparticle tethering to lipid bilayers enables the observation of hundreds of diffusing particles that are confined within a single field of view. A wide variety of materials ranging from plasmonic metals to soft matter can be stably tethered to the surface of a fluid bilayer by covalent or non-covalent means. The controlled environment of this experimental platform allows direct control over surface compositions and accurate tracking of nanoparticle interactions. This minireview will cover studies that use bilayer-tethered nanoparticles to investigate physical properties related to lipid mobility, biomolecule sensing, and surface interactions, as well as experiments to reversibly manipulate tethered nanoparticles by electric fields.

Dark-field-based observation of single-nanoparticle dynamics on a supported lipid bilayer for in situ analysis of interacting molecules and nanoparticles.

Observation of single plasmonic nanoparticles in reconstituted biological systems allows us to obtain snapshots of dynamic processes between molecules and nanoparticles with unprecedented spatiotemporal resolution and single-molecule/single-particle-level data acquisition. This Concept is intended to introduce nanoparticle-tethered supported lipid bilayer platforms that allow for the dynamic confinement of nanoparticles on a two-dimensional fluidic surface. The dark-field-based long-term, stable, real-time observation of freely diffusing plasmonic nanoparticles on a lipid bilayer enables one to extract a broad range of information about interparticle and molecular interactions throughout the entire reaction period. Herein, we highlight important developments in this context to provide ideas on how molecular interactions can be interpreted by monitoring dynamic behaviors and optical signals of laterally mobile nanoparticles.

Massively parallel and highly quantitative single-particle analysis on interactions between nanoparticles on supported lipid bilayer.

Observation of individual single-nanoparticle reactions provides direct information and insight for many complex chemical, physical, and biological processes, but this is utterly challenging with conventional high-resolution imaging techniques on conventional platforms. Here, we developed a photostable plasmonic nanoparticle-modified supported lipid bilayer (PNP-SLB) platform that allows for massively parallel in situ analysis of the interactions between nanoparticles with single-particle resolution on a two-dimensional (2D) fluidic surface. Each particle-by-particle PNP clustering process was monitored in real time and quantified via analysis of individual particle diffusion trajectories and single-particle-level plasmonic coupling. Importantly, the PNP-SLB-based nanoparticle cluster growth kinetics result was fitted well. As an application example, we performed a DNA detection assay, and the result suggests that our approach has very promising sensitivity and dynamic range (high attomolar to high femtomolar) without optimization, as well as remarkable single-base mismatch discrimination capability. The method shown herein can be readily applied for many different types of intermolecular and interparticle interactions and provide convenient tools and new insights for studying dynamic interactions on a highly controllable and analytical platform.