Sampling from Single Cells Actis, Paolo
Small methods,
March 13, 2018, Letnik:
2, Številka:
3
Journal Article
Recenzirano
The cell is the fundamental unit of biology. Major methodological advances in engineering and molecular biology have enabled the 'omics analysis of individual cells and supported biologists in ...understanding the deepest difference between health and disease. These advancements were based on the assumption that a single cell needs to be lysed or fixed before any in‐depth analysis can be performed. Here, a picture is painted of the innovative methods used for extracting the contents of living cells without affecting their viability. These novel methods are now empowering the biological community to repeatedly interrogate a single cell over time, thus giving a dynamic representation of the cell's 'omics rather than a snapshot at a particular time point.
The creative application of nanoprobe‐based methods for sampling the content of a single living cell without affecting its viability is reviewed. These novel methods are now empowering the biological community to repeatedly interrogate a single cell over time, thus giving a dynamic representation of the genotype of interest rather than a snapshot at a particular time point.
Abstract
The ability to detect low concentrations of biomarkers in patient samples is one of the cornerstones of modern healthcare. In general, biosensing approaches are based on measuring signals ...resulting from the interaction of a large ensemble of molecules with the sensor. Here, we report a biosensor platform using DNA origami featuring a central cavity with a target-specific DNA aptamer coupled with a nanopore read-out to enable individual biomarker detection. We show that the modulation of the ion current through the nanopore upon the DNA origami translocation strongly depends on the presence of the biomarker in the cavity. We exploit this to generate a biosensing platform with a limit of detection of 3 nM and capable of the detection of human C-reactive protein (CRP) in clinically relevant fluids. Future development of this approach may enable multiplexed biomarker detection by using ribbons of DNA origami with integrated barcoding.
Nanopore analysis of nucleic acid is now routine, but detection of proteins remains challenging. Here, we report the systematic characterization of the effect of macromolecular crowding on the ...detection sensitivity of a solid-state nanopore for circular and linearized DNA plasmids, globular proteins (β-galactosidase), and filamentous proteins (α-synuclein amyloid fibrils). We observe a remarkable ca. 1000-fold increase in the molecule count for the globular protein β-galactosidase and a 6-fold increase in peak amplitude for plasmid DNA under crowded conditions. We also demonstrate that macromolecular crowding facilitates the study of the topology of DNA plasmids and the characterization of amyloid fibril preparations with different length distributions. A remarkable feature of this method is its ease of use; it simply requires the addition of a macromolecular crowding agent to the electrolyte. We therefore envision that macromolecular crowding can be applied to many applications in the analysis of biomolecules by solid-state nanopores.
Fitness trackers are devices or applications for monitoring and tracking fitness-related metrics such as distance walked or run, calorie consumption, quality of sleep and heart rate. Since accurate ...heart rate monitoring is essential in fitness training, the objective of this study was to assess the accuracy and precision of the Fitbit Charge 2 for measuring heart rate with respect to a gold standard electrocardiograph. Fifteen healthy participants were asked to ride a stationary bike for 10 minutes and their heart rate was simultaneously recorded from each device. Results showed that the Fitbit Charge 2 underestimates the heart rate. Although the mean bias in measuring heart rate was a modest -5.9 bpm (95% CI: -6.1 to -5.6 bpm), the limits of agreement, which indicate the precision of individual measurements, between the Fitbit Charge 2 and criterion measure were wide (+16.8 to -28.5 bpm) indicating that an individual heart rate measure could plausibly be underestimated by almost 30 bpm.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Mitochondrial function is critical to continued cellular vitality and is an important contributor to a growing number of human diseases. Mitochondrial dysfunction is typically heterogeneous, ...mediated through the clonal expansion of mitochondrial DNA (mtDNA) variants in a subset of cells in a given tissue. To date, our understanding of the dynamics of clonal expansion of mtDNA variants has been technically limited to the single cell-level. Here, we report the use of nanobiopsy for subcellular sampling from human tissues, combined with next-generation sequencing to assess subcellular mtDNA mutation load in human tissue from mitochondrial disease patients. The ability to map mitochondrial mutation loads within individual cells of diseased tissue samples will further our understanding of mitochondrial genetic diseases.
The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a ...single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis.
Remote photoplethysmography (rPPG) allows contactless monitoring of human cardiac activity through a video camera. In this study, we assessed the accuracy and precision for heart rate measurements of ...the only consumer product available on the market, namely the FacereaderTM rPPG by Noldus, with respect to a gold standard electrocardiograph. Twenty-four healthy participants were asked to sit in front of a computer screen and alternate two periods of rest with two stress tests (i.e. Go/No-Go task), while their heart rate was simultaneously acquired for 20 minutes using the ECG criterion measure and the FacereaderTM rPPG. Results show that the FacereaderTM rPPG tends to overestimate lower heart rates and underestimate higher heart rates compared to the ECG. The Facereader™ rPPG revealed a mean bias of 9.8 bpm, the 95% limits of agreement (LoA) ranged from almost -30 up to +50 bpm. These results suggest that whilst the rPPG FacereaderTM technology has potential for contactless heart rate monitoring, its predictions are inaccurate for higher heart rates, with unacceptable precision across the entire range, rendering its estimates unreliable for monitoring individuals.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Single molecule delivery into living cells Chau, Chalmers C; Maffeo, Christopher M; Aksimentiev, Aleksei ...
Nature communications,
05/2024, Letnik:
15, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Controlled manipulation of cultured cells by delivery of exogenous macromolecules is a cornerstone of experimental biology. Here we describe a platform that uses nanopipettes to deliver defined ...numbers of macromolecules into cultured cell lines and primary cells at single molecule resolution. In the nanoinjection platform, the nanopipette is used as both a scanning ion conductance microscope (SICM) probe and an injection probe. The SICM is used to position the nanopipette above the cell surface before the nanopipette is inserted into the cell into a defined location and to a predefined depth. We demonstrate that the nanoinjection platform enables the quantitative delivery of DNA, globular proteins, and protein fibrils into cells with single molecule resolution and that delivery results in a phenotypic change in the cell that depends on the identity of the molecules introduced. Using experiments and computational modeling, we also show that macromolecular crowding in the cell increases the signal-to-noise ratio for the detection of translocation events, thus the cell itself enhances the detection of the molecules delivered.
Signal transduction by ion nano-gating (STING) technology is a label-free biosensor capable of identifying DNA and proteins. Based on a functionalized quartz nanopipette, the STING sensor includes ...specific recognition elements for analyte discrimination based on size, shape and charge density. A key feature of this technology is that it does not require any nanofabrication facility; each nanopipette can be easily, reproducibly, and inexpensively fabricated and tailored at the bench, thus reducing the cost and the turnaround time. Here, we show that STING sensors are capable of the ultrasensitive detection of HT-2 toxin with a detection limit of 100
fg/ml and compare the STING capabilities with respect to conventional sandwich assay techniques.
Manipulation and analysis of single cells is the next frontier in understanding processes that control the function and fate of cells. Herein we describe a single-cell injection platform based on ...nanopipettes. The system uses scanning microscopy techniques to detect cell surfaces, and voltage pulses to deliver molecules into individual cells. As a proof of concept, we injected adherent mammalian cells with fluorescent dyes.
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