Search

The spatial transcriptome has enabled researchers to resolve transcriptome expression profiles while preserving information about cell location to better understand the complex biological processes that occur in organisms. Due to technical limitations, the current high-throughput spatial transcriptome sequencing methods (known as next-generation sequencing with spatial barcoding methods or spot-based methods) cannot achieve single-cell resolution. A single measurement site, called a spot, in these technologies frequently contains multiple cells of various types. Computational tools for determining the cellular composition of a spot have emerged as a way to break through these limitations. These tools are known as deconvolution tools. Recently, a couple of deconvolution tools based on different strategies have been developed and have shown promise in different aspects. The resulting single-cell resolution expression profiles and/or single-cell composition of spots will significantly affect downstream data mining; thus, it is crucial to choose a suitable deconvolution tool. In this review, we present a list of currently available tools for spatial transcriptome deconvolution, categorize them based on the strategies they employ, and explain their advantages and limitations in detail in order to guide the selection of these tools in future studies.

Digital microfluidic (DMF) bioassays with the benefits of automation, addressability, integration and dynamic configuration ability for nucleic acids, proteins, immunoreaction and cell analysis are presented in this review.

Automated manipulation of discrete droplets by digital microfluidics (DMF) combined with chemiluminescence (CL) is promising to achieve automated and sensitive biosensing and bioanalysis.

Addressing the spread of coronavirus disease 2019 (COVID-19) has highlighted the need for rapid, accurate, and low-cost diagnostic methods that detect specific antigens for SARS-CoV-2 infection. Tests for COVID-19 are based on reverse transcription PCR (RT-PCR), which requires laboratory services and is time-consuming. Here, by targeting the SARS-CoV-2 spike protein, we present a point-of-care SERS detection platform that specifically detects SARS-CoV-2 antigen in one step by captureing substrates and detection probes based on aptamer-specific recognition. Using the pseudovirus, without any pretreatment, the SARS-CoV-2 virus and its variants were detected by a handheld Raman spectrometer within 5 min. The limit of detection (LoD) for the pseudovirus was 124 TU μL

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.

Accurate isolation of targeted extracellular vesicle (EV) is challenging due to the heterogeneity of EV subpopulations which are from different cell origins. Most EV subpopulations lack a single marker whose expression cleanly distinguish them from mixed populations of closely related EVs. Herein, we developed a modular platform capable of taking multiple binding events as input, logic computations, and producing two independent outputs for tandem microchips for EV subpopulation isolation. Taking advantages of the excellent selectivity of dual-aptamer recognition-mediated DNA computation and the sensitivity of tandem microchips, this method achieves, for the first time, sequential isolation of tumour PD-L1 EVs and non-tumour PD-L1 EVs. As a result, the developed platform can not only effectively distinguish cancer patients from healthy donors, but also provides new clues for assessing immune heterogeneity. Moreover, the captured EVs can be released through a DNA hydrolysis reaction with high efficiency, which is compatible with downstream mass spectrometry for EV proteome profiling. Overall, this strategy is expected to isolate different EV subpopulations, translate EVs into reliable clinical biomarkers, and accurately investigate the biological functions of different EV subsets.

Single-cell copy number variations (CNVs), major dynamic changes in humans, result in differential levels of gene expression and account for adaptive traits or underlying disease. Single-cell sequencing is needed to reveal these CNVs but has been hindered by single-cell whole-genome amplification (scWGA) bias, leading to inaccurate gene copy number counting. In addition, most of the current scWGA methods are labor intensive, time-consuming, and expensive with limited wide application. Here, we report a unique single-cell whole-genome library preparation approach based on d igital microfluidics for d igital counting of s ingle- c ell C opy N umber V ariation (dd-scCNV Seq). dd-scCNV Seq directly fragments the original single-cell DNA and uses these fragments as templates for amplification. These reduplicative fragments can be filtered computationally to generate the original partitioned unique identified fragments, thereby enabling digital counting of copy number variation. dd-scCNV Seq showed an increase in uniformity in the single-molecule data, leading to more accurate CNV patterns compared to other methods with low-depth sequencing. Benefiting from digital microfluidics, dd-scCNV Seq allows automated liquid handling, precise single-cell isolation, and high-efficiency and low-cost genome library preparation. dd-scCNV Seq will accelerate biological discovery by enabling accurate profiling of copy number variations at single-cell resolution.

Single-cell RNA-seq (scRNA-seq) analysis of multiple samples separately can be costly and lead to batch effects. Exogenous barcodes or genome-wide RNA mutations can be used to demultiplex pooled scRNA-seq data, but they are experimentally or computationally challenging and limited in scope. Mitochondrial genomes are small but diverse, providing concise genotype information. We developed “mitoSplitter”, an algorithm that demultiplexes samples using mitochondrial RNA (mtRNA) variants, and demonstrated that mtRNA variants can be used to demultiplex large-scale scRNA-seq data. Using affordable computational resources, mitoSplitter can accurately analyze 10 samples and 60,000 cells in 6 hours. To avoid the batch effects from separated experiments, we applied mitoSplitter to analyze the responses of five non-small cell lung cancer (NSCLC) cell lines to BET chemical degradation in a multiplexed fashion. We found the synthetic lethality ofTOP2Ainhibition and BET chemical degradation in BET inhibitor-resistant cells. The result indicates that mitoSplitter can accelerate the application of scRNA-seq assays in biomedical research.

Efficient molecular selection is a prerequisite for generating molecular tools used in diagnosis, pathology, vaccinology, and therapeutics. Selection efficiency is thermodynamically highly dependent on the dissociation equilibrium that can be reached in a single round. Extreme shifting of equilibrium towards dissociation favors the retention of high-affinity ligands over those with lower affinity, thus improving the selection efficiency. We propose to synergize dual effects by deterministic lateral-displacement microfluidics, including the collision-based force effect and the two-dimensional (2D) separation-based concentration effect, to greatly shift the equilibrium. Compared with previous approaches, this system can remove more low- or moderate-affinity ligands and maintain most high-affinity ligands, thereby improving affinity discrimination in selection. This strategy is demonstrated on phage display in both experiment and simulation, and two peptides against tumor markers ephrin type-A receptor 2 (EphA2) and CD71 were obtained with high affinity and specificity within a single round of selection, which offers a promising direction for discovery of robust binding ligands for a wide range of biomedical applications.

Unlike conventional bulk measurements, single-cell protein analysis permits quantification of protein expression in individual cells. This has shed light on the cell-to-cell variation in heterogeneous biological systems, such as solid tumors, brain tissues, and developing embryos. Herein, a microfluidic method is developed to profile protein expression in individual cells by performing single-cell intracellular protein immunoassay in picoliter paired droplets. The high sensitivity of single-cell protein analysis on a chip is achieved by the confined reaction volume of picoliter droplets, efficient kinetic characteristics of the immunoassay through active mixing, and minimum single-cell protein loss by integrated operations. The abundance of an intracellular prostate specific antigen at the single-cell level is measured, and then the platform is applied to identify cell types and investigate heterogeneity within cell populations. Overall, a paired chip for single-cell immunoassay establishes a foundation for parallel, sensitive, and integrated protein quantification at the single-cell level and will find wide applications in the field of single-cell proteomics.

The engineering strategy of artificial biointerfaces is vital for governing their performances in bioanalysis and diagnosis. Highly ordered arrangement of affinity ligands on the interface surface facilitates efficient interaction with target molecules, whereas biointerfaces aimed at drug delivery or rare cell isolation require sophisticated stimuli-response mechanisms. However, it is still challenging to facilely fabricate biointerfaces possessing the two features. Herein, we endow a biointerface with both reversibility and capability to orderly assemble affinity ligands by introducing boronic acid moieties alone. By boronate conjugation via glycosylation sites, avidin was well arranged at the surface of boronic acid-decorated carbon nitride nanosheets for the assembly of biotinylated aptamers. The ordered orientation of aptamers largely relieved their inactivation caused by inter-strand entanglement, facilitating significant increase in cell affinity for the isolation of circulating tumor cells (CTCs). The reversible boronate conjugation also facilitated mild release of CTCs by acid fructose with high cell viability. This engineered interface was capable of isolating CTCs from the peripheral blood of tumor-bearing mice and cancer patients. The successful utilization of the isolated CTCs in the downstream drug susceptibility test and mutation analysis demonstrated the clinical potential of this biointerface for the early diagnosis of cancers and precision medicine.

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.

Circulating tumor cells (CTCs), as important liquid biopsy target, can provide valuable information for cancer progress monitoring and individualized treatment. However, current isolation platforms incapable of balancing capture efficiency, specificity, cell viability, and gentle release have restricted the clinical applications of CTCs. Herein, inspired by the structure and functional merits of natural membrane interfaces, we established an antibody-engineered red blood cell (RBC-Ab) affinity interface on microfluidic chip for high-performance isolation and release of CTCs. The lateral fluidity, pliability, and anti-adhesion property of the RBC microfluidic interface enabled efficient CTCs capture (96.5%), high CTCs viability (96.1%), and high CTCs purity (average 4.2-log depletion of leukocytes). More importantly, selective lysis of RBCs by simply changing the salt concentration was utilized to destroy the affinity interface for efficient and gentle release of CTCs without nucleic acid contamination. Using this chip, CTCs were successfully detected in colon cancer samples with 90% sensitivity and 100% specificity (20 patients and 10 healthy individuals). After the release process, KRAS gene mutations of CTCs were identified from all the 5 cancer samples, which was consistent with the results of tissue biopsy. We expect this RBC interface strategy will inspire further biomimetic interface construction for rare cell analysis.

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.

Because of its high involvement in antibiotic therapy and the emergence of drug-resistance, the chemical structure and biosynthesis of bacterial peptidoglycan (PGN) have been some of the key topics in bacteriology for several decades. Recent advances in the development of fluorescent or bio-orthogonal stem peptide-mimicking probes for PGN-labeling have rekindled the interest of chemical biologists and microbiologists in this area. The structural designs, bio-orthogonal features and flexible uses of these peptide-based probes allow directly assessing, not only the presence of PGN in different biological systems, but also specific steps in PGN biosynthesis. In this review, we summarize the design rationales, functioning mechanisms, and microbial processes/questions involved in these PGN-targeting probes. Our perspectives on the limitations and future development of these tools are also presented.

Photothermal biosensors with advantages of speed and high sensitivity offer alternative and reliable solutions for real-time clinical diagnosis, food safety testing and environmental monitoring. Although metallic nanoparticles are usually used for photothermal biosensors, their poor photothermal stability and potential toxicity hinder clinical applications. Taking advantage of the low cytotoxicity and remarkable photothermal effect under the low laser power of polypyrrole-based organic nanoparticles, we developed a novel photothermal biosensor with a temperature and pressure dual readout. After the formation of immunoassay sandwich structures, polypyrrole as the photothermal agent is synthesized

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.

Applications in health diagnostics, community safety and environmental monitoring require selective and quantitatively accurate active viral load assessment. Herein, we report a microfluidic enrichment strategy to separate intact SARS-CoV-2 particles by the AND logic gate with the inputs of cholesterol oligonucleotides for the envelope and aptamers for the spike. Considering the unequal quantity of endogenous spike and lipid membrane on SARS-CoV-2, a dual-domain binding strate-gy based on two aptamers targeting two spike domains was applied to balance the spike-envelope stoichiometric ratio. With the stoichiometric balanced DNA computation and the promoted microscale mass transfer of herringbone chip, the devel-oped strategy enables sensitively detection of pseudotyped SARS-CoV-2 with a limit of detection as low as 34 active virion/μL, but also distinguishes from their inactive counterparts, other nontarget viruses and free spike. Moreover, the captured viral particles can be released through DNase I treatment with up to 90 % efficiency, which is fully compatible with virus culture and sequencing. Overall, the developed strategy not only 100% identifies healthy donors (n = 8) and patients (n = 14) with SARS-CoV-2 infection, but also provides a novel perspective on regulating of stoichiometric ratio to achieve a more biologically appropriate DNA computation.

BACKGROUND Analyses of immune cell subsets and immune indices are gaining in importance. By combining molecular probe and immunohistochemical (IHC) analysis with laser ablation, the imaging mass cytometry (IMC) offers spatially high-dimensional in situ measurements in tissue slides with a cell resolution of 1µm. The most critical challenge is how to implement stringent quality control strategies during application of IMC to facilitate reproducible data analysis. METHODS We discussed the multi-step experimental processing procedures of IMC, including specimen preparation, panel design, antibody selection, lanthanide metal labeling and data pre-processing workflows by IHC and fluorescent multiplex immunohistochemistry (mIHC) analysis. Based on IMC, we developed a standard operation and well-established agreement for 29 BC patients to identify a structured tumor immune microenvironment. RESULTS Metal labeling does not affect the antibodies target specificity (p > 0.05). The 3 µm-thick formalin-fixed and paraffin-embedded (FFPE) and fresh-frozen slides exhibited the least blur (p

Single-cell RNA sequencing (scRNA-seq) reveals the transcriptional heterogeneity of cells, but the static snapshots fail to reveal the time-resolved dynamics of transcription. Herein, we develop Well-TEMP-seq, a high-throughput, cost-effective, accurate, and efficient method for massively parallel profiling the temporal dynamics of single-cell gene expression. Well-TEMP-seq combines metabolic RNA labeling with scRNA-seq method Well-paired-seq to distinguish newly transcribed RNAs marked by T-to-C substitutions from pre-existing RNAs in each of thousands of single cells. The Well-paired-seq chip ensures a high single cell/barcoded bead pairing rate (~80%) and the improved alkylation chemistry on beads greatly alleviates chemical conversion-induced cell loss (~67.5% recovery). We further apply Well-TEMP-seq to profile the transcriptional dynamics of colorectal cancer cells exposed to 5-AZA-CdR, a DNA-demethylating drug. Well-TEMP-seq unbiasedly captures the RNA dynamics and outperforms the splicing-based RNA velocity method. We anticipate that Well-TEMP-seq will be broadly applicable to unveil the dynamics of single-cell gene expression in diverse biological processes.

Extracellular vesicle (EV)/exosome secretion is a dynamic process that tunes the cellular communication for response to internal and external cues. The selective enrichment of a newly synthesized EV/exosome has been hindered by the basic fact that all EVs/exosomes, new and old, share the similar inherent parameters and thus are indistinguishable. Here, we developed a method by cotranslational introduction of azide groups into EV/exosome proteins as a timestamp and label them with biotin tag by click chemistry, to separate the newly synthesized EVs/exosomes from preexisting populations by streptavidin-modified herringbone microfluidic chip. For mouse model of anti-PD-L1 immunotherapy, the level of newly synthesized PD-L1+ EVs detected by the developed approach was superior to the total PD-L1+ EVs from mixed time sources (quantified by classical method) for tumor progression. This method makes it possible to address the temporal characteristics of newly synthesized EVs/exosomes in cell and in vivo, for studying EV/exosome secretion to respond to specific stimuli.

MicroRNAs (miRNAs) have been extensively studied as non-invasive biomarkers for cancer diagnosis and prognosis, while the clinical application was constrained by the heterogeneous miRNA sources in plasma and the tedious assay processes. Here we developed a one-pot assay called dual-Surface-protein-guided Orthogonal Recognition of Tumor-derived Exosomes and in-situ profiling of microRNAs (SORTER) for rapid and precise diagnosis of prostate cancer. The SORTER utilizes the orthogonal barcoding of two allosteric aptamers against exosomal marker CD63 and tumor marker EpCAM to recognize and sort tumor-derived exosome subtypes. Furthermore, the labeled barcode on tumor-derived exosomes guided the targeted fusion with liposome miRNA detection probes, enabling in-situ profiling of tumor-derived exosomal miRNAs. With a signature of six miRNAs, SORTER differentiated prostate cancer and benign prostatic hyperplasia with a sensitivity, specificity, and accuracy of 100% in the training and validation cohorts. The SORTER provides a promising tool to advance the clinical adaptability of miRNA-based liquid biopsy.

Real-time localization and microbial activity information of indigenous gut microbiota over an extended period of time remains a challenge with existing visualizing methods. Here, we report a metabolic fluorine labeling (MEFLA)–based strategy for monitoring the dynamic gut microbiota via 19 F magnetic resonance imaging ( 19 F MRI). In situ labeling of different microbiota subgroups is achieved by using a panel of peptidoglycan-targeting MEFLA probes containing 19 F atoms of different chemical shifts, and subsequent real-time in vivo imaging is accomplished by multiplexed hotspot 19 F MRI with high sensitivity and unlimited penetration. Using this method, we realize extended visualization (>24 hours) of native gut microbes located at different intestinal sections and semiquantitative analysis of their metabolic dynamics modulated by various conditions, such as the host death and different β-lactam antibiotics. Our strategy holds great potential for noninvasive and real-time assessing of the metabolic activities and locations of the highly dynamic gut microbiota.

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.

Immune checkpoint therapy has provided a weapon against cancer, but its response rate has been extremely low due to the lack of effective predictors. Herein, we developed a F RET strategy based on l ectin for glycan labeling and an a ptamer for PD-L1 antigen recognition for visualization of PD-L1-specific g lycosylation (FLAG). The FLAG strategy combines the PD-L1 aptamer, which efficiently labels the PD-L1 polyantigen with smaller steric hindrance than the PD-L1 antibody, and metabolism-free lectin labeling for glycosylation. As a result, the FLAG strategy enables in situ visualization of PD-L1-specific glycosylation on the tissue section while maintaining the spatial context and tissue architecture. Due to nonmetabolic labeling, the FLAG strategy revealed that the tissue level of PD-L1-specific glycosylation is correlated with the efficacy of PD-1/PD-L1 therapy. Overall, the FLAG strategy provides a powerful tool for revealing the significance of PD-L1 glycosylation, offering the unprecedented potential for immunophenotypic differential analysis to predict the immunotherapy response.

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.

The multivalent effect is often used to engineer microfluidic affinity interfaces to improve the target separation efficiency. Currently, no design rules exist for thermodynamic and kinetic tuning of properly joining multiple ligands. Herein, we developed a thermodynamic and kinetic modulating strategy of the microfluidic affinity interface via a merit-complementary-heteromultivalent aptamers functionalized DNA nanoassembly. Our strategy is built on the two types of identified aptamers that bind to distinct sites of EpCAM. The aptamer binding of one type is more rapid but less tight, while the other is opposite. By assembling the two types of aptamers together with a tetrahedral DNA framework, we fully exploited these aptamers' merits for tight and rapid recognition of EpCAM, leading to target cell capture with high efficiency and throughput. Our strategy provides a perspective on engineering multivalent recognition molecules through thermodynamic and kinetic tuning.

The high resolution, deep penetration, and negligible biological background of