Small Methods

Spatial transcriptomics (ST) has emerged as a transformative tool for resolving the molecular heterogeneity of complex tissues within their native anatomical context. However, next-generation sequencing (NGS)-based ST platforms frequently encounter sensitivity bottlenecks arising from sub-optimal probe architectures on solid substrates. Conventional single-stranded DNA coupling methods often lead to disordered interfacial molecular conformations due to non-specific nucleobase-mediated surface tethering, which creates steric hindrance and inhibits the enzymatic efficiency of in situ library preparation. Here, we present Decomap (double-strand protected combinatorial barcoding microarray chip), a high-performance ST platform utilizing a triple-segment (dsZ-X-Y) fabrication strategy to achieve superior transcript capture efficiency. This structural optimization significantly enhances DNA ligation kinetics and subsequent polymerase-mediated extension, overcoming the fundamental limitations of traditional single-stranded coupling strategies. Decomap-seq achieved a median detection of 7,200 genes and 29,097 UMIs per 50 m-spot at a sequencing saturation of 50.1%. These results validate Decomap as a highly sensitive and robust tool for spatial transcriptomics, offering a powerful platform for advancing research in histopathology, developmental biology, and neuroscience.

The superior stealth properties and high information density make DNA a sought-after candidate in the field of molecular steganography. Here, we developed the InfinMark end-to-end DNA steganography framework for anti-counterfeiting applications by combining the characteristics of both the Internet of Things (IoT) and DNA-of-Things (DoT). InfinMark includes five modules: Information Transcoding, Fingerprint Writing, Nano-encapsulation, Invisible Marking, and Multi-level Rapid Authentication. It ensures precise anti-counterfeiting information reading and writing through a dynamic DNA-compatible transcoding algorithm, achieves seamless embedding by developing scalable nanoparticle manufacture methods, and supports cross-scenario on-site verification, ultimately granting it comprehensive anti-counterfeiting capabilities spanning from source labeling to terminal tracing. By addressing the bottlenecks in IoT and DoT integration, lifecycle tracking, as well on-site product authentication, this research constructs a full-chain bimodal anti-counterfeiting system, thereby showcasing the practical application of informational DNA nanoparticles in various aspects of production and daily life.

Persistent infection with high-risk human papillomavirus (HPV) is the primary cause of cervical cancer and other HPV-related malignancies. Effective screening and early detection of HPV, particularly in point-of-care (POC) settings, can reduce disease progression and associated mortality. Although PCR-based assays provide high sensitivity, their dependence on centralized laboratory infrastructure limits accessibility in POC settings. CRISPR-Cas diagnostics enable programmable, isothermal detection of HPV with lateral flow assay (LFA) readouts; however, visual interpretation of faint bands can be subjective and inconsistent. Our objective was to develop a machine learning (ML)-enhanced, smartphone-native CRISPR-LFA platform for highly sensitive and reliable detection of HPV DNA in plasma. A smartphone-based diagnostic system integrating CRISPR-LFA with a ML framework was developed using standardized image acquisition within a light-controlled enclosure. Radiomics-inspired strip features were extracted and analyzed using a multivariable logistic regression model. A total of 150 plasma samples were used for model development and 60 independent samples for validation. An optimized model was developed that had 96.7% sensitivity and 100% specificity for detection of HPV DNA. The smartphone-enabled CRISPR platform demonstrated higher sensitivity than visual interpretation, particularly for faint-band results, and reduced false positives. Validation in the independent cohort confirmed the robustness of the assay. Performance remained stable across smartphone models, lighting conditions, and operators, and on-device inference enabled reliable operation. In sum, the smartphone-integrated CRISPR-LFA platform can facilitate accurate and reliable detection of plasma HPV DNA in POC settings and has the potential to enhance early detection, prevention, and treatment of cervical cancer.

Over the past decades, the frequency of viral outbreaks has increased substantially worldwide, driven in part by global travel and resulting in millions of deaths each year. This trend underscores the urgent need for rapid, simple, and accessible diagnostic tools for infectious disease detection. Here, we present a nanofluidic digital chip (Nano-dChip) for point-of-care viral RNA detection that delivers results within 30 minutes at a cost of less than $0.50 per chip. The Nano-dChip employs reverse transcription loop-mediated isothermal amplification (RT-LAMP) for highly sensitive and specific target amplification. Reaction reagents are compartmentalized into numerous nanofluidic reservoirs, enabling highly quantitative detection while minimizing contamination risks. Using a single chip, we successfully detect both SARS-CoV-2 and Influenza H3 RNA with a detection limit of 10 fM, demonstrating the Nano-dChips potential as a rapid, low-cost, and scalable diagnostic platform for timely outbreak control.

Meningeal lymphatic vessels (mLVs) are vital for brain waste clearance, making them a promising therapeutic target. However, effective modulation strategies for mLVs with translational potential remain underdeveloped. Here, we develop a low-intensity focused ultrasound (LIFU) strategy that precisely targets the vault cranial meninges to non-invasively facilitate mLVs drainage. Using models of Alzheimers disease (AD) and aging, we demonstrate that this approach promotes CSF drainage, prevents cognitive decline, and reduces pathological biomarkers. Mechanistically, RNA sequencing combined with calcium imaging in vitro reveals that LIFU activates the Piezo1 ion channel in lymphatic endothelial cells, whereas pharmacological inhibition of Piezo1 abolishes LIFUs therapeutic effects. Compliant with FDA safety guidelines, this LIFU protocol demonstrates strong clinical translatability. If its efficacy is clinically confirmed, LIFU offers a promising therapy for neurodegenerative diseases triggered by waste accumulation.

Glycoproteins lining the luminal endothelial surface form the glycocalyx, composing the tripartite blood brain barrier. We explored the glycocalyx as a source of danger signals for complement lectin pathway after ischemic stroke. Our data indicate that hypoxic microvascular cells increased -D-mannosyl and N-acetylglucosaminyl exposure after re-oxygenation, favoring mannose binding lectin (MBL) pathogenic deposition, and overexpression of inflammatory genes (ICAM-1 and MMP-2). The hypoxia-conditioned medium induced neuronal damage (reduced MAP-2), microglia and astrocytic reactivity (increased/thickened ramifications) when applied to induced pluripotent stem cell-derived neurons, astrocytes and microglia co-cultures. All these effects were counteracted by mannose-capped gold nanoparticles (Man-GNPs), shown to bind and sequester MBL from the medium. We then tested the Man-GNPs in vivo, in an ischemic stroke model using humanized mice, knocked-in for human MBL. The ischemic mice (males:females 1:1) treated with Man-GNPs (3h after the ischemic onset) exhibited less anxiety at the elevated plus maze and reduced neuronal loss at 8d after ischemia compared to vehicle-treated. Thus, multivalent Man-GNPs represent a promising approach to take MBL away from its glycoproteic targets on the ischemic endothelium, hence preventing downstream pathogenesis. Moreover, these data support circulating MBL as a druggable pharmacological target to prevent the thrombo-inflammatory events following acute brain injury.

Osmotic pressure has been known to play essential roles in living systems from single cells to complex tissues. However, direct in-situ measurements of osmotic pressures in biosystems have remained challenging, especially in complicated heterogeneous systems in which osmotic pressure gradients could exist and induce directed forces. Bacterial biofilms -- organized communities of bacteria encased in a self-produced extracellular matrix -- are a major mode of bacterial life. It has, however, remained unexplored how the osmotic pressure is distributed in the biofilm and how this distribution contributes to biofilm growth and activity. Here, liposomal nano-sensors are developed for the in-situ mapping of osmotic pressures at an unprecedented microscale resolution in real time using Escherichia coli. biofilm as a model system that develops at the surface of a hydrogel containing the nutrients. The measurements reveal osmotic pressure gradients with a radially increasing trend from the inner regions to the outer regions of the biofilm, which is associated with biofilm formation, morphology, and metabolism. The gradients likely contribute to mechanical properties, internal stresses, and nutrient transport. The sensor readouts also show that there is an osmotic pressure difference between the biofilm and the adjacent medium, which may promote biofilm expansion through matrix swelling and bacteria growth via water and nutrient uptake from the surroundings. Our novel approach based on in-situ osmotic pressure mapping in a growing biofilm reveals a sophisticated spatial regulation of physical forces, which may inspire new models and approaches in the field of mechanobiology.

Liquid biopsy through circulating tumor DNA (ctDNA) analysis enables non-invasive detection of minimal residual disease (MRD) and early identification of cancer relapse, facilitating timely clinical intervention. However, detecting ctDNA in plasma samples with low tumor burden remains challenging due to the scarcity of mutant molecules, the background noise of sequencing errors and somatic mutations in normal cell-free DNA (cfDNA). Here, we present a mutation-informed fragmentomic framework and evaluate it on 90 stage III colorectal cancer patients with three years of follow-up. Using 712 serial whole-genome sequenced cfDNA samples (30x) with matched whole-genome sequencing of tumor tissue and germline DNA from buffycoat for each patient, we collected cfDNA fragments spanning tumor-derived somatic mutation positions and compared fragmentomic characteristics of mutation-bearing and non-mutated cfDNA fragments within the same sample. By leveraging fragment length and fragment end-motif patterns, our approach can distinguish cancer-positive from cancer-negative plasma samples without requiring model training or panel-of-normals calibration. The method achieved AUCs of 0.863 and 0.74 using fragment length and end motif features, respectively, and 0.871 when combined, outperforming tumor fraction estimates based on the frequency of mutated fragments (AUC=0.832). Integrating fragmentomic features with tumor fraction further improved performance, yielding an AUC of 0.873, indicating complementary signals between fragmentomic patterns and mutation burden. Aggregated analyses revealed ctDNA-specific patterns, including fragment shortening, motif enrichment of A/T ends, and depletion of C/G ends, directly linking fragmentomic features to tumor-derived cfDNA. Overall, mutation-informed fragmentomic profiling improves ctDNA detection beyond counting mutant reads and provides a scalable, training-free strategy for MRD assessment and early relapse detection while offering mechanistic insights into tumor-specific cfDNA biology.

In vitro models are increasingly critical for interrogating cancer biology and therapeutic response, however, accurately recapitulating the tumor microenvironment (TME) remains a persistent challenge, particularly in head and neck cancers (HNC) characterized by complex cell-matrix interactions and heterogeneity. Current models often lack independent tunability of biochemical and biophysical cues, limiting systematic investigation of microenvironmental cues in a high-throughput format. Here, we establish a 3D droplet-based bioprinting platform for the fabrication of customizable in vitro TME models using poly(ethylene glycol) (PEG) hydrogels. Human HNC cell lines (FaDu and 2A3) with differing HPV statuses were bioprinted into PEG matrices spanning physiologically relevant stiffnesses (0.7-4.8 kPa) and compositions, including non-functionalized PEG and peptide-functionalized PEG (PEGfnc: RGD, YIGSR, CNYYSNS) and cultured for 7 days. Cluster growth, cell viability, and cluster morphology were assessed across multiple time points, matrix compositions, and matrix stiffnesses. Proliferation and endpoint phenotype expression were visualized using confocal microscopy through immunofluorescence. Results indicated enhanced cell viability in PEGfnc matrices, compared to non-functionalized matrices, while effect of matrix stiffness was less prominent. Median cluster size reached 40-50 m by day 7, and linear mixed-effects modeling identified how changes in cluster surface area, volume, and tumoroid complexity varied with cell type, matrix, and stiffness. By decoupling and systematically varying key TME parameters, this approach provides a robust and scalable framework for dissecting tumor-matrix interactions and advancing physiologically relevant in vitro models for cancer research and therapeutic screening.

Accurate quantification of transplanted cardiac spheroids requires three-dimensional localisation within intact myocardium, yet this remains technically challenging. Optical clearing and light-sheet microscopy enable volumetric imaging of injection sites, but automated segmentation is difficult when transplanted spheroids and host tissue are labelled with the same fluorescent markers and cannot be separated by simple thresholding. We developed a random forest based pixel classification workflow for 3D detection of injected hiPSC derived cardiomyocyte and H9c2 spheroids in optically cleared rabbit myocardium. A supervised classifier trained on intensity, edge, and texture features generated a segmentation then grouped pixels via connected component analysis to reconstruct individual spheroids. The method showed good agreement with manual annotation and enabled automated extraction of spheroid size and spatial metrics. This accessible workflow enables reproducible three-dimensional quantification of transplanted spheroids in large light-sheet microscopy datasets and provides a practical route from volumetric imaging to spatial metrics in cardiac regeneration studies.

Chimeric antigen receptor T (CAR-T) cell therapy is transforming the treatment landscape of hematological malignancies. However, manufacturing with integrating viral vectors is costly, slow, and carries risks including insertional mutagenesis, pro-longed B cell aplasia, and other long-term toxicities. Expression of CAR with mRNA can reduce cost, manufacturing timelines, and improve safety. However, the short-lived expression necessitates frequent repeat dosing. Here, we describe a modified self-amplifying RNA (saRNA) platform for engineering CAR T cells with prolonged CAR expression and enhanced durability of tumor control relative to mRNA CAR T cells. In an acute lymphoblastic leukemia (ALL) xenograft model, saRNA CAR T cells achieve superior tumor suppression and prolong survival. Further, a single-strand modified saRNA supports the co-expression of multiple proteins, enabling the construction of advanced CAR systems, such as OR- and AND-gated logic CAR T cells. Together, these results highlight saRNA as a powerful and versatile platform for CAR T cell engi-neering with favorable safety, efficacy, and accessibility.

Rapid and highly selective sensing of ultra-low concentration protein biomarkers remains a critical challenge important for early disease diagnosis and monitoring. Here, we use conical SiO2 nanopore-based biosensing for the rapid detection of heart-type fatty acid binding protein (H-FABP). Antibodies were covalently immobilized on the nanopore surface through siloxane chemistry. The functionalized asymmetric nanopores generate a characteristic rectifying current-voltage response, which shows a distinct shift upon binding to the target protein due to partial neutralization of the negatively charged pore surface. The sensor exhibits excellent sensitivity in the attomolar to nanomolar concentration range with a detection limit (LOD) of [~]0.4 aM. Furthermore, the platform exhibits high selectivity, distinguishing H-FABP from non-target proteins (HSA and Hb) at concentrations six orders of magnitude higher. We also demonstrate that nanopores can be regenerated using sodium hypochloride and O2 plasma treatment, enabling repeated functionalization and reuse.

Extracellular vesicles (EVs) are lipid spheres released from cells. Research utilizing EVs has met several hurdles owing to the small size of the majority of EVs and other nanoparticles (<150 nm) and the lack of detection technologies capable of providing high-throughput single particle measurements at this scale. The use of high-throughput single particle measurements is critical for the assessment of EV heterogeneity and abundance which are features often used to assess the development of isolation protocols or particle characterization. The Coulter principle, known in the field as resistive pulse sensing (RPS), has been used for several decades to size and count cells. More recently, this technology has evolved to accommodate nanoparticle analysis. In the last decade a platform utilizing microfluidic resistive pulse sensing (MRPS) has been demonstrated for nanoparticles, offering ergonomic characterization of nanoparticles along with utilizing open format data. To date, assessment of MRPS accuracy and reporting standards have not been assessed. With the aim of increasing data accuracy, ergonomics, and reporting transparency, we developed a microfluidic resistive pulse sensing post-acquisition analysis software (RPSPASS) application for automated cohort calibration, population gating, statistical output, QC plot generation, alternative data file outputs, and standardized reporting templates.

Biocompatible fluorosurfactants are essential for many droplet microfluidic workflows but are often obtained from commercial sources because published syntheses of perfluoropolyether (PFPE)-based surfactants typically require acid chloride intermediates and chemistry-oriented purification methods. These requirements can limit access for biology and clinical laboratories seeking low-cost or customizable surfactant systems. Here we describe a practical method for preparing functional PFPE-based fluorosurfactant materials by direct carbodiimide coupling of functionalized PFPE carboxylic acids(Krytox 157 FSH) to amine-containing head groups under laboratory-accessible conditions. Using this approach, we prepared a PFPE-polyethylene-glycol (PFPE-PEG) material from Jeffamine ED900 and a PFPE-Tris material from Tris base. Because these products were not fully structurally characterized, we present them as functional reaction products and evaluate them by use in biomicrofluidic workflows rather than by definitive compositional assignment. PFPE-Tris was useful for generating relatively uniform small droplets, whereas the PFPE-PEG preparation supported a broader range of biological applications. These materials were used in genomic library screening for {beta}-glucosidase activity, thermocycling-associated droplet workflows, and protein crystallization experiments. In addition, the PFPE-PEG preparation improved emulsion behavior in many protein crystallization screens that were unstable with a commercial droplet oil used in our laboratory. This method reduces the practical barrier to in-house fluorosurfactant preparation and allows biology-focused laboratories to explore head-group chemistry, oil composition, and operating conditions without complete reliance on commercial reagents. The results support this workflow as a useful entry point for biomicrofluidics laboratories, while also highlighting the need for careful interpretation of thermocycled droplet assays and for future analytical characterization of the resulting materials. Significance statementDroplet microfluidics relies on fluorosurfactants that are often costly and difficult to synthesize outside of chemistry-focused settings. We describe a simple, biology-laboratory-compatible approach for generating functional perfluoropolyether-based fluorosurfactant materials using direct carbodiimide coupling and straightforward cleanup. The resulting materials supported multiple biomicrofluidic workflows in our laboratory, including enzymatic screening and protein crystallization, and provide a practical route for groups seeking lower-cost and more customizable surfactant systems.

Rare histologic variants of bladder carcinoma, such as squamous cell and neuroendocrine carcinoma, generally have a worse prognosis compared to pure urothelial carcinoma (PUC), but the underlying molecular determinants are not well understood. We developed a novel computational genomics framework to characterize the dynamics of tumor-stroma-immune interactions at tumor borders and interiors from spatial transcriptomics data. We profiled bladder carcinoma samples of different histological variants on the Visium platform. Differences in clonal phylogeny and spatial heterogeneity of major subclones between the samples suggested disparate clonal spatiotemporal dynamics and interaction with stromal and immune compartments - which was notably prominent at the tumor-stroma interface. Our framework captured immune heterogeneity in the tumor microenvironment, including variations in the presence and architecture of tertiary lymphoid structures. Our analyses further indicated that there are substantial histology-specific differences in cell type composition, clonal spatial heterogeneity, inter-cellular signaling, and cellular processes. These variations collectively suggest divergent mechanisms of microenvironment remodeling across bladder cancer histologies. Cell-free DNA profiling from liquid biopsy captured tumor and microenvironment signatures from tumor boundaries and interiors, potentially allowing for tracking clonal dominance non-invasively. Our method tracks the trajectory of neoplastic disease in bladder cancer samples while identifying aggressive features.

Endocavity ultrasound transducers, particularly transvaginal ultrasound (TVUS) probes, contain intricate structures such as notches, grooves, lens surfaces, and handle edges that are highly susceptible to microbial contamination yet difficult to disinfect using conventional high-level disinfection (HLD) methods. This study evaluated the efficacy of a novel ultraviolet-C light-emitting diode (UV-C LED) HLD system in eliminating microbial contamination from these complex probe surfaces. Two TVUS probes were sampled from predefined high-risk regions before and after disinfection following clinical use. Probe A was sampled at the top and bottom notches and both sides of the handle, while Probe B was assessed at the lens, edges, and bent groove regions. Microbial contamination was quantified using swab sampling, culture on agar plates, and identification via MALDI-TOF. Environmental sampling of examination and disinfection rooms was also performed. To assess this system robustness, probe sites were repeatedly inoculated with Bacillus subtilis spores and evaluated following UV-C treatment. Before UV-C treatment, contamination rates ranged from 25% to 57% across sampled regions, with microbial loads reaching up to 3.9 log CFU. Identified organisms included Staphylococcus epidermidis, Pseudomonas koreensis, Bacillus cereus, and Propionibacterium spp. Probe sheaths were also predominantly contaminated with Staphylococcus epidermidis., with counts reaching up to 4.3 log CFU, Environmental sampling revealed diverse microbiota, with higher contamination levels in examination rooms compared to disinfection areas. Following 90 seconds of UV-C exposure, no microbial growth was detected on any sampled site, indicating 100% decontamination. Additionally, UV-C treatment achieved >6.7 log reduction of B. subtilis spores across all tested regions. These findings demonstrate that UV-C LED technology provides rapid, effective, and consistent high-level disinfection of complex TVUS probe surfaces, supporting its potential as a rapid and reliable disinfection modality in clinical setting.

Genetic engineering experiments and therapies are constrained by the size of DNA integrations into human cells genomes. Existing AAV, lentiviral, and non-viral methods rapidly decrease in integration efficiency beyond [~]5kb of sequence. Through systematic evaluation of non-viral DNA template formats, we identified circular ssDNA and dsDNA as capable of mediating >5kb integrations. Large circular DNA delivery efficiency and its impacts on cell viability and payload expression could be significantly improved with small DNA "helper" plasmids, mRNA-encoded nucleases, and sequence design optimizations. Collectively, these modifications enabled ultra-large--up to 10 kb DNA--integrations at >20% efficiency in primary human T cells at the TRAC locus and at >60% efficiency in human iPSCs at the AAVS1 locus. Finally, we demonstrate that GMP clinical-manufactured T cells with ultra-large integrations are functional in vitro and in vivo. Overall, we identified optimal template architectures, delivery modes, and sequence design rules for ultra-large DNA integrations in both research and clinical settings to accelerate basic genetic research and next-generation cellular therapies.

Cathodoluminescence (CL) microscopy has the potential to achieve a key goal in biological imaging: the simultaneous visualization of proteins and cellular ultrastructure. This goal can be attained by tagging proteins of interest with spectrally distinct cathodoluminescent probes for detection in electron microscopy. To this end, lanthanide nanoparticles (LNPs) are promising probe candidates due to their stability under the electron beam and their distinct ion-dependent emission spectra suitable for multiplexed detection. However, the hydrophobic surface chemistry of LNPs limits their use in biological samples and requires surface functionalization compatible with aqueous environments and EM sample preparation protocols. Here, we use a DNA-based ligand exchange strategy that renders cathodoluminescent LNPs hydrophilic and compatible with further functionalization for specific protein labeling. We characterize the CL emission of DNA-functionalized LNPs following aqueous transfer and common EM preparation steps, including osmium tetroxide staining and drying protocols based on hexamethyldisilazane and critical point drying, and show that LNPs retain their CL emission under all tested conditions. Finally, we demonstrate multicolor CL imaging of spectrally distinct, DNA-functionalized LNPs on the surface of mammalian cells, enabling simultaneous visualization of cellular ultrastructure via secondary electrons and LNPs via multiple CL color channels.

How distinct regional identities emerge within a single developing brain remains poorly understood. Current in vitro models address this by fusing independently generated organoids, but this introduces variability in size, maturation state, and connectivity, confounding the study of regionalization itself. Here, we present a microfluidic platform that supports the co-development of different tissue identities within a single, continuous 3D culture domain. The device integrates controlled microfluidic flow with real-time fluorescence imaging, providing stable perfusion and high-resolution tracking of molecular transport without the need for embedded sensors or disruptive sampling. By delivering SAG, a Sonic hedgehog pathway agonist, to one surface of mouse forebrain organoids, we induced spatially segregated ventral (Nkx2.1+) and dorsal (Pax6+) domains within a unified tissue architecture. Controlled morphogen delivery is sufficient to drive region-specific fate specification without organoid fusion, offering a practical, scalable alternative for studying tissue regionalization in vitro.

The human vasculature is a complex, multiscale system comprising hierarchical networks of macroscale to microscopic vessels. Existing in vitro fabrication techniques often fail to bridge these disparate scales, as high-resolution methods like multiphoton ablation are too slow for replicating larger vessels, while 3D printing lacks the resolution for fine microscale features. Here, we report a "twisted wire templating" strategy capable of generating perfusable bifurcating hydrogel networks that seamlessly transition from the macro- to the micro-scale (2.3 mm to 140 {micro}m) through seven orders of bifurcations. By optimizing wire-twisting geometries and polyurethane dip-coating, we overcame instability-driven bead formation to ensure replication fidelity across the networks. Fabrication rigs were reconfigured from existing 2D planar layouts to 3D reconfigurable architectures to better replicate 3D vessel geometries which simultaneously reducing the laboratory footprint and fabrication times by 47%. Using a Taguchi orthogonal array, we further optimized surface chemistry and hydrogel composition to inhibit structural failure during template extraction, resulting in fully patent, perfusable networks. This method provides a robust, low-cost, and scalable foundation for creating physiologically representative vascular models for investigating multiscale disease mechanisms and organ-level tissue engineering.