Genetic and phenotypic profiling of single living circulating tumor cells from patients with microfluidics.

Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non-small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction.

Massively multiplexed nucleic acid detection with Cas13.

The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples1-3 while simultaneously testing for many pathogens4-6. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents7 self-organize in a microwell array8 to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health9-11.

Advancing Point-of-Care Applications with Droplet Microfluidics: From Single-Cell to Multicellular Analysis.

Recent advances in single-cell and multicellular microfluidics technology have provided powerful tools for studying cancer biology and immunology. The ability to create controlled microenvironments, perform high-throughput screenings, and monitor cellular interactions at the single-cell level has significantly advanced our understanding of tumor biology and immune responses. We discuss cutting-edge multicellular and single-cell microfluidic technologies and methodologies utilized to investigate cancer-immune cell interactions and assess the effectiveness of immunotherapies. We explore the advantages and limitations of the wide range of 3D spheroid and single-cell microfluidic models recently developed, highlighting the various approaches in device generation and applications in immunotherapy screening for potential opportunities for point-of-care approaches.

Multiomics Characterization of a Less Invasive Microfluidic-Based Cell Sorting Technique.

Fluorescence-activated cell sorting (FACS) is a specialized technique to isolate specific cell subpopulations with a high level of recovery and accuracy. However, the cell sorting procedure can impact the viability and metabolic state of cells. Here, we performed a comparative study and evaluated the impact of traditional high-pressure charged droplet-based and microfluidic chip-based sorting on the metabolic and phosphoproteomic profile of different cell types. While microfluidic chip-based sorted cells more closely resembled the unsorted control group for most cell types tested, the droplet-based sorted cells showed significant metabolic and phosphoproteomic alterations. In particular, greater changes in redox and energy status were present in cells sorted with the droplet-based cell sorter along with larger shifts in proteostasis. 13C-isotope tracing analysis on cells recovering postsorting revealed that the sorter-induced suppression of mitochondrial TCA cycle activity recovered faster in the microfluidic chip-based sorted group. Apart from this, amino acid and lipid biosynthesis pathways were suppressed in sorted cells, with minimum impact and faster recovery in the microfluidic chip-based sorted group. These results indicate microfluidic chip-based sorting has a minimum impact on metabolism and is less disruptive compared to droplet-based sorting.

Stabilizing and Accelerating Secondary Flow in Ultralong Spiral Channel for High-Throughput Cell Manipulation.

Efficient cell manipulation is essential for numerous applications in bioanalysis and medical diagnosis. However, the lack of stability and strength in the secondary flow, coupled with the narrow range of practical throughput, severely restricts the diverse applications. Herein, we present an innovative inertial microfluidic device that employs a spiral channel for high-throughput cell manipulation. Our investigation demonstrates that the regulation of Dean-like secondary flow in the microchannel can be achieved through geometric confinement. Introducing ordered microstructures into the ultralong spiral channel (>90 cm) stabilizes and accelerates the secondary flow among different loops. Consequently, effective manipulation of blood cells within a wide cell throughput range (1.73 × 108 to 1.16 × 109 cells/min) and cancer cells across a broad throughput range (0.5 × 106 to 5 × 107 cells/min) can be achieved. In comparison to previously reported technologies, our engineering approach of stabilizing and accelerating secondary flow offers specific performance for cell manipulation under a wide range of high-throughput manner. This engineered spiral channel would be promising in biomedical analysis, especially when cells need to be focused efficiently on large-volume liquid samples.

Automated Droplet Ejection from a Digital Microfluidics Sample Pretreatment Device Enables Batch-Mode Chemiluminescence Immunoassay.

Digital microfluidics (DMF) features programmed manipulation of fluids in multiple steps, making it a valuable tool for sample pretreatment. However, the integration of sample pretreatment with its downstream reaction and detection requires transferring droplets from the DMF device to the outside world. To address this issue, the present study developed a modified DMF device that allows automated droplet ejection out of the chip, facilitated by a tailor-designed interface. A double-layered DMF microchip with an oil-filled medium was flipped over, with a liquid infusion port and a liquid expulsion port accommodated on the top working PCB plate and the bottom grounded ITO plate, respectively, to facilitate chip-to-world delivery of droplets. Using chemiluminescent immunoassay (CLIA) as an illustrative application, the sample pretreatment was programmed on the DMF device, and CLIA droplets were ejected from the chip for signal reading. In our workflow, CLIA droplets can be ejected from the DMF device through the chip-to-world interface, freeing up otherwise occupied electrodes for more sample pretreatment and enabling streamlined droplet microreactions and batch-mode operation for bioanalysis. Integrated with these interfacing portals, the DMF system achieved a single-channel throughput of 17 samples per hour, which can be further upscaled for more productive applications by parallelizing the DMF modules. The results of this study demonstrate that the droplet ejection function that is innovated in a DMF sample pretreatment microsystem can significantly improve analytical throughput, providing an approach to establishing an automated but decentralized biochemical sample preparation workstation for large-scale and continuous bioanalysis.

Multicolor-Assay-on-a-Chip Processed by Robotic Operation (MACpro) with Improved Diagnostic Accuracy for Field-Deployable Detection.

The ability to deploy decentralized laboratories with autonomous and reliable disease diagnosis holds the potential to deliver accessible healthcare services for public safety. While microfluidic technologies provide precise manipulation of small fluid volumes with improved assay performance, their limited automation and versatility confine them to laboratories. Herein, we report the utility of multicolor assay-on-a-chip processed by robotic operation (MACpro), to address this unmet need. The MACpro platform comprises a robot-microfluidic interface and an eye-in-hand module that provides flexible yet stable actions to execute tasks in a programmable manner, such as the precise manipulation of the microfluidic chip along with different paths. Notably, MACpro shows improved detection performance by integrating the microbead-based antibody immobilization with enhanced target recognition and multicolor sensing via Cu2+-catalyzed plasmonic etching of gold nanorods for rapid and sensitive analyte quantification. Using interferon-gamma as an example, we demonstrate that MACpro completes a sample-to-answer immunoassay within 30 min and achieves a 10-fold broader dynamic range and a 10-fold lower detection limit compared to standard enzyme-linked immunosorbent assays (0.66 vs 5.2 pg/mL). MACpro extends the applications beyond traditional laboratories and presents an automated solution to expand diagnostic capacity in diverse settings.

In Situ Live Monitoring of Extracellular Acidosis near Cancer Cells Using Digital Microfluidics with an Integrated Optical pH Sensor Film.

We demonstrate the live monitoring of extracellular acidification on digital microfluidics using a chip-integrated fluorescent pH sensor film. The metabolism of various types of live cells including cancer and healthy cells were investigated through recording the extracellular pH (pHe) change. An optical pH sensor array was integrated onto a digital microfluidic (DMF) interface with a diameter of 2 mm per pH-sensing spot. Miniaturized, label-free, and noninvasive monitoring of extracellular acidosis on DMF was realized within a pH range of 5.0-8.0 with good sensitivity and rapid response. The pH sensitive probe fluorescein-5-isothiocyanate was covalently bound to poly-2-hydroxyethyl methacrylate and immobilized on a circularly exposed indium tin oxide interface on the DMF top plate. The surface of the fabricated pH sensor spots was modified with polydopamine via self-polymerization. Direct cell attachment on the sensor surfaces enabled rapid pH detection near the cell membranes. Automatic medium exchange on cell-attached pH sensing sites was achieved though solution passive dispensing on DMF. The developed DMF platform was used to monitor the pHe decrease during MCF-7 and A549 cancer cell proliferation due to abnormal glycolysis metabolism. A rapid pH decrease at the pH sensing area in the presence of cancer cells could be detected within 2 min after fresh medium exchange, while no obvious pHe change was observed with HUVEC healthy cells. Real-time detection of cell acidification and cellular response to different metabolic conditions such as higher glucose levels or administered anticancer drugs was possible.

Magnetic Nanoparticle-Based Microfluidic Platform for Automated Enrichment of High-Purity Extracellular Vesicles.

Extracellular vesicles (EVs) are available in various biological fluids and have highly heterogeneous sizes, origins, contents, and functions. Rapid enrichment of high-purity EVs remains crucial for enhancing research on EVs in tumors. In this work, we present a magnetic nanoparticle-based microfluidic platform (ExoCPR) for on-chip isolation, purification, and mild recovery of EVs from cell culture supernatant and plasma within 29 min. The ExoCPR chip integrates bubble-driven micromixers and immiscible filtration assisted by surface tension (IFAST) technology. The bubble-driven micromixer enhances the mixing between immunomagnetic beads and EVs, eliminating the need for manual pipetting or off-chip oscillatory incubation. The high-purity EVs were obtained after passing through the immiscible phase interface where hydrophilic or hydrophobic impurities nonspecifically bound to SIMI were removed. The ExoCPR chip had a capture efficiency of 75.8% and a release efficiency of 62.7% for model EVs. We also demonstrated the powerful performance of the ExoCPR in isolating EVs from biological samples (>90% purity). This chip was further employed in clinical plasma samples and showed that the number of GPC3-positive EVs isolated from hepatocellular carcinoma patients was significantly higher than that of healthy individuals. This ExoCPR chip may provide a promising tool for EV-based liquid biopsy and other fundamental research.

Microfluidic Digital Immunoassay for Point-of-Care Detection of NT-proBNP from Whole Blood.

The high prevalence and economic burden of heart failure remain a challenge to global health. This lifelong disease leads to a buildup of permanent heart damage, making early detection and frequent monitoring crucial for effective treatment. N-terminal proBNP (NT-proBNP) is an important biomarker for monitoring the disease state, but current commercial and research NT-proBNP assays require phlebotomy and bulky equipment or do not satisfy clinical requirements such as sensitivity and detection thresholds. Here, we report a point-of-care (POC) compatible microfluidic digital immunoassay that can quantify the NT-proBNP concentration in a small volume of whole blood. Our automated microfluidic device takes whole blood samples mixed with biotinylated detection antibodies and passes through a plasma filter to react with a capture antibody-functionalized sensor surface. Streptavidin-coated gold nanoparticles (GNPs) are then released to mark the surface-bound single NT-proBNP immunocomplexes and recorded with bright-field microscopy. NT-proBNP concentrations in the sample are quantified via a hybrid digital/analog calibration curve. Digital counts of bound GNPs are used as readout signal at low concentrations for high sensitivity detection, and GNP pixel occupancies are used at high concentrations for extended dynamic range. With this approach, we detected NT-proBNP in the range of 8.24-10 000 pg/mL from 7 µL of whole blood in 10 min, with a limit of detection of 0.94 pg/mL. Finally, the method was validated with 15 clinical serum samples, showing excellent linear correlation (r = 0.998) with Roche's Elecsys proBNP II assay. This evidence indicates that this method holds promise for decentralized monitoring of heart failure.

Coupling Microdroplet-Based Sample Preparation, Multiplexed Isobaric Labeling, and Nanoflow Peptide Fractionation for Deep Proteome Profiling of the Tissue Microenvironment.

There is increasing interest in developing in-depth proteomic approaches for mapping tissue heterogeneity in a cell-type-specific manner to better understand and predict the function of complex biological systems such as human organs. Existing spatially resolved proteomics technologies cannot provide deep proteome coverage due to limited sensitivity and poor sample recovery. Herein, we seamlessly combined laser capture microdissection with a low-volume sample processing technology that includes a microfluidic device named microPOTS (microdroplet processing in one pot for trace samples), multiplexed isobaric labeling, and a nanoflow peptide fractionation approach. The integrated workflow allowed us to maximize proteome coverage of laser-isolated tissue samples containing nanogram levels of proteins. We demonstrated that the deep spatial proteomics platform can quantify more than 5000 unique proteins from a small-sized human pancreatic tissue pixel (∼60,000 µm2) and differentiate unique protein abundance patterns in pancreas. Furthermore, the use of the microPOTS chip eliminated the requirement for advanced microfabrication capabilities and specialized nanoliter liquid handling equipment, making it more accessible to proteomic laboratories.

Portable Microfluidic Viscometer for Formulation Development and in Situ Quality Control of Protein and Antibody Solutions.

Viscosity of protein solutions is a critical product quality attribute for protein therapeutics such as monoclonal antibodies. Here we introduce a portable single-use analytical chip-based viscometer for determining the viscosity of protein solutions using low sample volumes of 10 µL. Through the combined use of a microfluidic viscometer, a smartphone camera for image capture, and an automated data processing algorithm for the calculation of the viscosity of fluids, we enable measurement of viscosity of multiple samples in parallel. We first validate the viscometer using glycerol-water mixtures and subsequently demonstrate the ability to perform rapid characterization of viscosity in four different monoclonal antibody formulations in a broad concentration (1 to 320 mg/mL) and viscosity (1 to 600 cP) range, showing excellent agreement with values obtained by a conventional cone-plate rheometer. Not only does the platform offer benefits of viscosity measurements using minimal sample volumes, but enables higher throughput compared to gold-standard methodologies owing to multiplexing of the measurement and single-use characteristics of the viscometer, thus showing great promise in developability studies. Additionally, as our platform has the capability of performing viscosity measurements at the point of sample collection, it offers the opportunity to employ viscosity measurement as an in situ quality control of therapeutic proteins and antibodies.

Multiparameter Mechanical Phenotyping for Accurate Cell Identification Using High-Throughput Microfluidic Deformability Cytometry.

Mechanical phenotyping has been widely employed for single-cell analysis over recent years. However, most previous works on characterizing the cellular mechanical properties measured only a single parameter from one image. In this paper, the quasi-real-time multiparameter analysis of cell mechanical properties was realized using high-throughput adjustable deformability cytometry. We first extracted 12 deformability parameters from the cell contours. Then, the machine learning for cell identification was performed to preliminarily verify the rationality of multiparameter mechanical phenotyping. The experiments on characterizing cells after cytoskeletal modification verified that multiple parameters extracted from the cell contours contributed to an identification accuracy of over 80%. Through continuous frame analysis of the cell deformation process, we found that temporal variation and an average level of parameters were correlated with cell type. To achieve quasi-real-time and high-precision multiplex-type cell detection, we constructed a back propagation (BP) neural network model to complete the fast identification of four cell lines. The multiparameter detection method based on time series achieved cell detection with an accuracy of over 90%. To solve the challenges of cell rarity and data lacking for clinical samples, based on the developed BP neural network model, the transfer learning method was used for the identification of three different clinical samples, and finally, a high identification accuracy of approximately 95% was achieved.

Reducing Cathodic Drift during Isoelectric Focusing Using Microscale Immobilized pH Gradient Gels.

Microfluidic analytical tools play an important role in miniaturizing targeted proteomic assays for improved detection sensitivity, throughput, and automation. Microfluidic isoelectric focusing (IEF) can resolve proteoforms in lysate from low-to-single cell numbers. However, IEF assays often use carrier ampholytes (CAs) to establish a pH gradient for protein separation, presenting limitations like pH instability in the form of cathodic drift (migration of focused proteins toward the cathode). Immobilized pH gradient (IPG) gels reduce cathodic drift by covalently immobilizing the pH buffering components to a matrix. To our knowledge, efforts to implement IPG gels at the microscale have been limited to glass microdevices. To adapt IEF using IPGs to widely used microfluidic device materials, we introduce a polydimethylsiloxane (PDMS)-based microfluidic device and compare the microscale pH gradient stability of IEF established with IPGs, CAs, and a hybrid formulation of IPG gels and CAs (mixed-bed IEF). The PDMS-based IPG microfluidic device (µIPG) resolved analytes differing by 0.1 isoelectric point within a 3.5 mm separation lane over a 20 min focusing duration. During the 20 min duration, we observed markedly different cathodic drift velocities among the three formulations: 60.1 µm/min in CA-IEF, 2.5 µm/min in IPG-IEF (∼24-fold reduction versus CA-IEF), and 1.4 µm/min in mixed-bed IEF (∼43-fold reduction versus CA-IEF). Lastly, mixed-bed IEF in a PDMS device resolved green fluorescent protein (GFP) proteoforms from GFP-expressing human breast cancer cell lysate, thus establishing stability in lysate from complex biospecimens. µIPG is a promising and stable technique for studying proteoforms from small volumes.

High-Density Microporous Drainage-Integrating Sheath Flow Generator for Streamlining Microfluidic Cell Sorting Systems.

Tremendous efforts have been made to develop practical and efficient microfluidic cell and particle sorting systems; however, there are technological limitations in terms of system complexity and low operability. Here, we propose a sheath flow generator that can dramatically simplify operational procedures and enhance the usability of microfluidic cell sorters. The device utilizes an embedded polydimethylsiloxane (PDMS) sponge with interconnected micropores, which is in direct contact with microchannels and seamlessly integrated into the microfluidic platform. The high-density micropores on the sponge surface facilitated fluid drainage, and the drained fluid was used as the sheath flow for downstream cell sorting processes. To fabricate the integrated device, a new process for sponge-embedded substrates was developed through the accumulation, incorporation, and dissolution of PMMA microparticles as sacrificial porogens. The effects of the microchannel geometry and flow velocity on the sheath flow generation were investigated. Furthermore, an asymmetric lattice-shaped microchannel network for cell/particle sorting was connected to the sheath flow generator in series, and the sorting performances of model particles, blood cells, and spiked tumor cells were investigated. The sheath flow generation technique developed in this study is expected to streamline conventional microfluidic cell-sorting systems as it dramatically improves versatility and operability.

An Integrated Inertial-Magnetophoresis Microfluidic Chip Online-Coupled with ICP-MS for Rapid Separation and Precise Detection of Circulating Tumor Cells.

Circulating tumor cells (CTCs) are recognized as promising targets for liquid biopsy, which play an important role in early diagnosis and efficacy monitoring of cancer. However, due to the extreme scarcity of CTCs and partial size overlap between CTCs and white blood cells (WBCs), the separation and detection of CTCs from blood remain a big challenge. To address this issue, we fabricated a microfluidic chip by integrating a passive contraction-expansion array (CEA) inertial sorting zone and an active magnetophoresis zone with the trapezoidal groove and online coupled it with inductively coupled plasma mass spectrometry (ICP-MS) for rapid separation and precise detection of MCF-7 cells (as a model CTC) in blood samples. In the integrated microfluidic chip, most of the small-sized WBCs can be rapidly removed in the circular CEA inertial sorter, while the rest of the magnetically labeled WBCs can be further captured in the trapezoidal groove under the magnetic field. As a result, the rapid separation of MCF-7 cells from blood samples was achieved with an average recovery of 91.6% at a sample flow rate of 200 µL min-1. The developed online integrated inertial-magnetophoresis microfluidic chip-ICP-MS system has been applied for the detection of CTCs in real clinical blood samples with a fast analysis speed (5 min per 1 mL blood). CTCs were detected in all 24 blood samples from patients with different types of cancer, exhibiting excellent application potential in clinical diagnosis.

Microfluidic Electrochemical Integrated Sensor for Efficient and Sensitive Detection of Candida albicans.

Traditional methods for the detection of pathogenic bacteria are time-consuming, less efficient, and sensitive, which affects infection control and bungles illness. Therefore, developing a method to remedy these problems is very important in the clinic to diagnose the pathogenic diseases and guide the rational use of antibiotics. Here, microfluidic electrochemical integrated sensor (MEIS) has been investigated, functionally for rapid, efficient separation and sensitive detection of pathogenic bacteria. Three-dimensional macroporous PDMS and Au nanotube-based electrode are successfully assembled into the modeling microchip, playing the functions of "3D chaotic flow separator" and "electrochemical detector," respectively. The 3D chaotic flow separator enhances the turbulence of the fluid, achieving an excellent bacteria capture efficiency. Meanwhile, the electrochemical detector provides a quantitative signal through enzyme-linked immunoelectrochemistry with improved sensitivity. The microfluidic electrochemical integrated sensor could successfully isolate Candida albicans (C. albicans) in the range of 30-3,000,000 CFU in the saliva matrix with over 95% capture efficiency and sensitively detect C. albicans in 1 h in oral saliva samples. The integrated device demonstrates great potential in the diagnosis of oral candidiasis and is also applicable in the detection of other pathogenic bacteria.

Separation of Activated T Cells Using Multidimensional Double Spiral (MDDS) Inertial Microfluidics for High-Efficiency CAR T Cell Manufacturing.

This study introduces a T cell enrichment process, capitalizing on the size differences between activated and unactivated T cells to facilitate the isolation of activated, transducible T cells. By employing multidimensional double spiral (MDDS) inertial sorting, our approach aims to remove unactivated or not fully activated T cells post-activation, consequently enhancing the efficiency of chimeric antigen receptor (CAR) T cell manufacturing. Our findings reveal that incorporating a simple, label-free, and continuous MDDS sorting step yields a purer T cell population, exhibiting significantly enhanced viability and CAR-transducibility (with up to 85% removal of unactivated T cells and approximately 80% recovery of activated T cells); we found approximately 2-fold increase in CAR transduction efficiency for a specific sample, escalating from ∼10% to ∼20%, but this efficiency highly depends on the original T cell sample as MDDS sorting would be more effective for samples possessing a higher proportion of unactivated T cells. This new cell separation process could augment the efficiency, yield, and cost-effectiveness of CAR T cell manufacturing, potentially broadening the accessibility of this transformative therapy and contributing to improved patient outcomes.

Development and Evaluation of a Noninvasive Microfluidic-Based Paper Analytical Device for Leptospirosis Diagnosis.

Leptospirosis is a re-emerging infectious disease that presents a diagnostic enigma for clinicians with frequent misdiagnosis due to lack of rapid and accurate diagnostic tests, as the current methods are encumbered by inherent limitations. The development of a diagnostic sensor with a sample-in-result-out capability is pivotal for prompt diagnosis. Herein, we developed a microfluidic paper-based analytical device (spin-µPAD) featuring a sample-in-result-out fashion for the detection of Leptospira specific urinary biomarker, sph2 sphingomyelinase, crucial for noninvasive point-of-care testing. Fabrication of paper devices involved precise photolithography techniques, ensuring a high degree of reproducibility and replicability. By optimizing the device's configuration and protein components, a remarkable sensitivity and specificity was achieved for detecting leptospiral sph2 in urine, even at low concentrations down to 1.5 fg/mL, with an assay time of 15 min. Further, the spin-µPAD was validated with 20 clinical samples, suspected of leptospirosis including other febrile illnesses, and compared with gold standard microscopic agglutination test, culture, Lepto IgM ELISA, darkfield microscopy, and Leptocheck WB spot test. In contrast to commercial diagnostic tools, the spin-µPAD was noninvasive, rapid, easy to use, specific, sensitive, and cost-effective. The results highlight the potential of this innovative spin-µPAD for an efficient and dependable approach to noninvasive leptospirosis diagnosis, addressing critical needs in the realms of public health and clinical settings.

Nanobody-Based Microfluidic Immunosensor Chip Using Tetraphenylethylene-Derived Covalent Organic Frameworks as Aggregation-Induced Electrochemiluminescence Emitters for the Detection of Thymic Stromal Lymphopoietin.

In this letter, a sensitive microfluidic immunosensor chip was developed using tetrakis(4-aminophenyl)ethene (TPE)-derived covalent organic frameworks (T-COF) as aggregation-induced electrochemiluminescence (AIECL) emitters and nanobodies as efficient immune recognition units for the detection of thymic stromal lymphopoietin (TSLP), a novel target of asthma. The internal rotation and vibration of TPE molecules were constrained within the framework structure, forcing nonradiative relaxation to convert into pronounced radiative transitions. A camel-derived nanobody exhibited superior specificity, higher residual activity and epitope recognition postcuring compared to monoclonal antibodies. Benefiting from the affinity between silver ions (Ag+) and cytosine (C), a double-stranded DNA (dsDNA) embedded with Ag+ was modified onto the surface of TSLP. A positive correlation was obtained between the TSLP concentration (1.00 pg/mL to 4.00 ng/mL) and ECL intensity, as Ag+ was confirmed to be an excellent accelerator of the generation of free radical species. We propose that utilizing COF to constrain luminescent molecules and trigger the AIECL phenomenon is another promising method for preparing signal tags to detect low-abundance disease-related markers.