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Nature Biotechnology Nov 2023Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop...
Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop a method that does not require specialized microfluidic devices, expertise or hardware. Our approach is based on particle-templated emulsification, which allows single-cell encapsulation and barcoding of cDNA in uniform droplet emulsions with only a vortexer. Particle-templated instant partition sequencing (PIP-seq) accommodates a wide range of emulsification formats, including microwell plates and large-volume conical tubes, enabling thousands of samples or millions of cells to be processed in minutes. We demonstrate that PIP-seq produces high-purity transcriptomes in mouse-human mixing studies, is compatible with multiomics measurements and can accurately characterize cell types in human breast tissue compared to a commercial microfluidic platform. Single-cell transcriptional profiling of mixed phenotype acute leukemia using PIP-seq reveals the emergence of heterogeneity within chemotherapy-resistant cell subsets that were hidden by standard immunophenotyping. PIP-seq is a simple, flexible and scalable next-generation workflow that extends single-cell sequencing to new applications.
Topics: Humans; Animals; Mice; Microfluidics; High-Throughput Nucleotide Sequencing; Single-Cell Analysis; Genomics; Transcriptome
PubMed: 36879006
DOI: 10.1038/s41587-023-01685-z -
SLAS Discovery : Advancing Life... Jan 2017Zebrafish ( Danio rerio) is an important vertebrate model organism in biomedical research, especially suitable for morphological screening due to its transparent body...
Zebrafish ( Danio rerio) is an important vertebrate model organism in biomedical research, especially suitable for morphological screening due to its transparent body during early development. Deep learning has emerged as a dominant paradigm for data analysis and found a number of applications in computer vision and image analysis. Here we demonstrate the potential of a deep learning approach for accurate high-throughput classification of whole-body zebrafish deformations in multifish microwell plates. Deep learning uses the raw image data as an input, without the need of expert knowledge for feature design or optimization of the segmentation parameters. We trained the deep learning classifier on as few as 84 images (before data augmentation) and achieved a classification accuracy of 92.8% on an unseen test data set that is comparable to the previous state of the art (95%) based on user-specified segmentation and deformation metrics. Ablation studies by digitally removing whole fish or parts of the fish from the images revealed that the classifier learned discriminative features from the image foreground, and we observed that the deformations of the head region, rather than the visually apparent bent tail, were more important for good classification performance.
Topics: Animals; Camptothecin; Deep Learning; Neural Networks, Computer; Zebrafish
PubMed: 27613194
DOI: 10.1177/1087057116667894 -
Frontiers in Bioengineering and... 2021Microbial resource mining of electroactive microorganism (EAM) is currently methodically hampered due to unavailable electrochemical screening tools. Here, we introduce...
Microbial resource mining of electroactive microorganism (EAM) is currently methodically hampered due to unavailable electrochemical screening tools. Here, we introduce an electrochemical microwell plate (ec-MP) composed of a 96 electrochemical deepwell plate and a recently developed 96-channel multipotentiostat. Using the ec-MP we investigated the electrochemical and metabolic properties of the EAM models and with acetate and lactate as electron donor combined with an individual genetic analysis of each well. Electrochemical cultivation of pure cultures achieved maximum current densities ( ) and coulombic efficiencies () that were well in line with literature data. The co-cultivation of and led to an increased current density of of 88.57 ± 14.04 µA cm (lactate) and of 99.36 ± 19.12 µA cm (lactate and acetate). Further, a decreased time period of reaching and biphasic current production was revealed and the microbial electrochemical performance could be linked to the shift in the relative abundance.
PubMed: 35242754
DOI: 10.3389/fbioe.2021.821734 -
International Journal of Molecular... Mar 2018Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints,... (Review)
Review
Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS). In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments.
Topics: Animals; Flow Cytometry; Humans; Microfluidics; Single-Cell Analysis
PubMed: 29534489
DOI: 10.3390/ijms19030807 -
International Journal of Biological... 2020Despite significant advances in parallel single-cell RNA sequencing revealing astonishing cellular heterogeneity in many tissue types, the spatial information in the... (Comparative Study)
Comparative Study Review
Despite significant advances in parallel single-cell RNA sequencing revealing astonishing cellular heterogeneity in many tissue types, the spatial information in the tissue context remains missing. Spatial transcriptome sequencing technology is designed to distinguish the gene expression of individual cells in their original location. The technology is important for the identification of tissue function, tracking developmental processes, and pathological and molecular detection. Encoding the position information is the key to spatial transcriptomics because different methods have different encoding efficiencies and application scenarios. In this review, we focus on the latest technologies of single-cell spatial transcriptomics, including technologies based on microwell plates, barcoded bead arrays, microdissection, hybridization, and barcode targeting, as well as mixed separation-based technologies. Moreover, we compare these encoding methods for use as a reference when choosing the appropriate technology.
Topics: Gene Expression Profiling; Genomics; Humans; Sequence Analysis, RNA; Single-Cell Analysis; Transcriptome
PubMed: 32792863
DOI: 10.7150/ijbs.43887 -
Pharmaceutics Jan 2021High-throughput light scattering instruments are widely used in screening of biopharmaceutical formulations and can be easily incorporated into processes by utilizing...
High-throughput light scattering instruments are widely used in screening of biopharmaceutical formulations and can be easily incorporated into processes by utilizing multi-well plate formats. High-throughput plate readers are helpful tools to assess the aggregation tendency and colloidal stability of biological drug candidates based on the diffusion self-interaction parameter (). However, plate readers evoke issues about the precision and variability of determined data. In this article, we report about the statistical evaluation of intra- and inter-plate variability (384-well plates) for the analysis of protein and peptide solutions. ANOVA revealed no significant differences between the runs. In conclusion, the reliability and precision of was dependent on the plate position of the sample replicates and value. Positive values (57.0 mL/g, coefficients of variation () 8.9%) showed a lower variability compared to negative values (-14.8 mL/g, 13.4%). The variability of was not reduced using more data points (120 vs. 30). A analysis exclusively based on center wells showed a lower (<2%) compared to edge wells (5-12%) or a combination of edge and center wells (2-5%). We present plate designs for analysis within the early formulation development, screening up to 20 formulations consuming less than 50 mg of active pharmaceutical ingredient (API).
PubMed: 33514069
DOI: 10.3390/pharmaceutics13020172 -
Journal of Microbiology and... Nov 2014Cellulase and xylanase are main hydrolysis enzymes for the degradation of cellulosic and hemicellulosic biomass, respectively. In this study, our aim was to develop and...
Cellulase and xylanase are main hydrolysis enzymes for the degradation of cellulosic and hemicellulosic biomass, respectively. In this study, our aim was to develop and test the efficacy of a rapid, high-throughput method to screen hydrolytic-enzyme-producing microbes. To accomplish this, we modified the 3,5-dinitrosalicylic acid (DNS) method for microwell plate-based screening. Targeted microbial samples were initially cultured on agar plates with both cellulose and xylan as substrates. Then, isolated colonies were subcultured in broth media containing yeast extract and either cellulose or xylan. The supernatants of the culture broth were tested with our modified DNS screening method in a 96-microwell plate, with a 200 μl total reaction volume. In addition, the stability and reliability of glucose and xylose standards, which were used to determine the enzymatic activity, were studied at 100°C for different time intervals in a dry oven. It was concluded that the minimum incubation time required for stable color development of the standard solution is 20 min. With this technique, we successfully screened 21 and 31 cellulase- and xylanase-producing strains, respectively, in a single experimental trial. Among the identified strains, 19 showed both cellulose and xylan hydrolyzing activities. These microbes can be applied to bioethanol production from cellulosic and hemicellulosic biomass.
Topics: Bacteria; Cellulase; Colorimetry; Endo-1,4-beta Xylanases; Enzyme Assays; Fungi; Salicylates
PubMed: 25085570
DOI: 10.4014/jmb.1405.05052 -
Communications Biology Nov 2023The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent...
The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent programming of light patterns in single wells of microwell plates. However, there is currently a lack of instrumentation to monitor such experiments in real time, necessitating repeated transfers of the samples to stand-alone analytical instruments, thus limiting the types of experiments that could be performed. Here we address this gap with the development of the optoPlateReader (oPR), an open-source, solid-state, compact device that allows automated optogenetic stimulation and spectroscopy in each well of a 96-well plate. The oPR integrates an optoPlate illumination module with a module called the optoReader, an array of 96 photodiodes and LEDs that allows 96 parallel light measurements. The oPR was optimized for stimulation with blue light and for measurements of optical density and fluorescence. After calibration of all device components, we used the oPR to measure growth and to induce and measure fluorescent protein expression in E. coli. We further demonstrated how the optical read/write capabilities of the oPR permit computer-in-the-loop feedback control, where the current state of the sample can be used to adjust the optical stimulation parameters of the sample according to pre-defined feedback algorithms. The oPR will thus help realize an untapped potential for optogenetic experiments by enabling automated reading, writing, and feedback in microwell plates through open-source hardware that is accessible, customizable, and inexpensive.
Topics: Optogenetics; Feedback; Escherichia coli; Algorithms; Spectrum Analysis
PubMed: 38001175
DOI: 10.1038/s42003-023-05532-4 -
Applied Microbiology and Biotechnology Jul 2023Since natural resources for the bioproduction of commodity chemicals are scarce, waste animal fats (WAF) are an interesting alternative biogenic residual feedstock. They...
Since natural resources for the bioproduction of commodity chemicals are scarce, waste animal fats (WAF) are an interesting alternative biogenic residual feedstock. They appear as by-product from meat production, but several challenges are related to their application: first, the high melting points (up to 60 °C); and second, the insolubility in the polar water phase of cultivations. This leads to film and clump formation in shake flasks and microwell plates, which inhibits microbial consumption. In this study, different flask and well designs were investigated to identify the most suitable experimental set-up and further to create an appropriate workflow to achieve the required reproducibility of growth and product synthesis. The dissolved oxygen concentration was measured in-line throughout experiments. It became obvious that the gas mass transfer differed strongly among the shake flask design variants in cultivations with the polyhydroxyalkanoate (PHA) accumulating organism Ralstonia eutropha. A high reproducibility was achieved for certain flask or well plate design variants together with tailored cultivation conditions. Best results were achieved with bottom baffled glass and bottom baffled single-use shake flasks with flat membranes, namely, >6 g L of cell dry weight (CDW) with >80 wt% polyhydroxybutyrate (PHB) from 1 wt% WAF. Improved pre-emulsification conditions for round microwell plates resulted in a production of 14 g L CDW with a PHA content of 70 wt% PHB from 3 wt% WAF. The proposed workflow allows the rapid examination of fat material as feedstock, in the microwell plate and shake flask scale, also beyond PHA production. KEY POINTS: • Evaluation of shake flask designs for cultivating with hydrophobic raw materials • Development of a workflow for microwell plate cultivations with hydrophobic raw materials • Production of polyhydroxyalkanoate in small scale experiments from waste animal fat.
Topics: Animals; Polyhydroxyalkanoates; Reproducibility of Results; Workflow; Bioreactors
PubMed: 37266584
DOI: 10.1007/s00253-023-12599-w -
Molecules (Basel, Switzerland) May 2023This study describes the development and validation of a new green and high-throughput microwell spectrophotometric assay (MW-SPA) for the determination of three...
Development and Validation of Green and High-Throughput Microwell Spectrophotometric Assay for the Determination of Selective Serotonin Reuptake Inhibitors in Their Pharmaceutical Dosage Forms.
This study describes the development and validation of a new green and high-throughput microwell spectrophotometric assay (MW-SPA) for the determination of three selective serotonin reuptake inhibitors (SSRIs) in their pharmaceutical dosage forms. These SSRIs are fluoxetine, fluvoxamine, and paroxetine, the most prescribed drugs for the treatment of depression. The proposed assay was based on the formation of orange-colored -substituted naphthoquinone derivatives upon the reaction of SSRIs with 1,2-naphthoquinone-4-sulphonate (NQS) in alkaline media. The assay was conducted in 96-microwell assay plates, and the absorbances of the reaction products were measured by a microplate reader at their maximum absorbance wavelengths. The optimum conditions of the reaction were refined and established. Under these conditions, calibration curves were generated, and linear regression equations were computed. The linear relations between the absorbances and drug concentrations were linear with good correlation coefficients (0.9992-0.9997) in the range of 2-80 µg/mL. The assay limits of detection were in the range of 1.5-4.2 µg/mL. The precision was satisfactory as the values of relative standard deviation did not exceed 1.70%. The accuracy of the assay was ≥98.2%. The proposed MW-SPA was successfully applied to the analysis of the SSRIs in their pharmaceutical dosage forms with acceptable accuracy and precision; the label claims were 99.2-100.5% (±0.96-1.35%). The results of the proposed MW-SPA were compared with those of the official/pre-validated assays by statistical analysis with respect to the accuracy (by -test) and precision (by F-test). No significant differences were found between the calculated and theoretical values of the t- and F-tests at the 95% confidence level, proving similar accuracy and precision in the determination of SSRIs by both assays. The greenness of the proposed assay was confirmed by two metric tools. In addition, the assay is characterized with a high-throughput property which enables the simultaneous analysis of many samples in a short time. Therefore, the assay is a valuable tool for rapid routine application in pharmaceutical quality control units for the determination of the investigated SSRIs.
Topics: Selective Serotonin Reuptake Inhibitors; Spectrophotometry; Fluoxetine; Fluvoxamine; Pharmaceutical Preparations
PubMed: 37241961
DOI: 10.3390/molecules28104221