The engineering of living cells represents a promising biomedical frontier that enables the design of cells with tailored functionalities for advanced therapeutic applications. Genetic manipulation serves as a primary approach in cell engineering, yet it faces inherent limitations, including the complexity of multigene editing and poor cross-species applicability, which restrict the development of cells with sophisticated functionalities. Therefore, flexible and versatile engineering strategies capable of functionalizing living cells to address diverse therapeutic requirements are highly desirable. Given its pivotal role in mediating cellular interactions, the cell surface is an attractive target for directing cell engineering. The diverse functional groups present in surface biomolecules offer abundant chemical modification sites, making them highly amenable to functionalization. Leveraging this inherent chemical accessibility, we have recently developed a flexible and versatile platform for surface functionalization of living cells through in situ dopamine polymerization that allows us to design personalized living cells with customizable functions by tuning the surface components. Here we provide a detailed protocol describing two distinct methods for bacterial functionalization. The first method uses dopamine polymerization-mediated mono-functionalization to construct mucus-penetrating bacteria that can reinforce intestinal mucosal barrier to prevent colitis. The second method uses dopamine polymerization-mediated dual-functionalization to generate synergy-immunoactivation bacteria that can simultaneously induce anticancer and antiviral immunity to treat cancer and prevent infection. Excluding bacterial culture, preparation of mucus-penetrating bacteria and synergy-immunoactivation bacteria takes ~3 h and 1 h, respectively. We anticipate that this protocol can offer valuable guidance for the engineering of living cells with designable and tailorable functionalities for innovative cell-based therapy.
The human cerebrovasculature is a critical yet historically understudied component of neurological health. Dysfunction of the diverse endothelial, mural, and perivascular cells that comprise cerebral vessels is central to diseases ranging from stroke to Alzheimer's disease. However, characterizing these cell populations at a molecular level has proven exceptionally challenging. Encased within a robust basement membrane, vascular cells resist standard dissociation methods, leading to their systematic depletion and underrepresentation in existing single-nucleus genomic atlases. This has created a major blind spot in neuroscience. To overcome this barrier, we developed vessel isolation and nucleus extraction for sequencing (VINE-seq) and its advanced iteration, MultiVINE-seq. The protocol provides a robust, reproducible workflow for the enrichment and high-resolution profiling of vascular, perivascular, and immune cells from fresh or frozen human and mouse brain tissue. First, intact vessels (predominantly capillaries and small arterioles/venules, 100 µm in diameter) are isolated from homogenized brain tissue via dextran-based density-gradient centrifugation, separating the vascular pellet from myelin and the parenchymal fraction. Second, the collected vessels are rigorously washed over a cell strainer to remove trapped contaminants. A critical innovation lies in the third stage: the optimized extraction of nuclei from purified vessels using enzymatic digestion. After extraction, the protocol uses fluorescence-activated cell sorting (FACS) to ensure collection of high-purity nuclei suitable for widely used droplet-based sequencing platforms (e.g., 10x Genomics single cell 3' or multiome). This protocol requires 4-5 h to complete and can be carried out by researchers with single-cell and flow cytometry training.
With the increasing progress in the emerging field of phase engineering of nanomaterials, transition metal dichalcogenides (TMDs) with metastable phases have attracted extensive research interest owing to their unique physicochemical properties and promising potential for various applications. Unlike their semiconducting counterparts, metastable 1T'-phase group VIB TMDs exhibit distinctive metallic behavior, making them highly promising for clean energy electrocatalysis (for example, hydrogen evolution), high-performance electronics and superconducting devices. However, conventional synthetic strategies usually suffer from low yields, poor phase purity, small crystal size and harsh experimental conditions, hindering their fundamental research and practical applications. Here we describe a salt-assisted method for the controlled preparation of various metastable 1T'-phase group VIB TMDs with high crystalline quality and high phase purity. In this context, 'salt-assisted' refers to the co-annealing of commercially available 2H-phase TMDs, chalcogen powders (for example, S or Se) and alkali metal salts (for example, K2C2O4·H2O or K2CO3). When heated in a tube furnace under an H2/Ar atmosphere, these alkali metal salts trigger a phase transformation of TMDs from the thermodynamically stable 2H phase to the metastable 1T' phase. This Protocol describes the details of experimental procedures to prepare a series of 1T'-TMDs, that is, MoS2, MoSe2, WS2, WSe2, MoS2xSe2(1-x) and WS2xSe2(1-x). It takes ~37 h 20 min and yields robust 1T'-TMD crystals with their size of up to hundreds of micrometers at a gram scale (up to ~2 g per batch), overcoming the longstanding synthetic bottlenecks and paving the way for advanced fundamental studies and the exploration of practical applications.
High-performance bioinspired composite materials require the precise alignment of synthetic nanosheets to mimic the mechanical properties of highly ordered laminated microstructures found in nature. Here, we detail a nanosheet superspreading alignment strategy for fabricating nanocomposite films that uses shear-flow forces at the interface between two immiscible phases to induce long-range, high-order alignment of the 2D nanosheets that enhances the films' mechanical properties. In situ interface crystallization or cross-linking follows the alignment and effectively locks the oriented configuration (the resulting orientation order parameter is >0.85). Subsequent solvent dewetting enables continuous film formation over large areas while maintaining well-defined microstructural integrity. This process overcomes the misorientation and aggregation typical of conventional alignment methods and can be scaled using a multi-nozzle extrusion setup compatible with commercial heating and film-collection components. The step-by-step procedures cover the nanosheet precursor preparation, the continuous-film fabrication and their microstructural characterization and require ≤23 days to complete. The procedures are applicable to a broad range of nanosheet materials, including graphene oxide, MXenes, transition-metal dichalcogenides and layered clays, and the resulting composite films exhibit enhanced mechanical strength, toughness and multifunctionality. Nanocomposites based on graphene oxide and clay nanosheets exhibit a tensile strength of up to 1,215 ± 80 MPa (mean ± s.d) and a Young's modulus of 198.8 ± 6.5 GPa, while clay-based nanocomposite films reach a toughness of 36.7 ± 3.0 MJ m-3. Superspreading alignment is a versatile and robust approach for the scalable fabrication of high-performance composites for materials science.
Here we present the pathology workflow of the Mutographs project, a 7-year, multicancer genomics initiative led by the Sanger Institute and funded by Cancer Research UK. Mutographs aims to elucidate global differences in cancer incidence through whole-genome sequencing and mutational signature analysis of selected cancers with unexplained variation of incidence rate worldwide. The Protocol was developed by the International Agency for Research on Cancer, where pathology assessment and paired tumor-blood DNA extraction were performed. We provide details for handling frozen cancer tissues for multistep pathology evaluation and quality control, tumor-enrichment methods (such as macrodissecion and laser-capture microdissection) for certain tumor types, and manual and automated DNA extraction. This Protocol has achieved an overall success rate of 88% in providing high-quality DNA for whole-genome sequencing, aiding in the decision to exclude low-quality or noneligible tissues. This Protocol provides a reproducible, fully integrated pathology-molecular pipeline in challenging conditions to ensure high-quality DNA and reliable data interpretation at an unprecedented multinational scale and could be considered a model for high-quality tissue processing in international genomics initiatives. It is based on routine practice of pathology and requires practical experience of processing frozen tissues and morphological assessment of frozen tissues by digital pathology.
Exposomics is an emerging field of research that aims to comprehensively investigate individuals' environmental exposures and how these exposures relate to health outcomes. Liquid chromatography-tandem mass spectrometry is widely used in exposomics studies. MetaboAnalyst ( https://www.metaboanalyst.ca/ ) is a widely used platform for statistical and functional analysis of metabolomics data. The current MetaboAnalyst 6.0 release incorporates updates to meet the needs of exposomics studies, including improved support for tandem mass spectrometry compound identification, exposome annotation, dose-response analysis and linking to genetics and functions. Here we extend our 2022 Nature Protocol by providing step-by-step instructions on how to use MetaboAnalyst 6.0 for exposomics data analysis, including: liquid chromatography-tandem mass spectrometry spectra processing and compound identification (Stage 1), exposomics data processing and exploratory analysis (Stage 2), dose-response modeling to study metabolic responses to exposure levels (Stage 3) and leveraging known genetic associations for causal inference (Stage 4). We demonstrate Stages 1-3 using data from a recent blood exposomics study concerning electronic waste exposure. Stage 4 is illustrated through an investigation of the potential causal link between ʟ-isoleucine and type 2 diabetes. Stage 1 may take ~2 h to complete depending on server load, and the remaining stages may be executed in a total of ~90 min.
The [FeFe]-hydrogenases are widely distributed enzymes that efficiently make and use hydrogen (H2). They have attracted intense interest as potential biocatalysts as well as models for biomimetic catalysis. Substrate turnover occurs at an Fe-based active site featuring an azadithiolate cofactor. The biosynthesis of the active site is remarkably elaborate. The production of these enzymes is greatly simplified by replacing the biosynthesis of this active site with reconstitution of the enzyme with chemically synthesized [Fe2[(μ-SCH2)2NH](CN)2(CO)4]2- ([1]2-), the subject of this protocol. Two approaches to [1]2- are described: a 'condensation route' and an 'Fmoc route'. Each of the two routes requires ~20 h of hands-on time. The condensation route entails the reaction of Fe2(μ-SH)2(CO)6 and N4(CH2)6 followed by cyanation to give [1]2-. The most demanding step in this route is the preparation of Fe2(μ-S2)(CO)6. The second route focuses on fluorenylmethoxycarbonyl (Fmoc)-protected derivatives of azadithiolate cofactor. This route proceeds via Fe2[(μ-SCH2)2NFmoc](CO)6. Both the condensation and Fmoc routes allow isotopic labeling of the active site, but 57Fe labeling is especially simple using the Fmoc route.
Optical metasurfaces represent a promising technology for compact, lightweight and multifunctional optical devices; yet, their practical implementation remains challenging because of the need for high-refractive-index (high-index) materials, complex fabrication processes and limited substrate compatibility. Conventional top-down approaches relying on deposition, electron-beam lithography and etching are expensive and typically restricted to rigid substrates, hindering scalability and integration with flexible platforms. Nanoimprint lithography is a cost-effective, high-throughput alternative for metasurface fabrication; nevertheless, the low refractive index of conventional imprint resins fundamentally limits device performance. Here, we describe the fabrication of optical metasurfaces using a printable high-index composite material known as nanoparticle-embedded resin (nanoPER). By embedding high-index nanoparticles into a curable resin matrix, nanoPER achieves an effective refractive index above 1.8 at the target wavelength and enables single-step replication of functional nanostructures on a wide range of substrates, including flexible and curved surfaces. The procedure focuses on TiO2 nanoPER and provides a reproducible and comprehensive guide covering resin formulation, nanoimprint lithography process parameters and optical characterization. By emphasizing scalability and versatility, this protocol supports the translation of metasurface research into real-world applications such as light detection and ranging, compact imaging and integrated photonics. The entire process can be completed within 1-2 days and can be performed by researchers with experience in nanofabrication and optical measurements.
The introduction of expansion microscopy (ExM), a decade ago, marked a shift in super-resolution imaging, by physically separating fluorophores to bypass the diffraction limit. Numerous ExM developments have extended the method's reach since, yet molecular-scale resolution remained inaccessible. We recently developed one-step nanoscale ExM, which combines ExM with fluctuation-based super-resolution analysis to enable the direct visualization of individual protein shapes, using conventional fluorescence microscopes, a capability that was previously limited to cryo-electron microscopy and averaging-based techniques. Here we provide detailed procedures for gel embedding, labeling, expansion, image acquisition and data analysis. We also introduce a stable, user-friendly software package for efficient fluctuation analysis. Although one-step nanoscale ExM is broadly applicable to a range of samples, including purified proteins, cells and tissues, its most distinctive contribution lies in making single-protein shape analysis accessible and reproducible. Overall, we provide a practical framework for protein imaging on conventional equipment.
Drug self-administration has the greatest construct and predictive validity of the preclinical models for substance use disorder, providing landmark insights into the neurobiology of addiction. However, these experiments have traditionally been performed in freely moving animals, which can prohibit the incorporation of emerging neurotechnologies that require or are greatly facilitated by head restraint. Recently, we developed and validated a head-restrained approach in mice for intravenous and oral self-administration of drug and nondrug rewards. Here we present a step-by-step protocol for these experiments, including custom equipment construction, open-source software implementation and adaptation, catheter implantation, and unique considerations for conducting head-fixed self-administration experiments. To ensure that each component can be implemented by a wide range of audiences, detailed descriptions are provided so that this Protocol may serve as a standalone guide for researchers with varying levels of experience.
Antioxidant nanozymes are nanomaterials with superoxide dismutase-like or catalase-like activities. They have emerged as promising therapeutics for oxidative stress-related disorders. However, the lack of standardized quantitative assays for activity characterization has limited cross-study reproducibility and direct comparison of reported performance. Here this protocol provides step-by-step procedures for the quantitative characterization of the superoxide dismutase-like and catalase-like activities of nanozymes. Superoxide dismutase-like activity is quantified via a self-prepared xanthine/xanthine oxidase/water-soluble tetrazolium salt-1 system, in which nanozymes catalyze the dismutation of superoxide anions (O2•-), thereby suppressing formazan formation. By tuning O2•- generation through controlled xanthine oxidase concentrations, the protocol enables the determination of Michaelis-Menten kinetics for the short-lived O2•-. Catalase-like activity is quantified by monitoring hydrogen peroxide (H2O2) decomposition at 240 nm via ultraviolet-visible spectrophotometry, which yields specific activity and kinetic constants, whereas a complementary 3,3',5,5'-tetramethylbenzidine oxidation assay excludes interference from peroxidase-like reactions. Representative nanozymes spanning carbon-based, metal oxide, noble metal and single-atom systems are used as case studies to demonstrate the robustness and versatility of this protocol. Functional validation in cellular models using flow cytometry further demonstrates the practical applicability of reactive oxygen species scavenging. This protocol enables reliable and reproducible evaluation of antioxidant nanozymes, facilitating direct comparison across materials, elucidation of structure-activity relationships and rational optimization for biomedical applications. The complete procedure can be performed by researchers with standard training in biochemistry and flow cytometric analysis within 10-12 h.
The stabilization of metal catalysts remains a major challenge in heterogeneous catalysis, particularly for single atoms and clusters that operate under demanding industrial conditions where sintering and agglomeration often lead to substantial deactivation. Here, to address the issue, this protocol presents a general method for synthesizing nanoisland catalysts in which nanoscale oxide islands are isolated on high-surface-area substrates to confine and stabilize metal species. The method uses strong electrostatic adsorption to deposit oxides such as CeOx, LaOx and InOx onto substrates such as SiO2 and Al2O3, resulting in small-sized, high-density and uniformly distributed nanoislands. Then, transition-metal precursors, including those of Pt, Pd and Ru, are introduced in a controlled manner such that single atoms or clusters are positioned preferentially on the nanoislands. These nanoislands have stronger interactions with metals than the substrates do, confining metal species within well-defined regions and preventing their migration and agglomeration. This confinement effect is crucial for maintaining the dispersion and activity of metal catalysts, especially at elevated temperatures. Compared with conventional impregnation or deposition-precipitation methods, this approach achieves more precise spatial control over metal deposition and creates strong confinement environments that effectively suppress sintering. This protocol enables the preparation of thermally stable small-sized metal catalysts suitable for fuel processing, exhaust treatment, chemical manufacturing and other uses. The complete workflow, which includes oxide deposition, metal loading and material characterization, typically requires about 4 d, and catalytic testing requires an additional 5-20 h depending on the system. The protocol can be implemented in laboratories equipped with standard wet-chemistry facilities and with prior experience in synthesizing inorganic nanomaterials.
New methods for recovering the energy and value from polyolefin plastic waste must account for all the hydrocarbons formed during a deconstruction reaction. Analysis of the reaction mixture distribution is key to determining a catalyst's performance (activity and selectivity) and evaluating the economic viability of a conversion process. The molecular species present in the reaction mixtures can range from H2 and CH4 to hyper-branched hydrocarbons above 100,000 g/mol and any hydrocarbon in between; therefore, multiple analytical techniques are required to quantify all of the products. Here we describe an optimized and validated workflow that uses integrated analytical gas chromatography for concurrent H2 and gas-phase hydrocarbon quantification of the headspace; complementary gas chromatography, liquid chromatography and multi-nuclear magnetic resonance spectroscopy to quantify the composition of soluble products, as well as gel permeation chromatography to determine of the molecular weight distribution of the residual insoluble polymeric material. Using polyolefin hydrogenolysis in an autoclave reactor as an example, we describe how to specifically adapt these techniques to polymer deconstruction experiments and fully quantify the entire hydrocarbon population, while resolving and assigning specific species and characterizing structures. The information from this comprehensive analysis is needed to study reaction kinetics and to evaluate the intrinsic activity of a catalyst and reactivity of polymers in upcycling experiments, enabling mechanistic investigations and providing data to link experiment and theoretical models. The comprehensive quantitative analysis in this protocol can be completed within 4 d.
Frontier large language models (LLMs), such as GPT-5, Claude 4.5, Gemini 3, Llama 4 and DeepSeek-R1, represent a transformative class of artificial intelligence tools capable of revolutionizing various aspects of healthcare by generating human-like responses across diverse contexts and adapting to novel tasks following human instructions. Their potential application spans a broad range of medical tasks, such as clinical documentation, matching patients to clinical trials and answering medical questions. Here in this Tutorial, we discuss an actionable set of best practices to help healthcare professionals utilize LLMs more effectively and efficiently. The overall workflow follows sequential phases from formulating the task, choosing the most appropriate LLMs, engineering the prompts, fine-tuning the requests and through to model deployment. We discuss a set of critical considerations in identifying medical tasks that align with the core capabilities of LLMs and selecting models based on the required task, data, performance and model interface. We then review the strategies, such as prompt engineering and fine-tuning, to adapt standard LLMs to specialized medical tasks. We then cover deployment considerations, including regulatory compliance, ethical guidelines and continuous monitoring for fairness and bias. By providing a structured step-by-step methodology, this entry-level tutorial aims to equip healthcare professionals with the tools necessary to effectively integrate LLMs into clinical practice, ensuring that these powerful technologies are applied in a safe, reliable, and impactful manner.
Ultrasound is the second most common clinical imaging modality. Ultrasound image quality often suffers from poor contrast resolution and thus can greatly benefit from appropriate contrast agents. While micron-sized gas core particles (microbubbles) are clinically utilized in this space for applications such as echocardiography, a smaller agent could be more broadly applicable to enhance the sensitivity and specificity of disease detection. To this end, lipid-shelled nanobubbles have recently emerged as robust ultrasound contrast agents for both diagnostic and therapeutic purposes. They have been used in preclinical applications ranging from oncology to endocrinology and are notably relevant in diseases that involve pathological vasculature. Owing to their shell composition, nanobubbles are versatile and can be functionalized with fluorophores, targeting agents and therapeutic moieties. Here we provide the steps needed to formulate plain, fluorophore-conjugated, ligand-conjugated, hydrophilic dye-integrated and drug-loaded nanobubbles. The typical formulation for lipid-shelled nanobubbles takes place via self-assembly driven by mechanical agitation, followed by differential centrifugation. The process is deceptively simple, yet there are many nuances in the formulation process that must be followed to produce consistently successful nanobubble batches without contamination from microbubbles. Even minor deviation from the standard protocol can substantially affect nanobubble yield, stability, acoustic performance and batch-to-batch reproducibility. This procedure also details common pitfalls and their potential consequences for bubble quality and performance. The procedure requires 3 h to complete the formulation and activation of the plain nanobubbles by users with basic laboratory expertise.
Cellular lipids shape health and disease through specific protein interactions, yet lipid–protein networks remain poorly defined. Despite rapid advances in functional lipid probes, the field still lacks a practical, dedicated protocol for conducting lipid–protein interaction studies. We describe detailed methods for determining lipid interactomes within cells using multifunctionalized lipid derivatives. We provide a protocol that details how to (i) treat cells with lipid derivatives and perform photochemistry to obtain lipid–protein conjugates, (ii) extract cellular lysates for downstream analysis, (iii) perform click chemistry on lysates with a fluorophore and observe lipid–protein conjugates by in-gel fluorescence and (iv) perform click chemistry on lysates with azide beads and prepare lipid–protein conjugates for proteomic analysis. We provide context on important parameters for each step and include guidelines for controls, as well as suggestions for troubleshooting based on common problems encountered during the preparation of this protocol. This protocol enables identification of proteins that bind to specific lipids across diverse biological systems and cellular states. The entire workflow from cell treatment to complete proteomic sample preparation requires ~15 h over 4 d, depending on the type of experimental readout (in-gel fluorescence or proteomics) and the usage of pause points. Practitioners are expected to be familiar with standard biochemical techniques, such as sterile sample handling and tissue culture and gel electrophoresis. Additional skills are needed for mass spectrometric analysis, and collaboration with a proteomics core facility is recommended. The described procedures uniquely enable the identification of the protein interactors (the interactome) of select lipid species, providing for a major advance in the characterization of the biological roles of lipids in cellular systems. Cellular lipids are known to have specific protein interactions. This protocol describes how to study the lipid–protein interactome using multifunctional lipid probes capable of photo-cross-linking and click chemistry by in-gel fluorescence or mass spectrometry.
Large-scale interrogation of genome structure is crucial for understanding how genomic organization influences cellular function, yet existing methods are limited by the low density of achievable modifications or the toxicity of methods. Here we address this gap by presenting a versatile approach that combines gene editing and recombinase technologies. The protocol serves two critical purposes: (1) facilitating the introduction of hundreds to thousands of precise genomic edits per cell and (2) enabling the creation of a controlled platform to systematically investigate the effects of induced genomic rearrangements. Specifically, the method leverages prime editing to insert recombinase recognition sites (for example, loxP) into repetitive genomic regions, such as LINE-1 elements, thereby enabling extensive genetic modifications in human cells. This scale of genome editing has not previously been attainable and supports a wide range of studies, including genome-wide functional analyses and essentiality mapping. Inducing controlled rearrangements with recombinase and tracking cell survival under selective conditions allows direct mapping of genome architecture to cellular fitness, opening new opportunities for genome-wide functional screens and rational synthetic genome design. Unlike methods that rely on double-strand breaks or random transposon insertion, this Protocol supports a programmable installation of thousands of recombination sites at repeat elements, offering denser and more predictable substrates for controlled genome rearrangement. The full protocol takes ~12–18 weeks to complete and requires intermediate to advanced expertise in genome editing, mammalian cell culture and sequencing analysis. This large-scale genome structure interrogation approach uses prime editing to insert recombinase recognition sites into repetitive genomic regions. It enables thousands of precise genomic edits to be installed per cell with systematic investigation of their effects.
The integration of biological and artificial systems promises the effective coupling of living cells with electronic devices. However, to create biomimetic platforms capable of bridging biological with artificial systems, it is necessary to first enhance cell adhesion and cell interactions with engineered surfaces via the integration of techniques from materials science, nanotechnology and synthetic biology, such as structural functionalization techniques, and chemical or biological surface modifications. In this Tutorial Review we cover the use of polymer-based semiconductors and micro- and nanofabrication methods for the integration of biologically relevant cell membrane models with chip-based devices. This integration enhances cell-device coupling and provides an approach for studying membrane-level interactions. Although cell membranes are essential for understanding biological mechanisms, including drug responses, existing technologies rely on simplified synthetic models which lack biological complexity. Advances in electrical impedance measurements enable the study of membrane protein activity, providing insight into drug interactions and biomolecular processes. In addition, exploiting these hybrid systems can result in improved adhesion and electrostatic interactions, facilitating functional coatings for microdevices and neuromorphic applications. We discuss the recent advances in biomembrane-electronic interfaces, device design, surface modification, electronic materials, biomembrane formation and measurement techniques in the context of applications in drug discovery, diagnostics and neuromorphic computing, along with future directions for the field.
Carbon quantum dots (CQDs) have emerged as promising materials for electroluminescent light-emitting diodes (LEDs) because of their photostability, low toxicity and tunable luminescence. However, their implementation in solid-state devices is often hindered by aggregation-induced quenching (AIQ), which leads to a substantial loss of photoluminescence quantum yield (PLQY) and poor device reproducibility. Here, we present a robust, reproducible and scalable protocol for the preparation of solid-state emissive CQDs (SSE-CQDs) and their integration into electroluminescent LEDs. The protocol uses a solvothermal reaction between aromatic aldehydes and aromatic nitriles, involving Knoevenagel-type condensation, dehydration and carbonization. This synthetic strategy enables the formation of non-planar conjugated architectures with incorporated long-chain electron-donating alkoxy groups, which effectively suppress intermolecular π-π interactions and mitigate AIQ in the solid state. Compared with existing approaches that rely on host matrices or multistep post-synthetic modification, this protocol produces intrinsic SSE-CQDs, simplifying processing and improving reproducibility. Using this approach, SSE-CQDs with PLQYs exceeding 40% under ambient conditions can be reproducibly obtained and processed using standard solution-based techniques. This protocol describes in detail the synthesis, purification and basic optical characterization of SSE-CQDs, followed by their incorporation into electroluminescent device architectures. The complete workflow, from CQD synthesis to LED fabrication, can be completed within ~41.5 h. The procedures are compatible with conventional laboratory equipment and are suitable for researchers with experience in nanomaterial synthesis and optoelectronic device fabrication. This protocol provides a general and transferable platform for developing CQD-based solid-state emitters and LEDs.
