Publications by authors named "Chaoyong Yang"

209 Publications

Microfluidic single-cell transcriptomics: moving towards multimodal and spatiotemporal omics.

Lab Chip 2021 Sep 20. Epub 2021 Sep 20.

The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.

Cells are the basic units of life with vast heterogeneity. Single-cell transcriptomics unveils cell-to-cell gene expression variabilities, discovers novel cell types, and uncovers the critical roles of cellular heterogeneity in biological processes. The recent advances in microfluidic technologies have greatly accelerated the development of single-cell transcriptomics with regard to throughput, sensitivity, cost, and automation. In this article, we review state-of-the-art microfluidic single-cell transcriptomics, with a focus on the methodologies. We first summarize six typical microfluidic platforms for isolation and transcriptomic analysis of single cells. Then the on-going trend of microfluidic transcriptomics towards multimodal omics, which integrates transcriptomics with other omics to provide more comprehensive pictures of gene expression networks, is discussed. We also highlight single-cell spatial transcriptomics and single-cell temporal transcriptomics that provide unprecedented spatiotemporal resolution to reveal transcriptomic dynamics in space and time, respectively. The emerging applications of microfluidic single-cell transcriptomics are also discussed. Finally, we discuss the current challenges to be tackled and provide perspectives on the future development of microfluidic single-cell transcriptomics.
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http://dx.doi.org/10.1039/d1lc00607jDOI Listing
September 2021

Reversible Immunoaffinity Interface Enables Dynamic Manipulation of Trapping Force for Accumulated Capture and Efficient Release of Circulating Rare Cells.

Adv Sci (Weinh) 2021 Sep 2:e2102070. Epub 2021 Sep 2.

The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.

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.
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http://dx.doi.org/10.1002/advs.202102070DOI Listing
September 2021

Imaging the in vivo growth patterns of bacteria in human gut Microbiota.

Gut Microbes 2021 Jan-Dec;13(1):1960134

Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

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 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 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.
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http://dx.doi.org/10.1080/19490976.2021.1960134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8386752PMC
August 2021

An electrochemical method for a rapid and sensitive immunoassay on digital microfluidics with integrated indium tin oxide electrodes coated on a PET film.

Analyst 2021 Jul 18;146(14):4473-4479. Epub 2021 Jun 18.

The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Pen-Tung Sah Institute of Micro-Nano Science and Technology, Xiamen University, Xiamen 361005, P.R. China.

Electrochemical detection is the simplest analytical tool to be integrated into digital microfluidics (DMF). It offers the advantages of small size, with detector electrodes incorporated into the device by patterning, and high compatibility with portable analytical instruments. Indium tin oxide (ITO) coated on glass has been commonly used for the top plate of DMF due to its good conductivity and transparency. However, instability and the low current response of ITO electrodes patterned on glass hindered their application for immunoassays. It has been reported that ITO coated on polyethylene terephthalate (PET) has better conductivity, owing to its higher carrier concentration, faster mobility and lower resistivity. Herein, we investigated the use of ITO electrodes patterned on PET film as the top plate of DMF for a simple and stable electrochemical immunoassay using square wave voltammetry (SWV), with an excellent peak resolution and high sensitivity. A magnetic bead-based immunoassay for H5N1 antigen was performed on a DMF platform with a limit of detection of 0.6 ng mL in buffer and 18 ng mL in human serum. These results showed the good electrochemical performance of ITO coated on a PET film, a lightweight, shock resistant and cost-effective material, which is promising for DMF fabrication and transparent electrodes for various electroanalytical methods.
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http://dx.doi.org/10.1039/d1an00513hDOI Listing
July 2021

Biodistributions of l,d-Transpeptidases in Gut Microbiota Revealed by Labeling with Peptidoglycan Analogs.

ACS Chem Biol 2021 07 29;16(7):1164-1171. Epub 2021 Jun 29.

Institute of Molecular Medicine, Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, China.

By catalyzing a 3-3 cross-link in peptidoglycan, l,d-transpeptidases (Ldts) can cause resistance to β-lactams in some pathogens . However, the prevalence of Ldt and Ldt-mediated responses to different β-lactams have never been explored. Here, we apply an metabolic labeling strategy to study their biodistributions and Ldt-induced bacterial responses to β-lactams in the mouse gut microbiota. A tetrapeptide-based fluorescent probe that functions as a substrate for Ldts in Gram-positive bacteria efficiently labels ∼18% of total gut bacteria after gavage, suggesting Ldts' high prevalence in gut microbiota. The cellular distributions of 3-3 cross-links on three gut bacterial species were then identified with the aid of fluorescence hybridization to identify the bacterial taxa. After oral administration of two β-lactams, ampicillin and meropenem, only the latter efficiently inhibits the tetrapeptide labeling, suggesting that Ldts may be able to cause resistance to some β-lactams in the mammalian gut.
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http://dx.doi.org/10.1021/acschembio.1c00346DOI Listing
July 2021

HCV poly U/UC sequence-induced inflammation leads to metabolic disorders in vulvar lichen sclerosis.

Life Sci Alliance 2021 08 18;4(8). Epub 2021 Jun 18.

Obstetrics and Gynecology Hospital, State Key Laboratory of Genetic Engineering, Institute of Metabolism and Integrative Biology and School of Life Sciences, Fudan University, Shanghai, China

Vulvar lichen sclerosis (VLS) is a dermatologic disorder that affects women worldwide. Women with VLS have white, atrophic papules on the vulva. They suffer from life-long intense pruritus. Corticosteroids are the first-line of treatments and the most effective medicines for VLS. Although VLS has been speculated as an autoimmune disease for a long time, its pathogenesis and the molecular mechanism is largely unknown. We performed a comprehensive multi-omics analysis of paired samples from VLS patients as well as healthy donors. From the RNA-seq analysis, we found that VLS is correlated to abnormal antivirus response because of the presence of Hepatitis C Virus poly U/UC sequences. Lipidomic and metabolomic analysis revealed that inflammation-induced metabolic disorders of fatty acids and glutathione were likely the reasons for pruritus, atrophy, and pigment loss in the vulva. Thus, the present study provides an initial interpretation of the pathogenesis and molecular mechanism of VLS and suggests that metabolic disorders that affect the vulva may serve as therapeutic targets for VLS.
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http://dx.doi.org/10.26508/lsa.202000906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8321666PMC
August 2021

A Fully Automated and Integrated Microfluidic System for Efficient CTC Detection and Its Application in Hepatocellular Carcinoma Screening and Prognosis.

ACS Appl Mater Interfaces 2021 Jun 18;13(25):30174-30186. Epub 2021 Jun 18.

Department of General Surgery, Department of Pathology, Department of Ultrasound, Xiang'an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen 361101, China.

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.
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http://dx.doi.org/10.1021/acsami.1c06337DOI Listing
June 2021

Visualizing the Growth and Division of Rat Gut Bacteria by D-Amino Acid-Based Labeling and FISH Staining.

Front Mol Biosci 2021 28;8:681938. Epub 2021 May 28.

Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

Rat is a widely used mammalian model for gut microbiota research. However, due to the difficulties of individual culture of many of the gut bacteria, much information about the microbial behaviors in the rat gut remains largely unknown. Here, to characterize the 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 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 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 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.
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http://dx.doi.org/10.3389/fmolb.2021.681938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8193097PMC
May 2021

Auto-Panning: a highly integrated and automated biopanning platform for peptide screening.

Lab Chip 2021 07;21(14):2702-2710

The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, Department of Chemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.

Biopanning, a common affinity selection approach in phage display, has evolved numerous ligands for diagnosis, imaging, delivery, and therapy applications. However, traditional biopanning has suffered from time-consuming processes, highly-repetitive procedures and labor-intensive manual operation. Herein, a highly integrated and automated biopanning platform (Auto-Panning) is proposed. Based on digital microfluidics (DMF), biopanning processes are integrated on a chip with highly reproducible, precise, automated liquid manipulation. Therefore, 3 rounds of Auto-Panning can be accomplished within 16 h, instead of nearly a week of complicated manual operations. Auto-Panning has been used to evolve a specific peptide against cancer biomarker EphA2 with excellent cellular penetrating ability and significant invasion suppression biofunction, successfully demonstrating the practicality of the platform. Overall, as an automated programmable molecular screening platform, Auto-Panning will further promote the discovery and applications of novel ligands.
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http://dx.doi.org/10.1039/d1lc00129aDOI Listing
July 2021

Analytical chemistry for infectious disease detection and prevention.

Anal Bioanal Chem 2021 07 5;413(18):4561-4562. Epub 2021 Jun 5.

Key Laboratory for Chemical Biology of Fujian Province, MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Collaborative Innovation Center of Chemistry for Energy Materials, State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.

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http://dx.doi.org/10.1007/s00216-021-03441-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8179694PMC
July 2021

In situ Raman enhancement strategy for highly sensitive and quantitative lateral flow assay.

Anal Bioanal Chem 2021 Jun 5. Epub 2021 Jun 5.

Key Laboratory for Chemical Biology of Fujian Province, MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Collaborative Innovation Center of Chemistry for Energy Materials, State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, Fujian, China.

As a paper-based analytical platform, lateral flow assay (LFA) gets benefit from the rapid analysis, low cost, high selectivity, good stability, and user-friendliness, and thus has been widely used in rapid screening or assisted diagnosis. Nevertheless, LFA still suffers from low detection sensitivity via the naked eye, limiting its applications to qualitative and semi-quantitative tests. To enhance the signal readout, various nanoparticle signal tags have been employed to replace traditional colloidal gold nanoparticles (AuNPs), such as fluorescent nanoparticles (FNPs), magnetic nanoparticles (MNPs), and Raman reporter-labeled nanoparticles. In particular, Raman reporter-labeled nanoparticles are extremely sensitive due to remarkable signal enhancement effect on metal surface. However, the application of LFA is still hampered by the poor stability of Raman reporter-labeled nanoparticles. Herein, we developed an in situ Raman enhancement strategy to create a surface-enhanced Raman scattering (SERS) signal on the AuNPs, shortened as "i-SERS," which not only preserves the original advantages of the colloidal gold strip (AuNPs-LFA), but also realizes highly sensitive and quantitative detection. We applied the i-SERS for procalcitonin (PCT) detection. The experimental process takes only 16 min, and the limit of detection (LOD) is 0.03 ng mL, far below the value using AuNPs-LFA. These results indicate that i-SERS assay was highly sensitive and suitable for the rapid detection of PCT.
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http://dx.doi.org/10.1007/s00216-021-03419-zDOI Listing
June 2021

Coupling Aptamer-based Protein Tagging with Metabolic Glycan Labeling for In Situ Visualization and Biological Function Study of Exosomal Protein-Specific Glycosylation.

Angew Chem Int Ed Engl 2021 08 9;60(33):18111-18115. Epub 2021 Jul 9.

The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the, Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian, 361005, China.

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.
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http://dx.doi.org/10.1002/anie.202103696DOI Listing
August 2021

A microfluidic-integrated lateral flow recombinase polymerase amplification (MI-IF-RPA) assay for rapid COVID-19 detection.

Lab Chip 2021 05;21(10):2019-2026

The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.

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.
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http://dx.doi.org/10.1039/d0lc01222jDOI Listing
May 2021

HUNTER-Chip: Bioinspired Hierarchically Aptamer Structure-Based Circulating Fetal Cell Isolation for Non-Invasive Prenatal Testing.

Anal Chem 2021 05 5;93(19):7235-7241. Epub 2021 May 5.

Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China.

Isolation and genetic analysis of circulating fetal cells from billions of maternal cells in peripheral blood are the cornerstone of fetal cell-based non-invasive prenatal testing. Inspired by the hierarchically multivalent architecture for enhanced capture of nature, an aptamer-based Hierarchically mUltivalent aNTibody mimic intERface (HUNTER) was designed with a tremendous avidity effect for highly efficient capture and non-destructive release of fetal cells. It was engineered by grafting Y-shaped DNA nanostructures to a linear polymer chain, creating a flexible polymer chain with bivalent aptamer side chains. This hierarchical arrangement of the aptamer ensures morphological complementarity, collective multiple-site interaction, and multivalent recognition between the aptamer and target cells. In combination with a deterministic lateral displacement (DLD)-patterned microdevice named as HUNTER-Chip, it achieves a binding affinity over 65-fold and a capture efficiency over 260%-fold due to the combination of hierarchically designed aptamers and frequent cell-ligand collision created by DLD. Moreover, a nuclease-assisted cell release strategy facilitates the release of fetal cells for gene analysis, such as fluorescence hybridization. With the advantages of high affinity, excellent capture efficiency, and compatible downstream analysis, the HUNTER-Chip holds great potential for non-invasive prenatal diagnosis.
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http://dx.doi.org/10.1021/acs.analchem.1c00330DOI Listing
May 2021

Imaging Commensal Microbiota and Pathogenic Bacteria in the Gut.

Acc Chem Res 2021 05 15;54(9):2076-2087. Epub 2021 Apr 15.

Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China.

As a newly discovered organ, gut microbiota has been extensively studied in the last two decades, with their highly diverse and fundamental roles in the physiology of many organs and systems of the host being gradually revealed. However, most of the current research heavily relies on DNA sequencing-based methodologies. To truly understand the complex physiological and pathological functions demonstrated by commensal and pathogenic gut bacteria, we need more powerful methods and tools, among which imaging strategies suitable for approaching this ecosystem in different settings are one of the most desirable. Although the phrase gut "dark matter" is often used in referring to the unculturability of many gut bacteria, it is also applicable to describing the formidable difficulties in visualizing these microbes in the intestines. To develop suitable and versatile chemical and biological tools for imaging bacteria in the gut, great efforts have been devoted in the past several years.In this Account, we highlight the recent progress made by our group and other laboratories in the development of visualization strategies for commensal microbiota and pathogenic bacteria in the gut. First, we summarize our efforts toward the development of derivatized antibiotic staining probes that directly bind to specific bacterial surface structures for selective labeling of different groups of gut bacteria. Next, metabolic labeling-based imaging strategies, using unnatural amino acids, unnatural sugars, and stable isotopes, for imaging gut bacteria on various scales and in different settings are discussed in detail. We then introduce nucleic acid staining-based bacterial imaging, using either general nucleic acid-binding reagents or selective-labeling techniques (e.g., fluorescence hybridization) to meet the diverse needs in gut microbiota research. This classical imaging strategy has witnessed a renaissance owing to a series of new technical advancements. Furthermore, despite the notorious difficulties of performing genetic manipulations in many commensal gut bacteria, great effort has been made recently in engineering gut bacteria with reporters like fluorescent proteins and acoustic response proteins.Our perspectives on the current limitations of the chemical tools and strategies and the future directions for improvement are also presented. We hope that this Account can offer valuable references to spark new ideas and invite new efforts to help decipher the complex biological and chemical interactions between commensal microbiota and pathogenic bacteria and the hosts.
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http://dx.doi.org/10.1021/acs.accounts.1c00068DOI Listing
May 2021

Multichannel Paper Chip-Based Gas Pressure Bioassay for Simultaneous Detection of Multiple MicroRNAs.

ACS Appl Mater Interfaces 2021 Apr 23;13(13):15008-15016. Epub 2021 Mar 23.

Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710119, China.

Simultaneous detection of multi-biomarkers not only enhances the accuracy of disease diagnosis but also improves detection efficiency and reduces cost. It is vital to achieve portable, simple, low-cost, and simultaneous detection of biomarkers for point-of-care (POC) diagnostics in a low-resource setting. Herein, a multichannel paper chip-based gas pressure bioassay was developed for the simultaneous detection of multiple biomarkers by combining multichannel paper chips with a portable gas pressure meter. Four DNA tetrahedral probes (DTPs) were used as capture probes and were immobilized in different detection zones of the paper chips to improve hybridization efficiency and reduce nonspecific adsorption. The formation of a sandwich structure between target microRNAs (miRNAs), the capture probe, and platinum nanoparticles (PtNPs)-modified complementary DNA (PtNPs-cDNA) transformed biomolecular recognition into quantitative detection of gas pressure. Four lung cancer-related miRNAs were detected simultaneously by a portable gas pressure meter. There is a good linear relationship between gas pressure and the logarithm of miRNA concentration in the range of 10 pM to 100 nM. Compared with single-stranded DNA capture probe, the signal-to-noise (S/N) of DNA tetrahedral probes improved more than 3 times. Using ring-oven washing, the unbound reagents in all channels of the paper chip were simultaneously and continuously washed away, leading to a more cheap, simple, and fast separation than magnetic separation. Therefore, it offers a promising multichannel paper chip-based gas pressure bioassay for portable and simultaneous detection of multiple biomarkers.
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http://dx.doi.org/10.1021/acsami.1c01568DOI Listing
April 2021

Stimulus-Responsive Microfluidic Interface Enables Efficient Enrichment and Cytogenetic Profiling of Circulating Myeloma Cells.

ACS Appl Mater Interfaces 2021 Apr 23;13(13):14920-14927. Epub 2021 Mar 23.

MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Key Laboratory for Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemical of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.

Minimal residual disease (MRD) provides an independent prognostic factor for multiple myeloma (MM) patients. However, clinical MRD assays suffer from highly invasive sampling, insufficient detection sensitivity, and high cost. Herein, a stiMulus-Responsive ligand-Decorated microfluidic chip (MRD-Chip) was developed for efficient capture and controlled release of circulating myeloma cells (CMCs) in the peripheral blood for noninvasive myeloma evaluation. The CD138 antibody-decorated herringbone chip with a disulfide linker was designed to enhance the collision probability between blood cells and capture antibodies, leading to high capture efficiency of CMCs. More importantly, the captured CMCs can be nondestructively released a thiol-exchange reaction, allowing them to be used for subsequent cellular and molecular analysis. By fluorescence hybridization assay, we successfully identified the cytogenetic abnormalities (chromosome 1q21 amplification and p53 deletion) of CMCs in clinical samples. Overall, with the merits of noninvasive sampling, high capture efficiency (70.93%), high throughput (1.5 mL/h), and nondestructive release of target cells (over 90% viability) for downstream analysis, our strategy provides new opportunities for myeloma evaluation, such as prognosis assessment, efficacy monitoring, and mechanism research of disease relapse and drug resistance.
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http://dx.doi.org/10.1021/acsami.1c00382DOI Listing
April 2021

Aptamer-Based Detection of Circulating Targets for Precision Medicine.

Chem Rev 2021 Mar 5. Epub 2021 Mar 5.

Institute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.

The past decade has witnessed ongoing progress in precision medicine to improve human health. As an emerging diagnostic technique, liquid biopsy can provide real-time, comprehensive, dynamic physiological and pathological information in a noninvasive manner, opening a new window for precision medicine. Liquid biopsy depends on the sensitive and reliable detection of circulating targets (e.g., cells, extracellular vesicles, proteins, microRNAs) from body fluids, the performance of which is largely governed by recognition ligands. Aptamers are single-stranded functional oligonucleotides, capable of folding into unique tertiary structures to bind to their targets with superior specificity and affinity. Their mature evolution procedure, facile modification, and affinity regulation, as well as versatile structural design and engineering, make aptamers ideal recognition ligands for liquid biopsy. In this review, we present a broad overview of aptamer-based liquid biopsy techniques for precision medicine. We begin with recent advances in aptamer selection, followed by a summary of state-of-the-art strategies for multivalent aptamer assembly and aptamer interface modification. We will further describe aptamer-based micro-/nanoisolation platforms, aptamer-enabled release methods, and aptamer-assisted signal amplification and detection strategies. Finally, we present our perspectives regarding the opportunities and challenges of aptamer-based liquid biopsy for precision medicine.
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http://dx.doi.org/10.1021/acs.chemrev.0c01140DOI Listing
March 2021

Sensitive, Rapid, and Automated Detection of DNA Methylation Based on Digital Microfluidics.

ACS Appl Mater Interfaces 2021 Feb 12;13(7):8042-8048. Epub 2021 Feb 12.

The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.

Biomarkers based on DNA methylation have attracted wide attention in biomedical research due to their potential clinical value. Therefore, a sensitive and accurate method for DNA methylation detection is highly desirable for the discovery and diagnostics of human diseases, especially cancers. Here, an integrated, low-cost, and portable point-of-care (POC) device is presented to analyze DNA methylation, which integrates the process of pyrosequencing in a digital microfluidic chip. Without additional equipment and complicated operation, droplets are manipulated by patterned electrodes with individually programmed control. The system exhibited an excellent sensitivity with a limit of detection (LOD) of 10 pg and a comparable checkout down to 5% methylation level within 30 min, which offered a potential substitute for the detection of DNA methylation. With the advantages of portability, ease of use, high accuracy, and low cost, the POC platform shows great potential for the analysis of tumor-specific circulating DNA.
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http://dx.doi.org/10.1021/acsami.0c21995DOI Listing
February 2021

Aptamer Blocking Strategy Inhibits SARS-CoV-2 Virus Infection.

Angew Chem Int Ed Engl 2021 04 10;60(18):10266-10272. Epub 2021 Mar 10.

The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian, 361005, China.

The COVID-19 pandemic caused by SARS-CoV-2 is threating global health. Inhibiting interaction of the receptor-binding domain of SARS-CoV-2 S protein (S ) and human ACE2 receptor is a promising treatment strategy. However, SARS-CoV-2 neutralizing antibodies are compromised by their risk of antibody-dependent enhancement (ADE) and unfavorably large size for intranasal delivery. To avoid these limitations, we demonstrated an aptamer blocking strategy by engineering aptamers' binding to the region on S that directly mediates ACE2 receptor engagement, leading to block SARS-CoV-2 infection. With aptamer selection against S and molecular docking, aptamer CoV2-6 was identified and applied to prevent, compete with, and substitute ACE2 from binding to S . CoV2-6 was further shortened and engineered as a circular bivalent aptamer CoV2-6C3 (cb-CoV2-6C3) to improve the stability, affinity, and inhibition efficacy. cb-CoV2-6C3 is stable in serum for more than 12 h and can be stored at room temperature for more than 14 days. Furthermore, cb-CoV2-6C3 binds to S with high affinity (K =0.13 nM) and blocks authentic SARS-CoV-2 virus with an IC of 0.42 nM.
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http://dx.doi.org/10.1002/anie.202100225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8014204PMC
April 2021

Highly paralleled emulsion droplets for efficient isolation, amplification, and screening of cancer biomarker binding phages.

Lab Chip 2021 03 8;21(6):1175-1184. Epub 2021 Feb 8.

The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, Department of Chemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.

Based on the linkage of genotype and phenotype, display technology has been widely used to generate specific ligands for profiling, imaging, diagnosis and therapy applications. However, due to the lack of effective monoclonal manipulation and affinity evaluation methods, traditional display technology has to undergo tedious steps of selection, clone isolation, amplification, sequencing, synthesis and characterization to obtain the binding sequences. To directly acquire high-affinity clones, we propose a double monoclonal display approach (dm-Display) for peptide screening based on highly paralleled monoclonal manipulation in emulsion droplets. dm-Display can monoclonally link the genotype, phenotype and affinity to realize integrated monoclonal separation, amplification, recognition and staining in one droplet so that discrete high-affinity clones can be quickly extracted. Monoclonal manipulations highly-parallelly occur in millions of droplets so that molecular screening of a highly diverse phage library is achieved. We have screened specific peptide ligands against CD71 and GPC1, proving the feasibility and generality of dm-Display. As a highly efficient ligand screening platform, dm-Display will promote the further development of molecular screening.
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http://dx.doi.org/10.1039/d0lc01146kDOI Listing
March 2021

Selection and applications of functional nucleic acids for infectious disease detection and prevention.

Anal Bioanal Chem 2021 Jul 28;413(18):4563-4579. Epub 2021 Jan 28.

The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, Fujian, China.

Infectious diseases caused by pathogenic microorganisms such as viruses and bacteria pose a great threat to human health. Although a significant progress has been obtained in the diagnosis and prevention of infectious diseases, it still remains challenging to develop rapid and cost-effective detection approaches and overcome the side effects of therapeutic agents and pathogen resistance. Functional nucleic acids (FNAs), especially the most widely used aptamers and DNAzymes, hold the advantages of high stability and flexible design, which make them ideal molecular recognition tools for bacteria and viruses, as well as potential therapeutic drugs for infectious diseases. This review summarizes important advances in the selection and detection of bacterial- and virus-associated FNAs, along with their potential prevention ability of infectious disease in recent years. Finally, the challenges and future development directions are concluded.
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http://dx.doi.org/10.1007/s00216-020-03124-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840224PMC
July 2021

Tracing Tumor-Derived Exosomal PD-L1 by Dual-Aptamer Activated Proximity-Induced Droplet Digital PCR.

Angew Chem Int Ed Engl 2021 03 25;60(14):7582-7586. Epub 2021 Feb 25.

The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.

Tumor-derived exosomal proteins have emerged as promising biomarkers for cancer diagnosis, but the quantitation accuracy is hindered by large numbers of normal cell-derived exosomes. Herein, we developed a dual-target-specific aptamer recognition activated in situ connection system on exosome membrane combined with droplet digital PCR (ddPCR) (TRACER) for quantitation of tumor-derived exosomal PD-L1 (Exo- ). Leveraging the high binding affinity of aptamers, excellent selectivity of dual-aptamer recognition, and the high sensitivity of ddPCR, this method exhibits significant sensitivity and selectivity for tracing tumor-derived Exo- in a wash-free manner. Due to the excellent sensitivity, the level of tumor-derived Exo- detected by TRACER can distinguish cancer patients from healthy donors, and for the first time was identified as a more reliable tumor diagnostic marker than total Exo- . The TRACER strategy holds great potential for converting exosomes into reliable clinical indicators and exploring the biological functions of exosomes.
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http://dx.doi.org/10.1002/anie.202015628DOI Listing
March 2021

Entropy subspace separation-based clustering for noise reduction (ENCORE) of scRNA-seq data.

Nucleic Acids Res 2021 02;49(3):e18

Institute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.

Single-cell RNA sequencing enables us to characterize the cellular heterogeneity in single cell resolution with the help of cell type identification algorithms. However, the noise inherent in single-cell RNA-sequencing data severely disturbs the accuracy of cell clustering, marker identification and visualization. We propose that clustering based on feature density profiles can distinguish informative features from noise. We named such strategy as 'entropy subspace' separation and designed a cell clustering algorithm called ENtropy subspace separation-based Clustering for nOise REduction (ENCORE) by integrating the 'entropy subspace' separation strategy with a consensus clustering method. We demonstrate that ENCORE performs superiorly on cell clustering and generates high-resolution visualization across 12 standard datasets. More importantly, ENCORE enables identification of group markers with biological significance from a hard-to-separate dataset. With the advantages of effective feature selection, improved clustering, accurate marker identification and high-resolution visualization, we present ENCORE to the community as an important tool for scRNA-seq data analysis to study cellular heterogeneity and discover group markers.
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http://dx.doi.org/10.1093/nar/gkaa1157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897472PMC
February 2021

Digital-WGS: Automated, highly efficient whole-genome sequencing of single cells by digital microfluidics.

Sci Adv 2020 Dec 9;6(50). Epub 2020 Dec 9.

Collaborative Innovation Center of Chemistry for Energy Materials, MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Engineering, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, P.R. China.

Single-cell whole-genome sequencing (WGS) is critical for characterizing dynamic intercellular changes in DNA. Current sample preparation technologies for single-cell WGS are complex, expensive, and suffer from high amplification bias and errors. Here, we describe Digital-WGS, a sample preparation platform that streamlines high-performance single-cell WGS with automatic processing based on digital microfluidics. Using the method, we provide high single-cell capture efficiency for any amount and types of cells by a wetted hydrodynamic structure. The digital control of droplets in a closed hydrophobic interface enables the complete removal of exogenous DNA, sufficient cell lysis, and lossless amplicon recovery, achieving the low coefficient of variation and high coverage at multiple scales. The single-cell genomic variations profiling performs the excellent detection of copy number variants with the smallest bin of 150 kb and single-nucleotide variants with allele dropout rate of 5.2%, holding great promise for broader applications of single-cell genomics.
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http://dx.doi.org/10.1126/sciadv.abd6454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725457PMC
December 2020

Nucleic Acids Analysis.

Sci China Chem 2020 Dec 2:1-33. Epub 2020 Dec 2.

Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1 Canada.

Nucleic acids are natural biopolymers of nucleotides that store, encode, transmit and express genetic information, which play central roles in diverse cellular events and diseases in living things. The analysis of nucleic acids and nucleic acids-based analysis have been widely applied in biological studies, clinical diagnosis, environmental analysis, food safety and forensic analysis. During the past decades, the field of nucleic acids analysis has been rapidly advancing with many technological breakthroughs. In this review, we focus on the methods developed for analyzing nucleic acids, nucleic acids-based analysis, device for nucleic acids analysis, and applications of nucleic acids analysis. The representative strategies for the development of new nucleic acids analysis in this field are summarized, and key advantages and possible limitations are discussed. Finally, a brief perspective on existing challenges and further research development is provided.
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http://dx.doi.org/10.1007/s11426-020-9864-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716629PMC
December 2020

Activation of Aptamers with Gain of Function by Small-Molecule-Clipping of Intramolecular Motifs.

Angew Chem Int Ed Engl 2021 03 27;60(11):6021-6028. Epub 2021 Jan 27.

The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian, 361005, China.

Click reactions have the advantages of high reactivity, excellent orthogonality, and synthetic accessibility. Inspired by click reactions, we propose the concept of "clipped aptamers", whose binding affinity is regulated by the "clip"-like specific interaction between a synthetic DNA-mismatch-binding small molecule (molecular glue, Z-NCTS) and the preset CGG/CGG sequences in nucleic acid sequences. In this study, we investigated a Z-NCTS-mediated de novo selection of clipped aptamers against epithelial cell adhesion molecule. The generated clipped aptamers can achieve the efficient transition from a binding-inactive state to an active state by clipping of Z-NCTS with two CGG sites, which otherwise would not hybridize. The experimental and simulation results showed that the clipped aptamer had ideal binding thermodynamics and the ability to regulate cellular adhesion. Because of this superior activated mechanism and structural diversity, clipped aptamers hold great potential in biosensing, imaging, conditional gene- and cellular behavior-regulation, and drug delivery.
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http://dx.doi.org/10.1002/anie.202013570DOI Listing
March 2021

3D-printed integrative probeheads for magnetic resonance.

Nat Commun 2020 11 13;11(1):5793. Epub 2020 Nov 13.

Department of Electronic Science, Fujian Provincial Key Laboratory of Plasma and Magnetic Resonance, Xiamen University, 361005, Xiamen, China.

Magnetic resonance (MR) technology has been widely employed in scientific research, clinical diagnosis and geological survey. However, the fabrication of MR radio frequency probeheads still face difficulties in integration, customization and miniaturization. Here, we utilized 3D printing and liquid metal filling techniques to fabricate integrative radio frequency probeheads for MR experiments. The 3D-printed probehead with micrometer precision generally consists of liquid metal coils, customized sample chambers and radio frequency circuit interfaces. We screened different 3D printing materials and optimized the liquid metals by incorporating metal microparticles. The 3D-printed probeheads are capable of performing both routine and nonconventional MR experiments, including in situ electrochemical analysis, in situ reaction monitoring with continues-flow paramagnetic particles and ions separation, and small-sample MR imaging. Due to the flexibility and accuracy of 3D printing techniques, we can accurately obtain complicated coil geometries at the micrometer scale, shortening the fabrication timescale and extending the application scenarios.
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http://dx.doi.org/10.1038/s41467-020-19711-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666178PMC
November 2020

Efficient Isolation and Phenotypic Profiling of Circulating Hepatocellular Carcinoma Cells via a Combinatorial-Antibody-Functionalized Microfluidic Synergetic-Chip.

Anal Chem 2020 11 30;92(22):15229-15235. Epub 2020 Oct 30.

The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.

As a malignant disease that seriously threatens human health, hepatocellular carcinoma (HCC) lacks effective early screening and prognostic assessment methods. Herein, we developed a method for efficient capture and multiphenotype analysis of circulating tumor cells (CTCs) of hepatocellular carcinoma. The anti-ASGPR antibody and the anti-EpCAM antibody were modified in parallel on a deterministic lateral displacement (DLD)-patterned microfluidic Synergetic-Chip to enhance capture efficiency by a complementary effect. CTCs were detected in 45 out of 45 (100%) HCC patients, with a sensitivity and specificity of 97.8 and 100%, respectively. Patients with more total CTCs and nonepithelial CTCs were in later stages of HCC and had more malignant progression. This strategy proposes a feasible approach for early diagnosis and prognosis of hepatocellular carcinoma.
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http://dx.doi.org/10.1021/acs.analchem.0c03936DOI Listing
November 2020

Three-Dimensional Quantitative Imaging of Native Microbiota Distribution in the Gut.

Angew Chem Int Ed Engl 2021 02 11;60(6):3055-3061. Epub 2020 Dec 11.

State Key Laboratory for Oncogenes & Related Genes and Bio-ID Center, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China.

Owing to the challenges to acquire detailed spatial information of gut bacteria in situ, three-dimensional (3D) microbiota distributions in the gut remain largely uncharted. Here, we propose a tissue clearing-based and D-amino acid labeling-facilitated (TiDaL) strategy that combines a novel microbiota in vivo labeling protocol, CUBIC-based tissue clearing and whole-mount tissue imaging, to achieve 3D imaging of indigenous gut microbiota. We demonstrate high-resolution 3D acquisition of their biogeography in different gut sections, and present quantitative spatial details in relation to the host epithelium. We unexpectedly observe microbiota in the small intestine crypts, which were thought to be bacteria-free. Significant bacterial overgrowth in the first two-thirds of the small intestine is detected in an enteritis model. We expect that this quantitative 3D imaging strategy for native gut microbiota will provide insightful information into the host-microbiota interactions.
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http://dx.doi.org/10.1002/anie.202010921DOI Listing
February 2021
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