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Cross-reactive sensor arrays are useful for discriminating multiple analytes in a complex sample. Herein, a portable and label-free gas pressure sensor array was proposed for multiplex analysis via a handheld gas pressure meter. It is based on the interaction diversity of analytes with catalase-like nanomaterials, including Pt nanoparticles (PtNP), Cosub3/subOsub4/subnanosheets (Cosub3/subOsub4/subNS), and Pt-Co alloy nanosheets (PtCoNS), respectively. Thus, the diverse influence of analytes on the catalase-like activity could be output as the difference in the gas pressure. By using principal component analysis, eight proteins were well distinguished by the gas pressure sensor array at the 10 nM level within 12 min. Moreover, different concentrations of proteins and mixtures of proteins could likewise be discriminated. More importantly, the effective discrimination of proteins in human serum and discrimination of five kinds of cells further confirmed the potential of the gas pressure sensor array. Therefore, it provides a portable, cheap, sensitive, and label-free gas pressure sensor array, which is totally different from the reported sensor arrays and holds great potential for portable and cheap discrimination of multiple analytes.

Aptamer-functionalized microfluidic interfaces hold great potential for liquid biopsies owing to their programmable nature. However, most previous studies have focused on development of multivalent aptamers to improve binding affinity, while ignoring aptamer orientation on microfluidic interfaces, resulting in suboptimal accessibility and affinity. Herein, we report a

The global spread of SARS-CoV-2 virus has severely affected human health, life, and work. Vaccine immunization is considered to be an effective means to protect the body from infection. Therefore, timely analysis of the antibody level is helpful to identify people with low immune response or attenuated antibodies so as to carry out targeted and precise vaccine booster immunization. Herein, we develop a magnetofluid-integrated multicolor immunochip, as a sample-to-answer system in a fully enclosed space, for visual analysis of neutralizing antibodies of SARS-CoV-2. Generally, this chip adopts an innovative three-dimensional two-phase system that utilizes mineral oil to block the connection between reagent wells in the vertical direction and provides a wide interface for rapid and nondestructive shuttle of magnetic beads during the immunoassay. In order to obtain visualized signal output, gold nanorods with a size-dependent color effect are used as the colorful chromogenic substrates for evaluation of the antibody level. Using this chip, the neutralizing antibodies were successfully detected in vaccine-immunized volunteers with 83.3% sensitivity and 100% specificity. Furthermore, changes in antibody levels of the same individual over time were also reflected by the multicolor assay. Overall, benefiting from simple operation, airtight safety, and nonrequirement of external equipment, this platform can provide a new point-of-care testing strategy for alleviating the shortage of medical resources and promoting epidemic control in underdeveloped areas.

Liquid biopsy capable of noninvasive and real-time molecular profiling is considered as a breakthrough technology, endowing an opportunity for precise diagnosis of individual patients. Extracellular vesicles (EVs) and circulating tumor cells (CTCs) consisting of substantial disease-related molecular information play an important role in liquid biopsy. Therefore, it is critically significant to exploit high-performance recognition ligands for efficient isolation and analysis of EVs and CTCs from complex body fluids. Aptamers exhibit extraordinary merits of high specificity and affinity, which are considered as superior recognition ligands for liquid biopsy. In this review, we first summarize recent advanced strategies for the evolution of high-performance aptamers and the construction of various aptamer-based recognition elements. Subsequently, we mainly discuss the isolation and analysis of EVs and CTCs based on the aptamer functioned biomaterials/biointerface. Ultimately, we envision major challenges and future direction of aptamer-based liquid biopsy for clinical utilities.

We describe a novel system for examining the mechanisms of AAV traversal of the BBB using a multi-chamber microplate wherein astrocytes and HBMECs are co-cultured, and subjected to shear fluid force similar to that under physiological conditions.

Gene mutation profiling of heterogeneous circulating tumor cells (CTCs) offers comprehensive and real-time molecular information of tumors for targeted therapy guidance, but the lack of efficient and multiplex genotyping techniques for single-CTC analysis greatly hinders its development and clinical application. This paper reports a single-CTC mass spectrometry analysis method for efficient and multiplex mutation profiling based on digital microfluidics. Digital microfluidics affords integrated single-CTC manipulation, from single-CTC isolation to high-performance whole genome amplification, via nanoliter droplet-based wettability trapping and hydrodynamic adjustment of cell distribution. Coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, multiplex mutation information of individual CTCs can be efficiently and accurately identified by the inherent mass differences of different DNA sequences. This platform achieves Kirsten rat sarcoma viral oncogene mutation profiling of heterogeneous CTCs at the single-cell level from cancer patient samples, offering new avenues for genotype profiling of single CTCs and cancer therapy guidance.

New neutralizing agents against SARS-CoV-2 and associated mutant strains are urgently needed for the treatment and prophylaxis of COVID-19. Herein, we develop a spherical cocktail neutralizing aptamer-gold nanoparticle (SNAP) to block the interaction between the receptor-binding domain (RBD) of SARS-CoV-2 and host ACE2. With the multivalent aptamer assembly as well as the steric hindrance effect of the gold scaffold, SNAP exhibits exceptional binding affinity against the RBD with a dissociation constant of 3.90 pM and potent neutralization against authentic SARS-CoV-2 with a half-maximal inhibitory concentration of 142.80 fM, about 2 or 3 orders of magnitude lower than that of the reported neutralizing aptamers and antibodies. More importantly, the synergetic blocking strategy of multivalent multisite binding and steric hindrance ensures broad neutralizing activity of SNAP, almost completely blocking the infection of three mutant pseudoviruses. Overall, the SNAP strategy provides a new direction for the development of antivirus agents against SARS-CoV-2 and other emerging coronaviruses.

Rapid and automated detection of acute myocardial infarction (AMI) at its developing stage is very important due to its high mortality rate. To quantitatively diagnose AMI, Myo, CK-MB, and cTnI are chosen as three biomarkers, which are usually detected through an immunosorbent assay, such as the enzyme-linked immunosorbent assay. However, the approach poses many drawbacks, such as long detection time, the cumbersome process, the need for professionals, and the difficulty of realizing automatic operation. Here, a multichannel digital microfluidic (DMF) thermal control chip integrated with a sandwich-based immunoassay strategy is proposed for the automated, rapid, and sensitive detection of AMI biomarkers. A miniaturized temperature control module is integrated on the back of the DMF chip, meeting the temperature requirement for the immunoassay. With this DMF thermal control chip, sample and reagent consumption are reduced to several microliters, significantly alleviating reagent consumption and sample dependence, and the automated and multichannel detection of biomarkers can be achieved. In this work, the simultaneously noninvasive detection of the human serum sample containing the three biomarkers of AMI is also achieved within 30 min, which improves the diagnostic accuracy of AMI. Due to the features of automation and miniaturization, the multichannel immunosensor can be used in community hospitals to increase the speed of diagnosis of patients with various acute diseases.

Multivalent display of SARS-CoV-2 RBDs (receptor-binding domains, prime proteins for viral infection and as vaccine immunogens) affects infectivity and as immunogens on a virus-like particle (VLP) can enhance immune response. However, the viral attachment and immune response initiated by the copy number and distribution pattern of SARS-CoV-2 RBDs remain poorly understood. Here, we organize SARS-CoV-2 RBDs on DNA nanoballs of ∼74 nm diameter by an aptamer-guided assembly for a systematic interrogation. We find that both the affinity and the rate of the DNA-based VLP binding to the host cell increase with the RBD number (10-90). In addition, a concentrated RBD distribution promotes faster and stronger interaction to the host cell than an even RBD distribution. Moreover, it is interesting to learn that the immunity activation does not increase linearly with RBD numbers on the VLP. As few as 20 evenly distributed RBDs per VLP can elicit up to 86% immunity of macrophage cells. Overall, the work provides a new tool to study SARS-CoV-2 infection and VLP-based immunity activation, which should deepen our understanding of viral infection and facilitate the development of highly effective antiviral vaccines.

Transmission of bacterial endospores between the environment and people and the following germinationiin vivo/iplay critical roles in both the deadly infections of some bacterial pathogens and the stabilization of the commensal microbiotas in humans. Our knowledge about the germination process of different bacteria in the mammalian gut, however, is still very limited due to the lack of suitable tools to visually monitor this process. We proposed a two-step labeling strategy that can image and quantify the endospores' germination in the recipient's intestines. Endospores collected from donor's gut microbiota were first labeled with fluorescein isothiocyanate and transplanted to mice via gavage. The recipient mice were then administered with Cyanine5-tagged D-amino acid to label all the viable bacteria, including the germinated endospores, in their intestinesiin situ/i. The germinated donor endospores could be distinguished by presenting two types of fluorescent signals simultaneously. The integrative use of cell-sorting, 16S rDNA sequencing, and fluorescenceiin situ/ihybridization (FISH) staining of the two-colored bacteria unveiled the taxonomic information of the donor endospores that germinated in the recipient's gut. Using this strategy, we investigated effects of different germinants and pre-treatment interventions on their germination, and found that germination of different commensal bacterial genera was distinctly affected by various types of germinants. This two-color labeling strategy shows its potential as a versatile tool for visually monitoring endospore germination in the hosts and screening for new interventions to improve endospore-based therapeutics.

Broad-spectrum anti-SARS-CoV-2 strategies that can inhibit the infection of wild-type and mutant strains would alleviate their threats to global public health. Here, we propose an icosahedral DNA framework for the assembly of up to 30 spatially arranged neutralizing aptamers (IDNA-30) to inhibit viral infection. Each triangular plane of IDNA-30 is composed of three precisely positioned aptamers topologically matching the SARS-CoV-2 spike trimer, thus forming a multivalent spatially patterned binding. Due to its multiple binding sites and moderate size, multifaced IDNA-30 induces aggregation of viruses. The rigid icosahedron framework afforded by four helixes not only forms a steric barrier to prevent the virus from binding to the host but also limits the conformational transformation of the SARS-CoV-2 spike trimer. Combining multivalent topologically patterned aptamers with structurally well-defined nanoformulations, IDNA-30 exhibits excellent broad-spectrum neutralization against SARS-CoV-2, including almost completely blocking the infection of Omicron pseudovirus. Overall, this multidimensional neutralizing strategy provides a new direction for the assembly of neutralizing reagents to enhance their inhibitory effect against SARS-CoV-2 infection and combat other disease-causing viruses.

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 in situ 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.

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 in situ 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.

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.

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.

Multiplexed protein secretion analysis of single cells is important to understand the heterogeneity of cellular functions and processes in healthy and disease states. However, current single-cell platforms, such as microwell-, microchamber-, or droplet-based assays, suffer from low single-cell occupancy, waste of reagents, limited sensitivity, or inability to perform necessary operations, etc. To overcome these drawbacks, we present an integrated droplet microfluidic device that interfaces with spatially patterned antibody barcodes for multiplexed single-cell secretome analysis. The trapping array of 100 picoliter-sized isolation chambers could achieve >80% single-cell capture efficiency with >90% viability. The single-cell analysis microchip was validated by the detection of four-plexed cytokines, including IL-8, MCP-1, MIP-1b, and TNF-a/IL-10, from unstimulated and lipopolysaccharide (LPS)-stimulated individual human macrophages. We also successfully applied the platform to profile protein secretions of human tumor cell lines and primary/metastatic cancer cells dissociated from cancer patients to observe the secretion heterogeneity among cells. This unique microfluidic platform enables multiplexed secretion assays for static droplet microfluidics, provides a reliable and straightforward workflow for protein secretion assays based on a low number of single cells in a short incubation time (∼4 h), and could have widespread applications for studying secretion-mediated cellular heterogeneity.

The most prevalent primary brain tumors are gliomas, which start in the glial cells. Although there have been significant technological advances in surgery and radio-chemotherapy, the prognosis and survival of patients with malignant gliomas remain poor. For routine diagnosis of glioma, computed tomography and magnetic resonance imaging primarily depend on anatomical changes and fail to detect the cellular changes that occur early in the development of malignant gliomas. Therefore, it is urgent to find effective molecular diagnostic tools to detect early stages of malignant gliomas. Currently, cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) technology is one effective tool to obtain DNA or RNA aptamers capable of differentiating the molecular signatures among different types of cell lines. Using cell-SELEX, we generated and characterized an aptamer, termed S6-1b, that can distinguish the molecular differences between glioma cell line SHG44 and human astrocytes. Under the conditions of 4 and 37 °C, respectively, the dissociation constants of aptamer-cell interaction were both measured in the low nanomolar range. The aptamer S6-1b also exhibited excellent selectivity, making it suitable for use in a complex biological environment. Furthermore, the aptamer can effectively target glioma cells for in vivo fluorescence imaging of tumors. The target type of aptamer S6-1b was identified as a cell membrane protein. Our work indicates that aptamer S6-1b has diagnostic and therapeutic potential to specifically deliver imaging or therapeutic agents to malignant gliomas.

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.

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.

Infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the rapid spread of coronavirus disease 2019 (COVID-19), has generated a public health crisis worldwide. The molecular mechanisms of SARS-CoV-2 infection and virus–host interactions are still unclear. In this study, we identified four unique microRNA-like small RNAs encoded by SARS-CoV-2. SCV2-miR-ORF1ab-1-3p and SCV2-miR-ORF1ab-2-5p play an important role in evasion of type I interferon response through targeting several genes in type I interferon signaling pathway. Particularly worth mentioning is that highly expressed SCV2-miR-ORF1ab-2-5p inhibits some key genes in the host innate immune response, such as IRF7, IRF9, STAT2, OAS1, and OAS2. SCV2-miR-ORF1ab-2-5p has also been found to mediate allelic differential expression of COVID-19-susceptible gene OAS1. In conclusion, these results suggest that SARS-CoV-2 uses its miRNAs to evade the type I interferon response and links the functional viral sequence to the susceptible genetic background of the host.

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.

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.

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.

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

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.

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.

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 via a thiol-exchange reaction, allowing them to be used for subsequent cellular and molecular analysis. By fluorescence in situ 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.

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.

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.

Liquid biopsy offers non-invasive and real-time molecular profiling of individual patients, and is thus considered a revolutionary technology in precision medicine. Exosomes have been acknowledged as significant biomarkers in liquid biopsy, as they play a central role in cell-cell communication and are closely related to the pathogenesis of most human malignancies. Nevertheless, in biofluids exosomes always co-exist with other particles, and the cargo components of exosomes are highly heterogeneous. Thus, the isolation and molecular characterization of exosomes are still technically challenging. Microfluidics technology effectively addresses this challenge by virtue of its inherent advantages, such as precise manipulation of fluids, low consumption of samples and reagents, and a high level of integration. Recent advances in microfluidics allow in situ exosome capture and molecular detection with unprecedented selectivity and sensitivity. In this review, the state-of-the-art developments in microfluidics-based exosome research, including exosome isolation approaches and molecular detection strategies, with highlights of the characterization of exosomal biomarkers in cancer liquid biopsy is summarized. The major challenges are also discussed and some perspectives for the future directions of exosome-based liquid biopsy in microfluidic systems are presented.

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-PD-L1 ). 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-PD-L1 in a wash-free manner. Due to the excellent sensitivity, the level of tumor-derived Exo-PD-L1 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-PD-L1 . The TRACER strategy holds great potential for converting exosomes into reliable clinical indicators and exploring the biological functions of exosomes.

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.

Here, a fully integrated multicolor immunosensor was developed for sensitive and reliable semiquantitative analysis of HIV-1 p24, which integrates the multistep reactions of horseradish peroxidase (HRP)-linked immunoassay and gold nanorod (AuNR)-based multicolor assay into a single microfluidic chip. The HRP-linked immunoassay functions by moving magnetic beads bound to a capture antibody through different aqueous phases containing immunoassay reagents. HRP-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) is used to mediate AuNRs etching for producing various color changes. Multiple etching processes can be activated by simple mixing of the reagents from the reagent storage reservoir. The fully integrated strategy with sample-in answer-out capability is initiated by simple chip manipulation and finally concluded by converting recognition of antigen-antibody into a vivid color variation for direct visualization and semiquantitative analysis. By bare eye observation, our integrated multicolor immunosensor allows sensitive and reliable semiquantitative analysis of HIV-1 p24 within 1 h. The microfluidic chip device demonstrated here simplifies the operation significantly and thus allows broader application of a multicolor immunosensor for point of care (POC) testing in low-resource settings.

The World Health Organization has declared the outbreak of a novel coronavirus (SARS-CoV-2 or 2019-nCoV) as a global pandemic. However, the mechanisms behind the coronavirus infection are not yet fully understood, nor are there any targeted treatments or vaccines. In this study, we identified high-binding-affinity aptamers targeting SARS-CoV-2 RBD, using an ACE2 competition-based aptamer selection strategy and a machine learning screening algorithm. The Kd values of the optimized CoV2-RBD-1C and CoV2-RBD-4C aptamers against RBD were 5.8 nM and 19.9 nM, respectively. Simulated interaction modeling, along with competitive experiments, suggests that two aptamers may have partially identical binding sites at ACE2 on SARS-CoV-2 RBD. These aptamers present an opportunity for generating new probes for recognition of SARS-CoV-2 and could provide assistance in the diagnosis and treatment of SARS-CoV-2 while providing a new tool for in-depth study of the mechanisms behind the coronavirus infection.

ScRNA-seq has the ability to reveal accurate and precise cell types and states. Existing scRNA-seq platforms utilize bead-based technologies uniquely barcoding individual cells, facing practical challenges for precious samples with limited cell number. Here, we present a scRNA-seq platform, named Paired-seq, with high cells/beads utilization efficiency, cell-free RNAs removal capability, high gene detection ability and low cost. We utilize the differential flow resistance principle to achieve single cell/barcoded bead pairing with high cell utilization efficiency (95%). The integration of valves and pumps enables the complete removal of cell-free RNAs, efficient cell lysis and mRNA capture, achieving highest mRNA detection accuracy (R = 0.955) and comparable sensitivity. Lower reaction volume and higher mRNA capture and barcoding efficiency significantly reduce the cost of reagents and sequencing. The single-cell expression profile of mES and drug treated cells reveal cell heterogeneity, demonstrating the enormous potential of Paired-seq for cell biology, developmental biology and precision medicine., Single-cell RNA-seq can reveal accurate and precise cell types and states. Here the authors present an scRNA-seq platform, Paired-seq, which uses differential flow resistance to achieve 95% cell utilisation efficiency for improved cell-free RNA removal and gene detection.

The dissociation constant (Kd) is a crucial parameter for characterizing binding affinity in molecular recognition, including antigen-antibody, DNA-protein, and receptor-ligand interactions. However, conventional methods for Kd characterization usually involve a multi-step process and time-consuming operations for incubation, washing, and detection, thus causing problems, such as time delays, microbead loss, degradation of sensitive molecules, and personal errors. Here we demonstrate an automated ligand binding affinity evaluation platform (Auto-affitech) using digital microfluidics (DMF), with individual droplets at the microliter level, programmed to rapidly perform the incubation and separation of target-beads and binding ligands. Because the loss of the beads influences the detection results, we propose a new strategy for magnetic bead separation using DMF, termed the bidirectional separation method. By splitting one droplet into two asymmetric droplets, high bead retention efficiency (89.57% ± 0.05%) and high washing efficiency (99.59% ± 0.17%, with four washings) were obtained. We demonstrate the determination of Kd of an aptamer-protein system (EpCAM and its corresponding aptamer SYL3C) and an antigen-antibody system (H5N1 antigen and antibody), proving the capability and universality of Auto-affitech in various receptor-ligand systems. Integrating all the sample processing procedures, the Auto-affitech not only saves manual labor and minimizes personal errors, but also conserves samples and shortens analysis time. Overall, this platform successfully demonstrates to be an automated approach for dissociation constant evaluation and exhibits great potential for highly efficient screening of ligands.

Circulating tumor cells (CTCs) undergoing epithelial mesenchymal transition (EMT) play an essential role in metastasis and have a better correlation with poor disease outcomes, but the most current affinity-based enrichment methods rely on targeting epithelial markers, which are less effective in capturing CTCs undergoing EMT. Herein, we identified and optimized an aptamer (ZY5C) sequence with high binding affinity and specificity against cell surface vimentin (CSV), which is overexpressed on the post-EMT CTCs. Not only can the hairpin-structured ZY5C aptamer specifically recognize a number of cancer cells with native CSV expression, but it can also bind to CSV expressed on EMT-cells. The Kd value of the ZY5C aptamer against CSV-positive T24 cells was found to be 38 ± 4 nM. Using the evolved ZY5C aptamer and multivalent ZY5C aptamer-functionalized chip, we were able to isolate CTCs from the blood of adenocarcinoma, sarcoma, and carcinosarcoma patients. Overall, this ZY5C aptamer and isolation method bring a fresh approach to CTCs analysis, which not only detects CTCs from nonepithelial origin, but also provides an efficient way to in-depth study the role of post-EMT CTCs in clinical application and metastasis mechanisms.

Minimal residual disease (MRD) offers a highly independent prognostic factor for leukemia patients. However, challenges confronting conventional MRD assays are high invasiveness, as well as limited detection sensitivity and clinical applicability. Inspired by the self-adaptive skeleton and multiple suckers or tendrils of climbing plants, a biomimetic Multivalent Aptamer Nanoclimber (MANC)-functionalized microfluidic chip (MANC-Chip) is reported for minimally invasive, highly sensitive and clinically applicable MRD detection in the peripheral blood of T-cell acute lymphoblastic leukemia patients. The MANCs are synthesized by a simple co-polymerization reaction. Due to their flexible structure and cooperative multivalent effect, MANCs dramatically enhance the binding affinity of aptamers targeting leukemia cells. A deterministic lateral displacement-patterned microfluidic chip is designed to further increase the collision probability between MANCs and leukemia cells. Benefiting from the synergistic effect of multivalent binding and enhanced collision, a high capture efficiency of 92.2% for leukemia cells is achieved. Moreover, the captured leukemia cells can be released with high efficiency of 88.9% and high viability of 93.8% via nuclease treatment prior to downstream analysis. Overall, the excellent features of MANC-Chip make it very useful for precise detection of MRD and better understanding of leukemia.

The ubiquitous biomembrane interface, with its dynamic lateral fluidity, allows membrane-bound components to rearrange and localize for high-affinity multivalent ligand-receptor interactions in diverse life activities. Inspired by this, we herein engineered a fluidic multivalent nanointerface by decorating a microfluidic chip with aptamer-functionalized leukocyte membrane nanovesicles for high-performance isolation of circulating tumor cells (CTCs). This fluidic biomimetic nanointerface with active recruitment-binding afforded significant affinity enhancement by 4 orders of magnitude, exhibiting 7-fold higher capture efficiency compared to a monovalent aptamer functionalized-chip in blood. Meanwhile, this soft nanointerface inherited the biological benefits of a natural biomembrane, minimizing background blood cell adsorption and maintaining excellent CTC viability (97.6%). Using the chip, CTCs were successfully detected in all cancer patient samples tested (17/17), suggesting the high potential of this fluidity-enhanced multivalent binding strategy in clinical applications. We expect this bioengineered interface strategy will lead to the design of innovative biomimetic platforms in the biomedical field by leveraging natural cell-cell interaction with a natural biomaterial.

Objective To ascertain whether en bloc resection could reduce the risk of seeding cancer cells into the circulation during the resection of non-muscle invasive bladder cancer (NMIBC). Methods Patients with primary NMIBC were enrolled in this prospective study from October 2017 to May 2018. Patients were allocated to receive conventional transurethral resection of the bladder (TURB) or retrograde en bloc resection technique of the bladder tumor (RERBT). Blood samples (1 ml) for circulating tumor cell (CTC) enumeration were drawn from the peripheral vein prior to resection (PV1), immediately after resection of the tumor base (PV2), and at 12 h after resection (PV3). Intra-group comparisons of the changes in the number of CTCs identified among the PV1, PV2, and PV3 blood samples were performed in each group. Results A total of 21 patients (12 in the RERBT group and 9 in the TURB group) were recruited. For patients receiving TURB, the level of CTCs identified in PV3 was significantly higher than that in PV1 (p = 0.047). However, there was no significant difference in CTC counts before and after resection in the RERBT group. Conclusion RERBT did not increase the number of tumor cells in the bloodstream.

Gas generation-based measurement is an attractive alternative approach for POC (Point-of-care) testing, which relies on the amount of generated gas to detect the corresponding target concentrations. In gas generation-based POC testing, the integration of a target recognition component and a catalyzed gas-generating reaction initiated by the target introduction can lead to greatly amplified signals, which can be highly sensitive measured via distance readout or simple hand-held devices. More importantly, numerous gas-generating reactions are environment-friendly since their products such as oxygen and nitrogen are nontoxic and odourless, which makes gas generation-based POC testing safe and secure for inexperienced staff. Researchers have demonstrated that gas generation-based measurements enable the rapid and highly sensitive POC detection of a variety of analytes. In this review, we focus on the recent developments in gas generation-based POC testing systems. The common types of gas-generating reactions are first listed and the translation of gas signals to different signal readouts for POC testing are then summarized, including distance readouts and hand-held devices. Moreover, we introduce gas bubbles as actuators to power microfluidic devices. We finally provide the applications and future perspective of gas generation-based POC testing systems.

The main function and biological processes of tissues are determined by the combination of gene expression and spatial organization of their cells. RNA sequencing technologies have primarily interrogated gene expression without preserving the native spatial context of cells. However, the emergence of various spatially-resolved transcriptome analysis methods now makes it possible to map the gene expression to specific coordinates within tissues, enabling transcriptional heterogeneity between different regions, and for the localization of specific transcripts and novel spatial markers to be revealed. Hence, spatially-resolved transcriptome analysis technologies have broad utility in research into human disease and developmental biology. Here, recent advances in spatially-resolved transcriptome analysis methods are summarized, including experimental technologies and computational methods. Strengths, challenges, and potential applications of those methods are highlighted, and perspectives in this field are provided.

Since the transferrin receptor (CD71 or TFRC) is known to be highly expressed in numerous cancers, CD71 has become an attractive target in cancer research. Acquiring specific molecular probes for CD71, such as small molecular ligands, aptamers, peptides, or antibodies, is of great importance for cancer cell recognition and capture. In this work, we chose CD71 as the target for phage display, and after four rounds of positive selection and one round of negative selection, the specific phage library was enriched. After verification and sequence analysis, six peptides were identified to be able to bind to CD71 with high specificity. The specific recognition of the CD71-positive cells was confirmed by flow cytometry and confocal microscopy. Competition experiments demonstrated that peptide Y1 and transferrin (TF) were bound to distinct sites on CD71, indicating that peptide Y1 could replace TF as a potential probe for cell imaging and drug delivery, thus avoiding competition by endogenous TF and side effects. Graphical abstract Six peptides were successfully isolated using in vitro biopanning against CD71 with high specificity and affinity. Peptides Y1 and Y2 would be powerful tools in biosensors and biomedicine due to their unique properties.

Enzyme-linked immunosorbent assay (ELISA) is a popular laboratory technique for detection of disease-specific protein biomarkers with high specificity and sensitivity. However, ELISA requires labor-intensive and time-consuming procedures with skilled operators and spectroscopic instrumentation. Simplification of the procedures and miniaturization of the devices are crucial for ELISA-based point-of-care (POC) testing in resource-limited settings. Here, we present a fully integrated, instrument-free, low-cost and portable POC platform which integrates the process of ELISA and the distance readout into a single microfluidic chip. Based on manipulation using a permanent magnet, the process is initiated by moving magnetic beads with capture antibody through different aqueous phases containing ELISA reagents to form bead/antibody/antigen/antibody sandwich structure, and finally converts the molecular recognition signal into a highly sensitive distance readout for visual quantitative bioanalysis. Without additional equipment and complicated operations, our integrated ELISA-Chip with distance readout allows ultrasensitive quantitation of disease biomarkers within 2h. The ELISA-Chip method also showed high specificity, good precision and great accuracy. Furthermore, the ELISA-Chip system is highly applicable as a sandwich-based platform for the detection of a variety of protein biomarkers. With the advantages of visual analysis, easy operation, high sensitivity, and low cost, the integrated sample-in-answer-out ELISA-Chip with distance readout shows great potential for quantitative POCT in resource-limited settings.