Ultra-sensitive and rapid detection of Salmonella enterica and Staphylococcus aureus to single-cell level by aptamer-functionalized carbon nanotube field-effect transistor biosensors.

Foodborne diseases caused by Salmonella enterica (S. enterica) and Staphylococcus aureus (S. aureus) significantly impact public health, underscoring the imperative for highly sensitive, rapid, and accurate detection technologies to ensure food safety and prevent human diseases. Nanomaterials hold great promise in the development of high-sensitivity transistor biosensors. In this work, field-effect transistor (FET) comprising high-purity carbon nanotubes (CNTs) were fabricated and modified with corresponding nucleic acid aptamers for the high-affinity and selective capture of S. enterica and S. aureus. The aptamer-functionalized CNT-FET biosensor demonstrated ultra-sensitive and rapid detection of these foodborne pathogens. Experimental results indicated that the biosensor could detect S. enterica at a limit of detection (LOD) as low as 1 CFU in PBS buffer, and S. aureus at an LOD of 1.2 CFUs, achieving single-cell level detection accuracy with exceptional specificity. The biosensor exhibited a rapid response time, completing single detections within 200 s. Even in the presence of interference from six complex food matrices, the biosensor maintained its ultra-sensitive (3.1 CFUs) and rapid response (within 200 s) characteristics for both pathogens. The developed aptamer-functionalized CNT-FET biosensor demonstrates a capability for low-cost, ultra-sensitive, label-free, and rapid detection of low-abundance S. enterica and S. aureus in both buffer solutions and complex environments. This innovation holds significant potential for applying this detection technology to on-site rapid testing scenarios, offering a promising solution to the pressing need for efficient and reliable pathogen monitoring in various settings.

Dielectrophoresis-Assisted Self-Digitization Chip for High-Efficiency Single-Cell Analysis.

Single-cell analysis of cell phenotypic information such as surface protein expression and nucleic acid content is essential for understanding heterogeneity within cell populations. Here the design and use of a dielectrophoresis-assisted self-digitization (SD) microfluidics chip is described; it captures single cells in isolated microchambers with high efficiency for single-cell analysis. The self-digitization chip spontaneously partitions aqueous solution into microchambers through a combination of fluidic forces, interfacial tension, and channel geometry. Single cells are guided to and trapped at the entrances of microchambers by dielectrophoresis (DEP) due to local electric field maxima created by an externally applied AC voltage. Excess cells are flushed away, and trapped cells are released into the chambers and prepared for in situ analysis by turning off the external voltage, by running reaction buffer through the chip, and by sealing the chambers with a flow of an immiscible oil phase through the surrounding channels. The use of this device in single-cell analysis is demonstrated by performing single-cell nucleic acid quantitation based on loop-mediated isothermal amplification (LAMP). This platform provides a powerful new tool for single-cell research pertaining to drug discovery. For example, the single-cell genotyping of cancer-related mutant gene observed from the digital chip could be useful biomarker for targeted therapy.

Discrimination of Multidrug Resistance in Cancer Cells Achieved Using Single-Cell Analysis.

The biophysical signatures of single cells, such as multidrug resistance (MDR), may easily change during their various disease states. Therefore, there is an ever-growing need for advanced methods to study and analyze the response of cancer cells to therapeutic intervention. To determine the cancer cells and responses to various cancer therapies, from a cell mortality perspective, we report a label-free and real-time method to monitor the in situ responses of ovarian cancer cells using a single-cell bioanalyzer (SCB). The SCB instrument was used to detect different ovarian cancer cells, such as NCI/ADR-RES cells, which are multidrug resistant (MDR), and non-MDR OVCAR-8 cells. The discrimination of ovarian cells has been achieved at the single-cell level by measuring drug accumulation quantitatively in real time, in which the accumulation is high in non-MDR single cells without drug efflux but is low in MDR single cells which are not efflux-free. The SCB was constructed as an inverted microscope for optical imaging and fluorescent measurement of a single cell that was retained in a microfluidic chip. The single ovarian cancer cell retained in the chip offered sufficient fluorescent signals for the SCB to measure the accumulation of daunorubicin (DNR) in the single cell in the absence of cyclosporine A (CsA). The same cell allows us to detect the enhanced drug accumulation due to MDR modulation in the presence of CsA, which is the MDR inhibitor. The measurement of drug accumulation in a cell was achieved after it was captured in the chip for one hour, with the correction of background interference. The detection of accumulation enhancement due to MDR modulation by CsA was determined in terms of either the accumulation rate or enhanced concentration of DNR in the single cell (same cell, p < 0.01). It showed that with the effectiveness of efflux blocking by CsA, the intracellular DNR concentration in a single cell increased by threefold against its same cell control. This single-cell bioanalyzer instrument has the ability to discriminate MDR in different ovarian cells due to drug efflux in them by eliminating the interference of background fluorescence and by using the same cell control.

Single-Cell Analysis of circRNA Using ddPCR.

Droplet digital polymerase chain reaction (ddPCR) is a new quantitative PCR method based on water-oil emulsion droplet technology. ddPCR enables highly sensitive and accurate quantification of nucleic acid molecules, especially when their copy numbers are low. In ddPCR, a sample is fractionated into ~20,000 droplets, and every nanoliter-sized droplet undergoes PCR amplification of the target molecule. The fluorescence signals of droplets are then recorded by an automated droplet reader. Circular RNAs (circRNAs) are single-stranded, covalently closed RNA molecules that are ubiquitously expressed in animals and plants. CircRNAs are promising as biomarkers for cancer diagnosis and prognosis and as therapeutic targets or agents to inhibit oncogenic microRNAs or proteins (Kristensen LS, Jakobsen T, Hager H, Kjems J, Nat Rev Clin Oncol 19:188-206, 2022). In this chapter, the procedures for the quantitation of a circRNA in single pancreatic cancer cells using ddPCR are described.

Cellular microarrays for assessing single-cell phenotypic changes in vascular cell populations.

Microengineering technologies provide bespoke tools for single-cell studies, including microarray approaches. There are many challenges when culturing adherent single cells in confined geometries for extended periods, including the ability of migratory cells to overcome confining cell-repellent surfaces with time. Following studies suggesting clonal expansion of only a few vascular smooth muscle cells (vSMCs) contributes to plaque formation, the investigation of vSMCs at the single-cell level is central to furthering our understanding of atherosclerosis. Herein, we present a medium throughput cellular microarray, for the tracking of single, freshly-isolated vSMCs as they undergo phenotypic modulation in vitro. Our solution facilitates long-term cell confinement (> 3 weeks) utilising novel application of surface functionalisation methods to define individual culture microwells. We demonstrate successful tracking of hundreds of native vSMCs isolated from rat aortic and carotid artery tissue, monitoring their proliferative capacity and uptake of oxidised low-density lipoprotein (oxLDL) by live-cell microscopy. After 7 days in vitro, the majority of viable SMCs remained as single non-proliferating cells (51% aorta, 78% carotid). However, a sub-population of vSMCs demonstrated high proliferative capacity (≥ 10 progeny; 18% aorta, 5% carotid), in line with reports that a limited number of medial SMCs selectively expand to populate atherosclerotic lesions. Furthermore, we show that, when exposed to oxLDL, proliferative cells uptake higher levels of lipoproteins, whilst also expressing greater levels of galectin-3. Our microwell array approach enables long-term characterisation of multiple phenotypic characteristics and the identification of new cellular sub-populations in migratory, proliferative adherent cell types.

Automated and Miniaturized Pico-Liter Metabolite Extraction System for Single-Cell Mass Spectrometry.

OBJECTIVE: Mass spectrometry has become the method of choice for single cell analysis due to its high sensitivity of detection and capability in analyzing a large number of metabolites simultaneously. For a long time, an automated and miniaturized system capable of extracting cellular contents from single cells at the pico-liter level for pico-ESI analysis has been lacking. METHODS: This paper presents a first-of-its-kind automated and miniaturized pico-liter extraction system for single-cell MS. The key modules, including imaging, bus controller, and fluidic driving are customized to achieve satisfactory performance at affordable costs, resulting in a miniaturized system movable on a trolley and connectable with the MS. To enable automation, a single cell trapping device, new image-based one-pixel accuracy positioning methods for cells and micropipette, and a surface-tension-based 1-pL accuracy volume control scheme are developed. RESULTS: The system is able to control the solvent loading at 1.97 ± 0.05 nL, solvent dispensing at 14-15 pL, and solvent evaporation at 689±48 pL. MS experiments demonstrate a throughput of 20 cells/h. CONCLUSION: The system has achieved better performance in consistency (∼21%), sensitivity (∼28%), and success rate (up to 40%) than manual operation. SIGNIFICANCE: This automated and miniaturized system lays a solid basis for applying pico-ESI MS analysis in the automated and high-throughput single cell MS analysis, such as single-cell metabolomics and lipidomics.

A single-cell mass cytometry platform to map the effects of preclinical drugs on cartilage homeostasis.

No disease-modifying drug exists for osteoarthritis (OA). Despite success in animal models, candidate drugs continue to fail in clinical trials owing to the unmapped interpatient heterogeneity and disease complexity. We used a single-cell platform based on cytometry by time-of-flight (cyTOF) to precisely outline the effects of candidate drugs on human OA chondrocytes. OA chondrocytes harvested from patients undergoing total knee arthroplasty were treated with 2 drugs, an NF-κB pathway inhibitor, BMS-345541, and a chondroinductive small molecule, kartogenin, that showed preclinical success in animal models for OA. cyTOF conducted with 30 metal isotope-labeled antibodies parsed the effects of the drugs on inflammatory, senescent, and chondroprogenitor cell populations. The NF-κB pathway inhibition decreased the expression of p-NF-κB, HIF2A, and inducible NOS in multiple chondrocyte clusters and significantly depleted 4 p16ink4a-expressing senescent populations, including NOTCH1+STRO1+ chondroprogenitor cells. While kartogenin also affected select p16ink4a-expressing senescent clusters, there was a less discernible effect on chondroprogenitor cell populations. Overall, BMS-345541 elicited a uniform drug response in all patients, while only a few responded to kartogenin. These studies demonstrate that a single-cell cyTOF-based drug screening platform can provide insights into patient response assessment and patient stratification.

Confined Nanopipet as a Versatile Tool for Precise Single Cell Manipulation.

The precise manipulation of single cells plays a fundamental role for single cell measurement, which is crucial for understanding the diverse cellular mechanisms. Unusual single cell behavior could thus be identified by integrating with advanced analytical methods such as single cell omics, unraveling the intrinsic cellular heterogeneity hidden in ensemble measurements. Herein, this technical note reports a nanopipet-based versatile method for manipulation of an ultrasmall volume of liquid, which further enables the precise manipulation of single cells. Femtoliter volumes of cytoplasm were extracted from single living cells and analyzed by time-of-flight secondary ion mass spectrometry. Moreover, several kinds of exogenous components were injected simultaneously into a cell, offering a delicate tool for multi-imaging in single living cells.

Upstart DNA sequencers could be a 'game changer'.

Increasing potential for fast, cheap genomes may break open biology's bottleneck and broaden clinical uses.

Molecular mechanisms governing circulating immune cell heterogeneity across different species revealed by single-cell sequencing.

BACKGROUND: Immune cells play important roles in mediating immune response and host defense against invading pathogens. However, insights into the molecular mechanisms governing circulating immune cell diversity among multiple species are limited. METHODS: In this study, we compared the single-cell transcriptomes of immune cells from 12 species. Distinct molecular profiles were characterized for different immune cell types, including T cells, B cells, natural killer cells, monocytes, and dendritic cells. RESULTS: Our data revealed the heterogeneity and compositions of circulating immune cells among 12 different species. Additionally, we explored the conserved and divergent cellular crosstalks and genetic regulatory networks among vertebrate immune cells. Notably, the ligand and receptor pair VIM-CD44 was highly conserved among the immune cells. CONCLUSIONS: This study is the first to provide a comprehensive analysis of the cross-species single-cell transcriptome atlas for peripheral blood mononuclear cells (PBMCs). This research should advance our understanding of the cellular taxonomy and fundamental functions of PBMCs, with important implications in evolutionary biology, developmental biology, and immune system disorders.

High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip.

Global quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements. However, single-cell proteomics is still immature and confronts many technical challenges. Herein we describe a nested nanoPOTS (N2) chip to improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip reduces reaction volume to <30 nL and increases capacity to >240 single cells on a single microchip. The tandem mass tag (TMT) pooling step is simplified by adding a microliter droplet on the nested nanowells to combine labeled single-cell samples. In the analysis of ~100 individual cells from three different cell lines, we demonstrate that the N2 chip-based scProteomics platform can robustly quantify ~1500 proteins and reveal membrane protein markers. Our analyses also reveal low protein abundance variations, suggesting the single-cell proteome profiles are highly stable for the cells cultured under identical conditions.

Advances in single-cell sequencing: insights from organ transplantation.

Single-cell RNA sequencing (scRNA-seq) is a comprehensive technical tool to analyze intracellular and intercellular interaction data by whole transcriptional profile analysis. Here, we describe the application in biomedical research, focusing on the immune system during organ transplantation and rejection. Unlike conventional transcriptome analysis, this method provides a full map of multiple cell populations in one specific tissue and presents a dynamic and transient unbiased method to explore the progression of allograft dysfunction, starting from the stress response to final graft failure. This promising sequencing technology remarkably improves individualized organ rejection treatment by identifying decisive cellular subgroups and cell-specific interactions.

A Fluorescent Real-Time Plaque Assay Enables Single-Cell Analysis of Virus-Induced Cytopathic Effect by Live-Cell Imaging.

Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to countable plaques that are visualized with standard techniques such as crystal violet, neutral red, or immunolabeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA fluorescent dyes with differential cell permeability to visualize them by live-cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identification using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This fluorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern fluorescence microscopy and image analysis approaches for future applications in virology.

Single-cell immunoblotting resolves estrogen receptor-α isoforms in breast cancer.

An array of isoforms of the nuclear estrogen receptor alpha (ER-α) protein contribute to heterogeneous response in breast cancer (BCa); yet, a single-cell analysis tool that distinguishes the full-length ER-α66 protein from the activation function-1 deficient ER-α46 isoform has not been reported. Specific detection of protein isoforms is a gap in single-cell analysis tools, as the de facto standard immunoassay requires isoform-specific antibody probes. Consequently, to scrutinize hormone response heterogeneity among BCa tumor cells, we develop a precision tool to specifically measure ER-α66, ER- α46, and eight ER-signaling proteins with single-cell resolution in the highly hetero-clonal MCF-7 BCa cell line. With a literature-validated pan-ER immunoprobe, we distinguish ER-α66 from ER-α46 in each individual cell. We identify ER-α46 in 5.5% of hormone-sensitive (MCF-7) and 4.2% of hormone-insensitive (MDA-MB-231) BCa cell lines. To examine whether the single-cell immunoblotting can capture cellular responses to hormones, we treat cells with tamoxifen and identify different sub-populations of ER-α46: (i) ER-α46 induces phospho-AKT at Ser473, (ii) S6-ribosomal protein, an upstream ER target, activates both ER-α66 and ER-α46 in MCF-7 cells, and (iii) ER-α46 partitions MDA-MB-231 subpopulations, which are responsive to tamoxifen. Unlike other single-cell immunoassays, multiplexed single-cell immunoblotting reports-in the same cell-tamoxifen effects on ER signaling proteins and on distinct isoforms of the ER-α protein.

High-precision targeting workflow for volume electron microscopy.

Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)-SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.

A design and optimization of a high throughput valve based microfluidic device for single cell compartmentalization and analysis.

The need for high throughput single cell screening platforms has been increasing with advancements in genomics and proteomics to identify heterogeneity, unique cell subsets or super mutants from thousands of cells within a population. For real-time monitoring of enzyme kinetics and protein expression profiling, valve-based microfluidics or pneumatic valving that can compartmentalize single cells is advantageous by providing on-demand fluid exchange capability for several steps in assay protocol and on-chip culturing. However, this technique is throughput limited by the number of compartments in the array. Thus, one big challenge lies in increasing the number of microvalves to several thousand that can be actuated in the microfluidic device to confine enzymes and substrates in picoliter volumes. This work explores the design and optimizations done on a microfluidic platform to achieve high-throughput single cell compartmentalization as applied to single-cell enzymatic assay for protein expression quantification. Design modeling through COMSOL Multiphysics was utilized to determine the circular microvalve's optimized parameters, which can close thousands of microchambers in an array at lower sealing pressure. Multiphysical modeling results demonstrated the relationships of geometry, valve dimensions, and sealing pressure, which were applied in the fabrication of a microfluidic device comprising of up to 5000 hydrodynamic traps and corresponding microvalves. Comparing the effects of geometry, actuation media and fabrication technique, a sealing pressure as low as 0.04 MPa was achieved. Applying to single cell enzymatic assay, variations in granzyme B activity in Jurkat and human PBMC cells were observed. Improvement in the microfluidic chip's throughput is significant in single cell analysis applications, especially in drug discovery and treatment personalization.

Dielectric nanohole array metasurface for high-resolution near-field sensing and imaging.

Dielectric metasurfaces support resonances that are widely explored both for far-field wavefront shaping and for near-field sensing and imaging. Their design explores the interplay between localised and extended resonances, with a typical trade-off between Q-factor and light localisation; high Q-factors are desirable for refractive index sensing while localisation is desirable for imaging resolution. Here, we show that a dielectric metasurface consisting of a nanohole array in amorphous silicon provides a favourable trade-off between these requirements. We have designed and realised the metasurface to support two optical modes both with sharp Fano resonances that exhibit relatively high Q-factors and strong spatial confinement, thereby concurrently optimizing the device for both imaging and biochemical sensing. For the sensing application, we demonstrate a limit of detection (LOD) as low as 1 pg/ml for Immunoglobulin G (IgG); for resonant imaging, we demonstrate a spatial resolution below 1 µm and clearly resolve individual E. coli bacteria. The combined low LOD and high spatial resolution opens new opportunities for extending cellular studies into the realm of microbiology, e.g. for studying antimicrobial susceptibility.

3D mechanical characterization of single cells and small organisms using acoustic manipulation and force microscopy.

Quantitative micromechanical characterization of single cells and multicellular tissues or organisms is of fundamental importance to the study of cellular growth, morphogenesis, and cell-cell interactions. However, due to limited manipulation capabilities at the microscale, systems used for mechanical characterizations struggle to provide complete three-dimensional coverage of individual specimens. Here, we combine an acoustically driven manipulation device with a micro-force sensor to freely rotate biological samples and quantify mechanical properties at multiple regions of interest within a specimen. The versatility of this tool is demonstrated through the analysis of single Lilium longiflorum pollen grains, in combination with numerical simulations, and individual Caenorhabditis elegans nematodes. It reveals local variations in apparent stiffness for single specimens, providing previously inaccessible information and datasets on mechanical properties that serve as the basis for biophysical modelling and allow deeper insights into the biomechanics of these living systems.

Rolling angle recovery of flowing cells in holographic tomography exploiting the phase similarity.

Holographic tomography allows the 3D mapping of the refractive index of biological samples thanks to reconstruction methods based on the knowledge of illumination directions or rotation angles of the imaged sample. Recently, phase contrast tomographic flow cytometry by digital holography has been demonstrated to reconstruct the three-dimensional refractive index distribution of single cells while they are flowing along microfluidic channels. In this system, the illumination direction is fixed while the sample's rotation is not deterministically known a priori but induced by hydrodynamic forces. We propose here a technique to retrieve the rolling angles, based on a new phase images similarity metric that is capable of identifying a cell's orientations from its 3D positioning while it is flowing along the microfluidic channel. The method is experimentally tested and also validated through appropriate numerical simulations. We provide demonstration of concept by achieving reconstruction of breast cancer cells tomography.