Biology subjects

Wan, Z.

Publications and source records attributed to Wan, Z..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Evolutionary Genomics Guides Scalable Coral Probiotics for Climate Resilience

A universal bottleneck limiting probiotic efficacy in medicine, aquaculture, agriculture, and wildlife conservation is uncertain long-term colonization, necessitating repeated administration. We present an evolution-guided framework for probiotic identification based on genomic hallmarks of emerging host dependency, including widespread pseudogenization and insertion sequence proliferation driving genomic restructuring. Applied to coral reefs, we screened over 1,200 coral-associated bacterial isolates and identified Ruegeria MC10 as exhibiting these signatures. Its presence was associated with increased thermal tolerance of a model cnidarian. Following nursery application, MC10 persisted in reef corals for an 8-month monitoring period through a natural bleaching event, improving color retention and retaining algal photosynthesis performance. This work establishes a predictive, scalable pipeline for selecting persistent probiotics, directly addressing a central constraint on microbiome-based interventions across host systems.

microbiology↗

bioRxiv2025

Neural organoids protect engineered heart tissues from glucolipotoxicity by transferring versican in a co-culture system

Metabolic disorders could cause dysregulated glucose and lipid at the systemic level, but how inter-tissue/organ communications contribute to glucolipotoxicity is difficult to dissect in animal models. To solve this problem, myocardium and nerve tissues were modeled by 3D engineered heart tissues (EHTs) and neural organoids (NOs), which were co-cultured in a generalized medium with normal or elevated glucose/fatty acid contents. Morphology, gene expression, cell death and functional assessments detected no apparent alterations of EHTs and NOs in co-culture under normal conditions. By contrast, NOs significantly ameliorated glucolipotoxicity in EHTs. Transcriptomic and protein secretion assays identified the extracellular matrix protein versican as a key molecule that was transferred from NOs into EHTs in the high-glucose/fatty acid condition. Recombinant versican protein treatment was sufficient to reduce glucolipotoxicity in EHTs. Adeno-associated virus-delivered versican overexpression was sufficient to ameliorate cardiac dysfunction in a murine model of diabetic cardiomyopathy. These data provide the proof-of-concept evidence that inter-tissue/organ communications exist in the co-culture of engineered tissues and organoids, which could be systemically studied to explore potential pathological mechanisms and therapeutic strategies for multi-organ diseases in vitro.

cell biology↗

bioRxiv2025

Remodeling of self-assembled microvascular networks under long term flow

The incorporation of a functional perfusable microvascular network (MVN) is a common requirement for most organ on-chip-models. Long-term perfusion of MVNs is often required for the maturation of organ phenotypes and disease pathologies and to model the transport of cells and drugs entering organs. In our microphysiological system, we observe that flow can recover perfusion in regressed MVNs and maintain perfusable MVNs for at least 51 days. Throughout the 51 days, however, the MVNs are continuously remodeling to align with the direction of bulk flow and only appear to attain morphological homeostasis with the use of maintenance medium without growth factors. We observed that the flow resistance of the MVNs decreases over time, and using a computational model, we show that stable vessels have higher flow rates and velocities compared to regressing vessels. Cytokine analysis suggests that static conditions generate an inflammatory state, and that continuous flow reduces inflammation over an extended period. Finally, through bulk RNA sequencing we identify that both the endothelial and fibroblast cells are actively engaged in vascular and matrix remodeling due to flow and that these effects persist for at least 2 weeks. This MPS can be applied to study hemodynamically driven processes, such as metastatic dissemination or drug distribution, or to model long-term diseases previously not captured by MPS, such as chronic inflammation or aging-associated diseases.

biophysics↗

bioRxiv2025

A Hyperbolic Discrete Diffusion 3D RNA Inverse Folding Model for functional RNA design

Generative design of functional RNAs presents revolutionary opportunities for diverse RNA-based biotechnologies and biomedical applications. To this end, RNA inverse folding is a promising strategy for generatively designing new RNA sequences that can fold into desired topological structures. However, three-dimensional (3D) RNA inverse folding remains highly challenging due to limited availability of experimentally derived 3D structural data and unique characteristics of RNA 3D structures. In this study, we propose RIdiffusion, a hyperbolic denoising diffusion generative RNA inverse folding model, for 3D RNA design tasks. By embedding geometric features of RNA 3D structures and topological properties into hyperbolic space, RIdiffusion efficiently recovers the distribution of nucleotides for targeted RNA 3D structures based on limited training samples using a discrete diffusion model. We perform extensive evaluations on RIdiffusion using different datasets and strict data-splitting strategies and the results demonstrate that RIdiffusion consistently outperforms baseline generative models for RNA inverse folding. This study introduces RIdiffusion as a powerful tool for the generative design of functional RNAs, even in structure-data-scarce scenarios. By leveraging geometric deep learning, RIdiffusion enhances performance and holds promise for diverse downstream applications.

bioinformatics↗

bioRxiv2025

BuzzWatch: uncovering multi-scale temporal patterns in mosquito behavior through continuous long-term monitoring

Understanding the temporal dynamics of mosquito behavior is essential for developing effective interventions against pathogen transmission. However, limited knowledge exists about the environmental, physiological, and genetic factors influencing mosquito activity patterns. This knowledge gap is partly due to a lack of tools to accurately quantify the behavior of free-flying mosquitoes over extended periods. Here, we introduce BuzzWatch, an open-source, low-cost platform designed to continuously monitor mosquito flight behavior over several weeks with high temporal resolution. BuzzWatch records videos of mosquitoes freely flying in a transparent cage and automates the extraction, analysis, and visualization of behavioral data, including flight trajectories and population-level flight and sugar-feeding statistics. Using BuzzWatch, we quantified the daily rhythms of 10 Aedes aegypti populations from various geographic origins. Globally invasive Ae. aegypti showed increased sugar feeding and flight activity during midday compared to native African populations. Our platform further revealed subtle, long-lasting effects of blood feeding on activity patterns and a complex response to extended daylight periods. By integrating a host-seeking module in BuzzWatch to deliver CO2 and heat pulses, we observed a twofold increase in Ae. aegyptis response to host-associated cues during the daytime compared to nighttime. Combined, these results demonstrate BuzzWatchs potential to investigate responses to host cues over seconds, natural variability in daily rhythms over hours, and phenotypic plasticity over days. BuzzWatch offers a novel perspective on mosquito behavior over multiple timescales, paving the way for advanced ecological and epidemiological studies that can inform targeted and effective vector control strategies. Short SummaryThis study introduces a new experimental platform to monitor multiple features of mosquito behavior (flight activity, sugar feeding, short-range host-seeking) with high temporal resolution over several weeks. HighlightsO_LIContinuous month-long tracking of mosquito flight activity in a laboratory setting. C_LIO_LIMultiscale analysis of flight behavior, from seconds to weeks. C_LIO_LIMapping of natural variation in daily rhythm of Aedes aegypti populations. C_LIO_LIQuantifying the long-lasting effect of physiological (blood-meal digestion) and environmental (photoperiod increase) perturbations. C_LIO_LIAutomatically monitoring response to short-range host-seeking cues at specific times of the day. C_LI

animal behavior and cognition↗

bioRxiv2024

miR126-mediated impaired vascular integrity in Rett syndrome

Rett syndrome (RTT) is a neurodevelopmental disorder that is caused by mutations in melty-CpG binding protein 2 (MeCP2). MeCP2 is a non-cell type-specific DNA binding protein, and its mutation influences not only neural cells but also non-neural cells in the brain, including vasculature associated with endothelial cells. Vascular integrity is crucial for maintaining brain homeostasis, and its alteration may be linked to the pathology of neurodegenerative disease, but a non-neurogenic effect, especially the relationship between vascular alternation and Rett syndrome pathogenesis, has not been shown. Here, we recapitulate a microvascular network using Rett syndrome patient-derived induced pluripotent stem (iPS) cells that carry MeCP2[R306C] mutation to investigate early developmental vascular impact. To expedite endothelial cell differentiation, doxycycline (DOX)-inducible ETV2 expression vectors were inserted into the AAVS1 locus of Rett syndrome patient-derived iPS cells and its isogenic control by CRISPR/Cas9. With these endothelial cells, we established a disease microvascular network (Rett-dMVNs) and observed higher permeability in the Rett-dMVNs compared to isogenic controls, indicating altered barrier function by MeCP2 mutation. Furthermore, we unveiled that hyperpermeability is involved in the upregulation of miR126-3p in Rett syndrome patient-derived endothelial cells by microRNA profiling and RNAseq, and rescue of miR126-3p level can recover their phenotype. We discover miR126-3p-mediated vascular impairment in Rett syndrome patients and suggest the potential application of these findings for translational medicine.

cell biology↗

bioRxiv2024

Regulation of alternative polyadenylation isoforms of Timp2 is an effector event of RAS signaling in cell transformation

Alternative polyadenylation (APA) generates mRNA isoforms with different lengths of the 3 untranslated region (3 UTR). The tissue inhibitor of metalloproteinase 2 (TIMP2) plays a key role in extracellular matrix remodeling under various developmental and disease conditions. Both human and mouse genes encoding TIMP2 contain two highly conserved 3UTR APA sites, leading to mRNA isoforms that differ substantially in 3UTR size. APA of Timp2 is one of the most significantly regulated events in multiple cell differentiation lineages. Here we show that Timp2 APA is highly regulated in transformation of NIH3T3 cells by the oncogene HRASG12V. Perturbations of isoform expression with long 3UTR isoform-specific knockdown or genomic removal of the alternative UTR (aUTR) region indicate that the long 3UTR isoform contributes to the secreted Timp2 protein much more than the short 3UTR isoform. The short and long 3UTR isoforms differ in subcellular localization to endoplasmic reticulum (ER). Strikingly, Timp2 aUTR enhances secreted protein expression but no effect on intracellular proteins in reporter assays. Furthermore, downregulation of Timp2 long isoform mitigates gene expression changes elicited by HRASG12V. Together, our data indicate that regulation of Timp2 protein expression through APA isoform changes is an integral part of RAS-mediated cell transformation and 3UTR isoforms of Timp2 can have distinct impacts on expression of secreted vs. intracellular proteins.

molecular biology↗

bioRxiv2024

Soft-lithographically defined template for arbitrarily patterned acoustic bioassembly

Acoustic bioassembly is recently regarded as a highly efficient biofabrication tool to generate functional tissue mimics. Despite their capacity of directly patterning live cells with close intercellular proximity, most acoustic bioassembly techniques are currently limited to generate some specific simple types of periodic and symmetric patterns, which represents an urgent challenge to emulate geometrically complex cytoarchitecture in human tissue. To address this challenge, we herein demonstrate a soft-lithographically defined acoustic bioassembly (SLAB) technique that enables to assemble live cells into geometrically defined arbitrary multicellular structures. Particularly, we employed a widely accessible soft lithography technique to fabricate a PDMS construct that works as an amplitude modulation template to define the pressure distribution of near-field acoustic waves. We found that zero pressure areas of the near-field acoustic waves at the PDMS surface distribute above the air-filling regions of the PDMS construct when both the PDMS top layer and air layer are approximately one-tenth of the acoustic wavelength. Using this technique, bioparticles can be assembled into symmetrical or asymmetrical patterns. Specifically, we have demonstrated the SLAB of endothelial spheroids and hepatic cells into liver tissue mimics (LTMs). The functional analysis further indicates that the formed LTMs displayed liver-specific functions, including albumin secretion, urea synthesis, glucose metabolism, and lipid storage. We expect this SLAB technique will be broadly used to construct complex functional tissues for tissue engineering and regenerative medicine.

bioengineering↗

bioRxiv2024

A 3D in vitro assay to study combined immune cell infiltration and cytotoxicity

Immune cell-mediated killing of cancer cells in a solid tumor is prefaced by a multi-step infiltration cascade of invasion, directed migration, and cytotoxic activities. In particular, immune cells must invade and migrate through a series of different extracellular matrix (ECM) boundaries and domains before reaching and killing their target tumor cells. These infiltration events are a central challenge to the clinical success of CAR T cells against solid tumors. The current standard in vitro cell killing assays measure cell cytotoxicity in an obstacle-free, two-dimensional (2D) microenvironment, which precludes the study of 3D immune cell-ECM interactions. Here, we present a 3D combined infiltration/cytotoxicity assay based on an oil-in-water microtechnology. This assay measures stromal invasion following extravasation, migration through the stromal matrix, and invasion of the solid tumor in addition to cell killing. We compare this 3D cytotoxicity assay to the benchmark 2D assay through tumor assembloid cocultures with immune cells and engineered immune cells. This assay is amenable to an array of imaging techniques, which allows direct observation and quantification of each stage of infiltration in different immune and oncological contexts. We establish the 3D infiltration/cytotoxicity assay as an important tool for the mechanistic study of immune cell interactions with the tumor microenvironment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/586980v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@6a161dorg.highwire.dtl.DTLVardef@d1e5d3org.highwire.dtl.DTLVardef@47b87corg.highwire.dtl.DTLVardef@a0e051_HPS_FORMAT_FIGEXP M_FIG The 3D combined infiltration/cytotoxicity assay captures three important steps of immune cell infiltration into the solid tumor microenvironment: (1) circulating immune cells extravasate and invade the stromal matrix, (2) immune cells migrate through the stromal matrix to reach the tumor core, and (3) immune cells that successfully navigate the stroma must cross a basement membrane boundary secreted by the cancer cells to contact and kill the cancer cells within a solid tumor. C_FIG

cancer biology↗

bioRxiv2024

Ether lipids influence cancer cell fate by modulating iron uptake

Cancer cell fate has been widely ascribed to mutational changes within proteincoding genes associated with tumor suppressors and oncogenes. In contrast, the mechanisms through which the biophysical properties of membrane lipids influence cancer cell survival, dedifferentiation and metastasis have received little scrutiny. Here, we report that cancer cells endowed with high metastatic ability and cancer stem celllike traits employ ether lipids to maintain low membrane tension and high membrane fluidity. Using genetic approaches and lipid reconstitution assays, we show that these ether lipid-regulated biophysical properties permit non-clathrin-mediated iron endocytosis via CD44, resulting in significant increases in intracellular redox-active iron and enhanced ferroptosis susceptibility. Using a combination of in vitro threedimensional microvascular network systems and in vivo animal models, we show that loss of ether lipids from plasma membranes also strongly attenuates extravasation, metastatic burden and cancer stemness. These findings illuminate a mechanism whereby ether lipids in carcinoma cells serve as key regulators of malignant progression while conferring a unique vulnerability that can be exploited for therapeutic intervention.

cancer biology↗

bioRxiv2024

Charting Single Cell Lineage Dynamics and Mutation Networks via Homing CRISPR

Single cell lineage tracing, essential for unraveling cellular dynamics in disease evolution is critical for developing targeted therapies. CRISPR-Cas9, known for inducing permanent and cumulative mutations, is a cornerstone in lineage tracing. The novel homing guide RNA (hgRNA) technology enhances this by enabling dynamic retargeting and facilitating ongoing genetic modifications. Charting these mutations, especially through successive hgRNA edits, poses a significant challenge. Our solution, LINEMAP, is a computational framework designed to trace and map these mutations with precision. LINEMAP meticulously discerns mutation alleles at single-cell resolution and maps their complex interrelationships through a mutation evolution network. By utilizing a Markov Process model, we can predict mutation transition probabilities, revealing potential mutational routes and pathways. Our reconstruction algorithm, anchored in the Markov models attributes, reconstructs cellular lineage pathways, shedding light on the cells evolutionary journey to the minutiae of single-cell division. Our findings reveal an intricate network of mutation evolution paired with a predictive Markov model, advancing our capability to reconstruct single-cell lineage via hgRNA. This has substantial implications for advancing our understanding of biological mechanisms and propelling medical research forward.

bioinformatics↗

bioRxiv2023

Engineering self-propelled tumor-infiltrating CAR T cells using synthetic velocity receptors

Chimeric antigen receptor (CAR) T cells express antigen-specific synthetic receptors, which upon binding to cancer cells, elicit T cell anti-tumor responses. CAR T cell therapy has enjoyed success in the clinic for hematological cancer indications, giving rise to decade-long remissions in some cases. However, CAR T therapy for patients with solid tumors has not seen similar success. Solid tumors constitute 90% of adult human cancers, representing an enormous unmet clinical need. Current approaches do not solve the central problem of limited ability of therapeutic cells to migrate through the stromal matrix. We discover that T cells at low and high density display low- and high-migration phenotypes, respectively. The highly migratory phenotype is mediated by a paracrine pathway from a group of self-produced cytokines that include IL5, TNF, IFN{gamma}, and IL8. We exploit this finding to "lock-in" a highly migratory phenotype by developing and expressing receptors, which we call velocity receptors (VRs). VRs target these cytokines and signal through these cytokines cognate receptors to increase T cell motility and infiltrate lung, ovarian, and pancreatic tumors in large numbers and at doses for which control CAR T cells remain confined to the tumor periphery. In contrast to CAR therapy alone, VR-CAR T cells significantly attenuate tumor growth and extend overall survival. This work suggests that approaches to the design of immune cell receptors that focus on migration signaling will help current and future CAR cellular therapies to infiltrate deep into solid tumors.

immunology↗

bioRxiv2023

Patient-Specific Vascularized Tumor Model: Blocking TAM Recruitment with Multispecific Antibodies Targeting CCR2 and CSF-1R

Tumor-associated inflammation drives cancer progression and therapy resistance, with the infiltration of monocyte-derived tumor-associated macrophages (TAMs) associated with poor prognosis in diverse cancers. Targeting TAMs holds potential against solid tumors, but effective immunotherapies require testing on immunocompetent human models prior to clinical trials. Here, we develop an in vitro model of microvascular networks that incorporates tumor spheroids or patient tissues. By perfusing the vasculature with human monocytes, we investigate monocyte trafficking into the tumor and evaluate immunotherapies targeting the human tumor microenvironment. Our findings demonstrate that macrophages in vascularized breast and lung tumor models can enhance monocyte recruitment via TAM-produced CCL7 and CCL2, mediated by CSF-1R. Additionally, we assess a novel multispecific antibody targeting CCR2, CSF-1R, and neutralizing TGF-{beta}, referred to as CSF1R/CCR2/TGF-{beta} Ab, on monocytes and macrophages using our 3D models. This antibody repolarizes TAMs towards an anti-tumoral M1-like phenotype, reduces monocyte chemoattractant protein secretion, and effectively blocks monocyte migration. Finally, we show that the CSF1R/CCR2/TGF-{beta} Ab inhibits monocyte recruitment in patient-specific vascularized tumor models. Overall, this vascularized tumor model offers valuable insights into monocyte recruitment and enables functional testing of innovative therapeutic antibodies targeting TAMs in the tumor microenvironment (TME).

bioengineering↗

bioRxiv2023

High-affinity binding to the SARS-CoV-2 spike trimer by a nanostructured, trivalent protein-DNA synthetic antibody

Multivalency enables nanostructures to bind molecular targets with high affinity. Although IgG antibodies can be generated against a wide range of antigens, their shape and size cannot be tuned to match a given target. DNA nanotechnology provides an attractive approach for designing customized multivalent scaffolds due to the addressability and programmability of the nanostructure shape and size. Here, we use computational simulation to guide the design and synthesis of a DNA nanostructure-based synthetic antibody ("nano-synbody"). The nano-synbody is comprised of a three-helix bundle DNA nanostructure with three identical arms terminating in a mini-binder protein that targets the SARS-CoV-2 spike protein. The structure was designed to match the valence and distance between the three receptor binding domains (RBDs) in the spike trimer, in order to enhance binding through avidity effects. Moreover, the design allowed for the display of one, two, or three protein-displaying arms, thereby systematically probing the effect of multivalency on binding affinity. The binding strength of the nano-synbody increased with the increasing number of arms, yielding 11.2 pM affinity ([~]100-fold enhancement over monovalent binding) for the wild-type spike protein for the three-arm structure. Moreover, the multivalency was able to yield a 95 pM affinity for the Omicron variant, a mutant against which the monovalent protein was ineffective. The nano-synbody could also block infection of a spike protein-bearing pseudovirus, and similarly demonstrated effective inhibition of the Omicron variant when trimerized. The structure of the three-arm nano-synbody bound to the Omicron variant spike trimer was solved by negative-stain transmission electron microscopy reconstruction, and shows the protein-DNA nanostructure with all three arms bound to the RBD domains, confirming the intended trivalent attachment. Finally, nano-synbody binding could be reversed by removing one, two, or three arms in a programmable fashion, via toehold-mediated strand displacement. The ability to tune the size and shape of the nano-synbody, as well as its potential ability to attach (and then remove) two or more different binding ligands, will enable the high-affinity binding of a range of proteins, and pave the way towards their manipulation using DNA-based nano-robotic devices.

bioengineering↗

bioRxiv2023

An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

Systemic Lupus Erythematosus (SLE) is characterized by autoreactive B cell activation, upregulation of Type I Interferon (IFN) and widespread inflammation. Mitochondrial nucleic acids (NAs) are increasingly recognized as triggers of IFN1. Thus, defective removal of mitochondria from mature red blood cells (Mito+ RBCs), a feature of SLE, contributes to IFN production by myeloid cells2. Here we identify blood monocytes (Mo) that have internalized RBCs and co-express IFN-stimulated genes (ISGs) and interleukin-1{beta} (IL-1{beta}) in SLE patients with active disease. We show that ISG expression requires the interaction between Mito+ RBC-derived mitochondrial DNA (mtDNA) and cGAS, while IL-1{beta} production entails Mito+ RBC-derived mitochondrial RNA (mtRNA) triggering of RIG-I-like receptors (RLRs). This leads to the cytosolic release of Mo-derived mtDNA that activates the NLRP3 inflammasome. Importantly, IL-1{beta} release depends on the IFN-inducible myxovirus resistant protein 1 (MxA), which enables the translocation of this cytokine into a trans-Golgi network (TGN)-mediated unconventional secretory pathway. Our study highlights a novel and synergistic pathway involving IFN and the NLRP3 inflammasome in SLE.

immunology↗

bioRxiv2022

Transcriptomic landscape and potential therapeutic targets for human testicular aging revealed by single-cell RNA sequencing

BackgroundTesticular aging is known to cause male age-related fertility decline and hypogonadism, but the underlying molecular mechanisms remain unclear. MethodsWe survey the single-cell transcriptomic landscape of testes from young and old men and examine age-related changes in germline and somatic niche cells. ResultsIn-depth evaluation of the gene expression dynamics of germline cells reveals that disturbance of base-excision repair pathway is a major feature of aging spermatogonial stem cells (SSCs), suggesting that defective DNA repair of SSCs may serve as a potential driver for increased de novo germline mutations with age. Further analysis of aging-associated transcriptional changes shows that stress-related changes and apoptotic signaling pathway accumulate in aged somatic cells. We identify age-related impairment of redox homeostasis in aged Leydig cells and find that pharmacological treatment with antioxidants alleviate this cellular dysfunction of Leydig cells and promote testosterone production. Lastly, our results reveal that decreased pleiotrophin (PTN) signaling is a contributing factor for testicular aging. ConclusionsThese findings provide a comprehensive understanding of the cell-type-specific mechanisms underlying human testicular aging at a single-cell resolution, and suggest potential therapeutic targets that may be leveraged to address age-related male fertility decline and hypogonadism. FundingThis work was supported by the National Key Research and Development Program of China (2018YFA0107200, 2018YFA0801404), the National Natural Science Foundation of China (32130046, 82171564, 82101669, 81871110, 81971759), the Key Research and Development Program of Guangdong Province (2019B020234001), the Natural Science Foundation of Guangdong Province, China (2022A1515010371), the Major Project of Medical Science and Technology Development Research Center of National Health Planning Commission, China (HDSL202001000), the Open Project of NHC Key Laboratory of Male Reproduction and Genetics (Family Planning Research Institute of Guangdong Province) (KF202001), the Guangdong Province Regional Joint Fund-Youth Fund Project (2021A1515110921), the China Postdoctoral Science Foundation (2021M703736).

developmental biology↗

bioRxiv2022

Rapid Regulation of Local Temperature and TRPV1 Ion Channels with Wide-Field Plasmonic Thermal Microscopy

Plasmonic absorption of light can create significant local heat and has become a promising tool for rapid temperature regulation in diverse fields, from biomedical technology to optoelectronics. Current plasmonic heating usually relies on specially designed nanomaterials randomly distributed in the space and hardly provides uniform temperature regulation in a wide field. Herein we report a rapid temperature regulation strategy on a plain gold-coated glass slip using the plasmonic scattering microscopy, which can be referred to as wide-field plasmonic thermal microscopy (W-PTM). We calibrated the W-PTM by monitoring the phase transition of the temperature-sensitive polymer solutions, showing that it can provide a temperature regulation range of 33-80 {degrees}C. Moreover, the W-PTM provides imaging capability, thus allowing the statistical analysis of the phase-transitioned polymeric nanoparticles. Finally, we demonstrated that W-PTM can be used for noninvasive and local regulation of the transient receptor potential vanilloid 1 (TRPV1) ion channels in the living cells, which can be monitored by simultaneous fluorescence imaging of calcium influx. With the nondestructive local temperature-regulating and concurrent fluorescence imaging capability, we anticipate that W-PTM can be a powerful tool to study cellular activities associated with cellular membrane temperature changes.

bioengineering↗

bioRxiv2022

Multiplexed protein detection and parallel binding kinetics analysis with label-free digital single-molecule counting

Multiplexed protein detection is critical for improving the drug and biomarkers screening efficiency. Here we show that multiplexed protein detection and parallel protein interaction analysis can be realized by evanescent scattering microscopy with label-free digital single-molecule counting. We implemented an automatic single-molecule counting strategy with high temporal resolution to precisely determine the binding time, which improves the counting efficiency and accuracy. We show that digital single-molecule counting can recognize proteins with different molecular weights, thus making it possible to monitor the protein binding processes in the solution by real time tracking the numbers of free and bound proteins landing on the sensor surface. Furthermore, we show that this strategy can simultaneously analyze the kinetics of two different protein interaction processes on the surface and in the solution. This work may pave a way to investigate complicated protein interactions, such as the competition of biomarker-antibody binding in biofluid with biomarker-protein binding on the cellular membrane.

biochemistry↗