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Circulating fetal nucleated cells (CFCs) carrying whole genomic coding of the fetus in maternal blood have been pursued as ideal biomarkers for noninvasive prenatal testing (NIPT). However, a significant limitation is the need to enrich sufficient cells in quantity and purity for fetal genetic disorder diagnosis. This study for the first time demonstrates a stimuli-responsive ligand enabling interface on array patterned microfluidic chip (NIPT-Chip) for high efficient isolation and release of CFCs in untreated whole blood. Deterministic lateral displacement (DLD)-array was patterned in the chip to increase collision frequency between CFCs and surface-anchored antibody to achieve high efficient cell capture. More importantly, the stimuli-responsive interface enables gentle release of captured CFCs through a thiol exchange reaction for downstream gene analysis of NIPT. With the advantages of simple processing, efficient isolation, and gentle release, NIPT-Chip offers great potential for clinical translation of circulating fetal cell-based NIPT.

DNA bricks build nanotube transistors Semiconducting carbon nanotubes (CNTs) are an attractive platform for field-effect transistors (FETs) because they potentially can outperform silicon as dimensions shrink. Challenges to achieving superior performance include creating highly aligned and dense arrays of nanotubes as well as removing coatings that increase contact resistance. Sun et al. aligned CNTs by wrapping them with single-stranded DNA handles and binding them into DNA origami bricks that formed an array of channels with precise intertube pitches as small as 10.4 nanometers. Zhao et al. then constructed single and multichannel FETs by attaching the arrays to a polymer-templated silicon wafer. After adding metal contacts across the CNTs to fix them to the substrate, they washed away all of the DNA and then deposited electrodes and gate dielectrics. The FETs showed high on-state performance and fast on-off switching. Science , this issue p. 874 , p. 878

Herein, we propose a metabolic d-amino acid-based labeling and in situ hybridization-facilitated (MeDabLISH) strategy for the quantitative analysis of the indigenous metabolic status of gut bacteria. The fluorescent d-amino acid (FDAA)-based labeling intensities of bacteria were found to highly correlate with their temporal and steady-state metabolic status. Then, after taxonomic identification of bacterial genera in the in vivo FDAA-labeled mouse gut microbiota, by corresponding fluorescence in situ hybridization (FISH) probes, the metabolic activities of different gut bacterial genera are quantified by flow cytometry, using FISH signals to differentiate genera and FDAA signals to indicate their basal metabolic levels. It was found that Gram-negative genera in the mouse microbiota have stronger metabolic activities during the daytime, and Gram-positive genera have higher activities at the night. Our strategy will be instrumental in deepening our understanding of the highly complex microbiota.

The concentration of macromolecules in cells can reach up to 50–400 mg/mL. They occupy 40%(volume fraction) of the whole cellar space, known as molecular crowding. The diluted solution condition in vitro is different from the crowded physiological condition in vivo. Therefore, the simulation of the physiological condition is necessary for obtaining the reliable results. It has been reported that G-quadruplex can bind to hemin to enhance its catalytic function for generating oxygen radicals, which can oxidize the lipids, proteins and DNA, thus leading to the damage of cells and tissues. In this paper, we chose PEG400 as molecular crowding reagent to simulate the molecular crowding environment in vivo. The catalytic characteristics of G-quadruplex-hemin complex in H2O2-ABTS system have been investigated[ABTS=2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate)]. The results showed that the binding affinity of G-quadruplex and hemin was decreased with the increasing of PEG400 concentration. They even lose their binding affinity in the presence of 40% PEG400. As a result, the peroxidase activity of G-quadruplex-hemin also reduced. Therefore, in physiological condition, hemin might not bind to G-quadruplex and it might not be the main reason to cause the damages of cells and tissues.

Recent advances in structural DNA nanotechnology, including DNA origami and DNA bricks, have enabled arbitrarily complexed nanopatterns. However, most of these DNA structures are limited with sub-100 nm dimensions because of the limits from the length of scaffold strand, as well as the sequence library. This review will focus on different strategies for scaling-up DNA self-assembly, including the hierarchical assembly of the preformed DNA building blocks both in solution and on surface, the scaffolded assembly of finite sized DNA structures, the nonhierarchical assembly of single-stranded DNA bricks, and the seed-mediated algorithmic assembly. The design criteria, the building blocks, and the key assembly conditions for each assembly strategy are described. In addition, the future challenges, as well as application potentials of large-area DNA structures, are discussed.

Liquid biopsy allows noninvasive and real-time analysis of tumor-derived circulating targets in body fluids, holding great value for cancer diagnosis, prognosis, personalized therapy, as well as the understanding of mechanisms underlying tumorigenesis and progression. However, the low concentration and heterogeneity of circulating targets and the complexity of body fluids bring daunting technical challenges to liquid biopsy. Aptamers with many unique properties are considered as promising recognition ligands for liquid biopsy. Their facile modification and affinity regulation afford numerous platforms for efficient isolation and release of circulating targets. With versatile structural design and engineering, aptamers also offer many strategies for sensitive detection and analysis of circulating targets. In this review, recent progress in aptamer-based liquid biopsy is summarized, with a focus on the isolation and detection of circulating tumor cells (CTCs) and extracellular vesicles (EVs). First, recently emerged strategies for evolving aptamers against circulating targets are introduced, and existing aptamers successfully applied in the liquid biopsy are comprehensively summarized, including their targets, sequences, affinities, etc. Then, aptamer-based approaches for CTC isolation, release, and analysis are summarized. Additionally, aptamer-mediated signal transduction and amplification for the detection of EVs are also reviewed. Finally, the challenges and future perspectives in this field are discussed.

The enzyme-linked immunosorbent assay (ELISA) is one of the most commonly implemented clinical diagnostic tools for the detection and quantification of protein biomarkers. However, conventional ELISA tests require sophisticated infrastructure, expensive reagents, long assay time, and expertise for operation, which are not often easily accessible in resource-limited settings. Microfluidic ELISA-chip based point-of-care (POC) testing allows miniaturization and integration of complex functions that facilitate their usage in limited-resource settings. The current work demonstrates a simple, portable, low cost and equipment-free paper/poly(methyl methacrylate) (PMMA) integrated microfluidic ELISA-chip as a POC device with a visual distance-based readout for quantitative detection of clinical biomarkers. The integrated paper/PMMA ELISA-chip utilizes the movement of immunoassay complexes with magnetic beads by a permanent magnet in a PMMA part of the compartment. The target concentration is translated into a visual distance signal readout for quantitative detection of biomarkers in a μPAD. Because it does not require sophisticated instruments and has the added advantages of low cost, easy operation, and disposability with quantitative visual readout, the paper/PMMA ELISA-chip holds great promise for portable detection of target bioanalytes as a POC diagnostic tool in resource-limited setups.

Recently, the SARS-CoV-2 Omicron has spread very quickly worldwide. Several studies have indicated that the Omicron variant causes a substantial evasion of the humoral immune response and the majority of existing SARS-CoV-2 neutralizing antibodies. Here we address this challenge by applying a spherical cocktail neutralizing aptamer-gold nanoparticle (SNAP) to block the interaction of Omicron receptor binding domain (RBD) and host Angiotensin-Converting Enzyme 2 (ACE2). With the synergetic blocking strategy based on multivalent multisite aptamer binding and steric hindrance by the size-matched gold scaffold, the SNAP conjugate tightly binds to Omicron RBD with a dissociation constant of 13.6 pM, almost completely blocking the infection of Omicron pseudovirus with a half-maximal inhibitory concentration of 35.9 pM. Overall, the SNAP strategy not only fills the gap of the humoral immune evasion caused by clustered mutations on Omicron, but also provides a clue for the development of new broad neutralizing reagents against future variants.

Aptamer selection against novel infections is a complicated and time-consuming approach. Synergy can be achieved by using computational methods together with experimental procedures. This study aims to develop a reliable methodology for a rational aptamer in silico et vitro design. The new approach combines multiple steps: (1) Molecular design, based on screening in a DNA aptamer library and directed mutagenesis to fit the protein tertiary structure; (2) 3D molecular modeling of the target; (3) Molecular docking of an aptamer with the protein; (4) Molecular dynamics (MD) simulations of the complexes; (5) Quantum-mechanical (QM) evaluation of the interactions between aptamer and target with further analysis; (6) Experimental verification at each cycle for structure and binding affinity by using small-angle X-ray scattering, cytometry, and fluorescence polarization. By using a new iterative design procedure, structure- and interaction-based drug design (SIBDD), a highly specific aptamer to the receptor-binding domain of the SARS-CoV-2 spike protein, was developed and validated. The SIBDD approach enhances speed of the high-affinity aptamers development from scratch, using a target protein structure. The method could be used to improve existing aptamers for stronger binding. This approach brings to an advanced level the development of novel affinity probes, functional nucleic acids. It offers a blueprint for the straightforward design of targeting molecules for new pathogen agents and emerging variants.

Single-cell RNA-sequencing (scRNA-seq) has become a powerful tool for biomedical research by providing a variety of valuable information with the advancement of computational tools. Lineage analysis based on scRNA-seq provides key insights into the fate of individual cells in various systems. However, such analysis is limited by several technical challenges. On top of the considerable computational expertise and resources, these analyses also require specific types of matching data such as exogenous barcode information or bulk assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) data. To overcome these technical challenges, we developed a user-friendly computational algorithm called "LINEAGE" (label-free identification of endogenous informative single-cell mitochondrial RNA mutation for lineage analysis). Aiming to screen out endogenous markers of lineage located on mitochondrial reads from label-free scRNA-seq data to conduct lineage inference, LINEAGE integrates a marker selection strategy by feature subspace separation and de novo "low cross-entropy subspaces" identification. In this process, the mutation type and subspace-subspace "cross-entropy" of features were both taken into consideration. LINEAGE outperformed three other methods, which were designed for similar tasks as testified with two standard datasets in terms of biological accuracy and computational efficiency. Applied on a label-free scRNA-seq dataset of BRAF-mutated cancer cells, LINEAGE also revealed genes that contribute to BRAF inhibitor resistance. LINEAGE removes most of the technical hurdles of lineage analysis, which will remarkably accelerate the discovery of the important genes or cell-lineage clusters from scRNA-seq data.

Acute myeloid leukemia (AML) requires close monitoring of remission status for timely disease management. Liquid biopsy serves as a noninvasive approach for evaluating treatment response and guiding therapeutic modifications. Herein, we designed a non-invasive Leukemic stem cell Specific Capture Chip (LSC-Chip) with reversible recognition interface for AML remission status monitoring and prognosis prediction. A stem cell marker CD34 antibody coated herringbone chip with disulfide linkers was designed to capture and release leukemic stem cells (LSCs) in peripheral blood for efficient LSC enumeration and downstream single-cell analysis. Samples from 32 AML patients and 10 healthy donors were recruited for LSC enumeration and prognosis-associated subtyping with panels of official LSC markers (CD34

Controllable assembly and disassembly of recognition interface are vital for bioanalysis. Herein, a strategy of dynamic manipulation of trapping force by engineering a dynamic and reversible immunoaffinity microinterface (DynarFace) in a herringbone chip (DynarFace‐Chip) for liquid biopsy is proposed. The DynarFace is assembled by magnetically attracting immunomagnetic beads (IMBs) on chip substrate, with merits of convenient operation and reversible assembly. The DynarFace allows accumulating attachment of IMBs on circulating rare cell (CRC) surfaces during hydrodynamically enhanced interface collision, where accumulatively enhanced magnetic trapping force improves capture efficiency toward CRCs with medium expression of biomarkers from blood samples by 134.81% compared with traditional non‐dynamic interfaces. Moreover, magnet withdrawing‐induced disappearance of trapping force affords DynarFace disassembly and CRC release with high efficiency (>98%) and high viability (≈98%), compatible with downstream in vitro culture and gene analysis of CRCs. This DynarFace strategy opens a new avenue to accumulated capture and reversible release of CRCs, holding great potential for liquid biopsy‐based precision medicine., A dynamic and reversible immunoaffinity microinterface is engineered in a microfluidic chip, with merits of easy operation and reversible assembly. It enables dynamic manipulation of trapping force for accumulated capture and reversible release of circulating rare cells, holding great potential in liquid biopsy‐based precision medicine.

How to study the unculturable bacteria in the laboratory is one of the major challenges in human gut microbiota research. The resulting lack of microbiology knowledge of this “dark matter” greatly hinders further understanding of our gut microbiota. Here, to characterize the in vivo growth and division of human gut bacteria, we report the integrative use of STAMP (sequential tagging with D-amino acid–based metabolic probes) and fluorescence in situ hybridization (FISH) in a human microbiota-associated mouse model. After stable colonization of the human fecal microbiotas in germ-free mice, two fluorescent D-amino acid probes were sequentially administered by gavage, and the dually labeled peptidoglycan of the bacteria provided a chronological recording of their cell wall syntheses. Following taxonomic identification with FISH staining, the growth patterns of 32 species, including 5 currently unculturables, were identified. Surprisingly, we found that many bacterial species in the human microbiota were significantly shorter than those in the mouse gut microbiota. An imaging database for gut bacteria – Microbiome Atlas was built for summarizing STAMP imaging of bacteria from different microbiotas, which can be contributed by the microbiota research community worldwide. This integrative imaging strategy and the database will promote our understanding of the bacterial cytology in gut microbiotas and facilitate communications among cellular microbiologists.

Lipidomics is a younger member of the "omics" family. It aims to profile lipidome alterations occurring in biological systems. Similar to the other "omics", lipidomic data is highly dimensional and contains a massive amount of information awaiting deciphering and data mining. Currently, the available bioinformatic tools targeting lipidomic data processing and lipid pathway analysis are limited. A few tools designed for lipidomic analysis perform only basic statistical analyses, and lipid pathway analyses rely heavily on public databases (KEGG, Reactome, and HMDB). Due to the inadequate understanding of lipid signaling and metabolism, the use of public databases for lipid pathway analysis can be biased and misleading. Instead of using public databases to interpret lipidomic ontology, the authors introduce an intra-omic integrative correlation strategy for lipidomic data mining. Such an intra-omic strategy allows researchers to unscramble and predict lipid biological functions from correlated genomic ontological results using statistical approaches. To simplify and improve the lipidomic data processing experience, they designed an interactive web-based tool: LINT-web (http://www.lintwebomics.info/) to perform the intra-omic analysis strategy, and validated the functions of LINT-web using two biological systems. Users without sophisticated statistical experience can easily process lipidomic datasets and predict the potential lipid biological functions using LINT-web.

As a well-studied biochemical reduction process in environmental microbiology, extracellular electron transfer (EET) was recently discovered in bacteria closely related to human health, and orthologues of a flavin-based EET gene were found in the genomes of many species across Firmicutes, a major phylum in mammalian gut microbiota. However, EET has not yet been confirmed to occur in mammalian gut, the presence of which may have broad physiological influences. Toward this end, here we first confirmed the occurrence of EET in mouse gut microbiotas cultured in vitro. Cyclic voltammetry analysis was then performed by directly inserting electrodes into the mouse cecum under anaerobic conditions, and a characteristic catalytic wave was observed in the gut of conventional but not germ-free mouse, proving the existence of in vivo bacterial EET. We also detected similar catalytic waves in the cecal microbiotas of rat and guinea pig in vivo, suggesting EET's high prevalence in mammalian intestines. Our finding on the bacterial electron production in mammalian guts offers a new bioelectrochemical scope for deciphering the complex microbiology of gut bacteria and its effects on host physiology.

The analysis of circulating tumor cells (CTCs) holds great significance for cancer diagnosis, prognosis, and personalized therapy. However, the rarity, vulnerability and heterogeneity of CTCs bring daunting technical challenges to their isolation, release and analysis. Recent exciting advances in microfluidics have greatly promoted CTC isolation and analysis due to its merits of precise control of fluid behavior, integration, and automation. Especially, aptamer-based microfluidic chip is considered as promising platform, because aptamers as recognition ligands have inherent superiority in CTC isolation and release for their convenient modification and controllable recognition ability. This review focuses on recent progresses in aptamer-based microfluidics for isolation, release and analysis of CTCs. First, existing CTC-related aptamers and their selection methods are briefly introduced. Strategies for conjugating aptamers onto microfluidic chips are also reviewed. Then, aptamer-based microfluidic chips for CTC isolation, release and analysis are summarized. Finally, future research directions and challenges in this field are discussed.

Accurate cancer theranostics is important to improve the survival rate and life quality. Circulating tumor cell (CTC) is a type of tumor cells that spread through peripheral blood after detaching from a solid tumor, which is the most promising target in liquid biopsy. It offers non-invasive sampling for cancer diagnosis and real-time monitor of biological tumor information to study the mechism of tumor metastasis. However, the extreme rarity and heterogeneity of CTC pose a huge technical challenge for CTC profiling. Various technologies have been proposed. This review aims to provide in-depth insights into CTC related research work from Yang’s group, including CTC targeting molecule discovery, microfluidic-based enrichment and high-throughput single cell analysis.

Glioma stem cells (GSCs), a particular group of cells from gliomas, are capable of infinite proliferation and differentiation. Recent studies have shown that GSCs may be the root of tumor recurrence, metastasis, and resistance. Early detection and targeted therapy of GSCs may significantly improve the survival rate of glioma patients. Therefore, molecular ligands capable of selectively recognizing GCSs are of great importance. The objective of this study is to generate DNA aptamers for selective identification of the molecular signature of GSCs using cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX). GSCs were used as the positive selection target, while U87 cells were used in negative cycles for removal of DNA molecules binding to common glioma cell lines. Finally, we successfully identified one aptamer named W5-7 with a Kd value of 4.9 ± 1.4 nM. The sequence of the aptamer was further optimized, and its binding target was identified as a membrane protein. The aptamer W5-7 was stable in cerebral spinal fluid over 36 h and could also effectively detect glioma stem cells in cerebral spinal fluid samples. With its superb targeting properties and functional versatility, W5-7 holds great potential for use as a molecular probe for detecting and isolating GSCs.

As the third-generation nucleic acid amplification technology, digital polymerase chain reaction (PCR) has been widely adopted in the analysis of nucleic acids. However, further application of this powerful technology is hindered due to the limitation of surfactants. Here, for the first time, we propose the use of colloidosomes self-assembled from fluorinated silica nanoparticle for digital PCR to address this limitation. A one-step fluorinated silica nanoparticle synthesis method is proposed, which is much more convenient and reproducible compared with the synthesis of conventional fluorine-based surfactants. Fluorinated silica nanoparticles facilitate the formation of colloidosomes with excellent stability capable of enduring the rapid temperature changes associated with the PCR and avoiding material exchange (cross-talk) between droplets for high-fidelity analysis. The colloidosome digital PCR method was developed using these colloidosomes as highly parallel reactors for single-copy nucleic acid amplification and rare mutant detection. The method is robust and accurate, and it offers possibilities for a great variety of applications, such as gene expression studies, single cell analysis, and circulating tumor DNA detection.

The CRISPR-Cas9 system enables facile and efficient genome engineering in living cells and organisms. Small molecule control of CRISPR-Cas9 would allow precise temporal control in applications of the technology. In this work, we developed s[combining low line]mall m[combining low line]olecule-activated allosteric a[combining low line]ptamer r[combining low line]egulat[combining low line]ing (SMART)-sgRNAs as general strategies to control programmable genome editing. With SMART-sgRNAs, temporal control of the CRISPR-Cas9 system against different targets can be achieved, which should facilitate its application in various fields, such as biomedical genome editing, drug screening and chromosome imaging.

In this study, catalase-linked immunosorbent pressure assay with a gas-generation reaction was established for quantitative detection of disease biomarker C-reactive protein (CRP) by a portable pressuremeter. The pressure-based detection system recognizes, transduces, and amplifies the target signal to a convenient target-correlated pressure signal reading in a closed chamber. Biotin molecules were modified on the surface of catalase in order to incorporate catalase into the pressure immunoassay by the streptavidin-biotin interaction. To improve the assay performance, the modification ratios of biotin molecules to catalase, and the concentrations of capture and detection antibodies were further optimized. The catalase-linked immunosorbent pressure assay allows portable and quantitation analysis of CRP with a limit of detection of 1.8 nM, which can satisfy the clinical needs for determining the risk of cardiovascular disease. The catalase-linked immunosorbent pressure assay also shows superior specificity and good accuracy. Compared to the previously reported assay catalyzed by PtNP nanozyme, catalase is not easily deactivated during storage and operation. With the merits of enzymatic efficiency, biocompatibility, low non-specific adsorption and facile modification, catalase can be reasonably used for reproducible, stable, simple quantitative detection of disease markers using a portable pressure-based assay in resource-limited settings.

The mechanical force between a virus and its host cell plays a critical role in viral infection. However, characterization of the virus-cell mechanical force at the whole-virus level remains a challenge. Herein, we develop a platform in which the virus is anchored with multivalence-controlled aptamers to achieve transfer of the virus-cell mechanical force to a DNA tension gauge tether (Virus-TGT). When the TGT is ruptured, the complex of binding module-virus-cell is detached from the substrate, accompanied by decreased host cell-substrate adhesion, thus revealing the mechanical force between whole-virus and cell. Using Virus-TGT, direct evidence about the biomechanical force between SARS-CoV-2 and the host cell is obtained. The relative mechanical force gap (10 pN) at the cellular level between the wild-type virus to cell and a variant virus to cell is measured, suggesting a possible positive correlation between virus-cell mechanical force and infectivity. Overall, this strategy provides a new perspective to probe the SARS-CoV-2 mechanical force.

Exosomal programmed cell death ligand 1 (exoPD-L1) has emerged as a promising biomarker for cancer diagnosis and immunotherapy outcome prediction. However, the existing quantitation methods are incapable of addressing the heterogeneity of exoPD-L1 glycosylation, which has been demonstrated to be the institutional basis for PD-L1/PD-1 interaction and the crucial participant in inhibiting the activity of CD8

By catalyzing a 3-3 cross-link in peptidoglycan, l,d-transpeptidases (Ldts) can cause resistance to β-lactams in some pathogens

Analysis of circulating tumor cells (CTCs) is regarded as a useful diagnostic index to monitor tumor development and guide precision medicine. Although the immunoassay is a common strategy for CTC identification and heterogeneity characterization, it is challenged by poor reaction efficiency and laborious manipulations in microdevices, which hinder the sensitivity, throughput, simplification, and applicability. To meet the need for rapid, sensitive, and simple CTC analysis, we developed an efficient CTC detection system by integrating a 3D printed off-chip multisource reagent platform, a bubble retainer, and a single CTC capture microchip, which can achieve CTC capture and identification within 90 min. Compared with traditional CTC identification methods, this system decreases immunostaining time and antibody consumption by 90% and performs the on-chip immunoassay in a fully automated manner. Using this system, CTCs from the peripheral blood of 19 patients with various cancers were captured, detected, and compared with clinical data. The system shows great potential for early screening, real-time monitoring, and precision medicine for hepatocellular carcinoma (HCC). With the advantages of automation, stability, economy, and user-friendly operation, the proposed system is promising for clinical scenarios.

The COVID-19 pandemic, caused by SARS-CoV-2, currently poses an urgent global medical crisis for which there remains a lack of affordable point-of-care testing (POCT). In particular, resource-limited areas need simple and easily disseminated testing solutions to manage the outbreak. In this work, a microfluidic-integrated lateral flow recombinase polymerase amplification (MI-IF-RPA) assay was developed for rapid and sensitive detection of SARS-CoV-2, which integrates the reverse transcription recombinase polymerase amplification (RT-RPA) and a universal lateral flow (LF) dipstick detection system into a single microfluidic chip. The single-chamber RT-RPA reaction components are mixed with running buffer, and then delivered to the LF detection strips for biotin- and FAM-labelled amplified analyte sequences, which can provide easily interpreted positive or negative results. Testing requires only a simple nucleic acid extraction and loading, then incubation to obtain results, approximately 30 minutes in total. SARS-CoV-2 armored RNA particles were used to validate the MI-IF-RPA system, which showed a limit of detection of 1 copy per μL, or 30 copies per sample. Chip performance was further evaluated using clinically diagnosed cases of COVID-19 and revealed a sensitivity of 97% and specificity of 100%, highly comparable to current reverse transcription-polymerase chain reaction (RT-PCR)-based diagnostic assays. This MI-IF-RPA assay is portable and comprises affordable materials, enabling mass production and decreased risk of contamination. Without the need for specialized instrumentation and training, MI-IF-RPA assay can be used as a complement to RT-PCR for low-cost COVID-19 screening in resource-limited areas.

Rat is a widely used mammalian model for gut microbiota research. However, due to the difficulties of individual in vitro culture of many of the gut bacteria, much information about the microbial behaviors in the rat gut remains largely unknown. Here, to characterize the in situ growth and division of rat gut bacteria, we apply a chemical strategy that integrates the use of sequential tagging with D-amino acid-based metabolic probes (STAMP) with fluorescence in situ hybridization (FISH) to rat gut microbiota. Following sequential gavages of two different fluorescent D-amino acid probes to rats, the resulting dually labeled gut bacteria provides chronological information of their in situ cell wall synthesis. After taxonomical labeling with FISH probes, most of which are newly designed in this study, we successfully identify the growth patterns of 15 bacterial species, including two that have not been cultured separately in the laboratory. Furthermore, using our labeling protocol, we record Butyrivibrio fibrisolvens cells growing at different growth stages of a complete cell division cycle, which offers a new scope for understanding basic microbial activities in the gut of mammalian hosts.

Exosomal glycoproteins play important roles in many physiological and pathological functions. Herein, we developed a dual labeling strategy based on a protein-specific aptamer tagging and metabolic glycan labeling for visualizing glycosylation of specific proteins on exosomes. The glycosylation of exosomal PD-L1 (exoPD-L1) was imaged in situ using intramolecular fluorescence resonance energy transfer (FRET) between fluorescent PD-L1 aptamers bound on exoPD-L1 and fluorescent tags on glycans introduced via metabolic glycan labeling. This method enables in situ visualization and biological function study of exosomal protein glycosylation. Exosomal PD-L1 glycosylation was confirmed to be required in interaction with PD-1 and participated in inhibiting of CD8+ T cell proliferation. This is an efficient and non-destructive method to study the presence and function of exosomal protein-specific glycosylation in situ, which provides a powerful tool for exosomal glycoproteomics research.