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1.
Nat Commun ; 11(1): 4384, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873796

RESUMO

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.

2.
Nano Lett ; 20(7): 5553-5561, 2020 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-32559088

RESUMO

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 (ß-galactosidase), and filamentous proteins (α-synuclein amyloid fibrils). We observe a remarkable ca. 1000-fold increase in the molecule count for the globular protein ß-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.

3.
Biochem Soc Trans ; 48(2): 357-365, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32267469

RESUMO

The manipulation of cultured mammalian cells by the delivery of exogenous macromolecules is one of the cornerstones of experimental cell biology. Although the transfection of cells with DNA expressions constructs that encode proteins is routine and simple to perform, the direct delivery of proteins into cells has many advantages. For example, proteins can be chemically modified, assembled into defined complexes and subject to biophysical analyses prior to their delivery into cells. Here, we review new approaches to the injection and electroporation of proteins into cultured cells. In particular, we focus on how recent developments in nanoscale injection probes and localized electroporation devices enable proteins to be delivered whilst minimizing cellular damage. Moreover, we discuss how nanopore sensing may ultimately enable the quantification of protein delivery at single-molecule resolution.

4.
PLoS One ; 14(11): e0225592, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31756239

RESUMO

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.


Assuntos
Frequência Cardíaca , Fotopletismografia/métodos , Adulto , Eletrocardiografia , Feminino , Humanos , Masculino , Adulto Jovem
5.
Nat Nanotechnol ; 14(1): 80-88, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30510280

RESUMO

Much of the functionality of multicellular systems arises from the spatial organization and dynamic behaviours within and between cells. Current single-cell genomic methods only provide a transcriptional 'snapshot' of individual cells. The real-time analysis and perturbation of living cells would generate a step change in single-cell analysis. Here we describe minimally invasive nanotweezers that can be spatially controlled to extract samples from living cells with single-molecule precision. They consist of two closely spaced electrodes with gaps as small as 10-20 nm, which can be used for the dielectrophoretic trapping of DNA and proteins. Aside from trapping single molecules, we also extract nucleic acids for gene expression analysis from living cells without affecting their viability. Finally, we report on the trapping and extraction of a single mitochondrion. This work bridges the gap between single-molecule/organelle manipulation and cell biology and can ultimately enable a better understanding of living cells.


Assuntos
Nanotecnologia , Pinças Ópticas , Análise de Célula Única , Animais , Axônios/metabolismo , Biópsia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , DNA/química , Eletricidade , Eletrodos , Fluorescência , Humanos , Camundongos , Mitocôndrias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Soluções
6.
PLoS One ; 13(2): e0192691, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29489850

RESUMO

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.


Assuntos
Actigrafia/métodos , Frequência Cardíaca , Monitorização Fisiológica , Humanos
10.
ACS Nano ; 9(4): 3587-95, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25794527

RESUMO

Understanding the behavioral properties of single molecules or larger scale populations interacting with single molecules is currently a hotly pursued topic in nanotechnology. This arises from the potential such techniques have in relation to applications such as targeted drug delivery, early stage detection of disease, and drug screening. Although label and label-free single molecule detection strategies have existed for a number of years, currently lacking are efficient methods for the controllable delivery of single molecules in aqueous environments. In this article we show both experimentally and from simulations that nanopipets in conjunction with asymmetric voltage pulses can be used for label-free detection and delivery of single molecules through the tip of a nanopipet with "on-demand" timing resolution. This was demonstrated by controllable delivery of 5 kbp and 10 kbp DNA molecules from solutions with concentrations as low as 3 pM.


Assuntos
DNA , Nanotecnologia/métodos , Eletricidade , Imagem Óptica , Água/química
11.
ACS Nano ; 8(1): 875-84, 2014 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-24377306

RESUMO

The measurement of key molecules in individual cells with minimal disruption to the biological milieu is the next frontier in single-cell analyses. Nanoscale devices are ideal analytical tools because of their small size and their potential for high spatial and temporal resolution recordings. Here, we report the fabrication of disk-shaped carbon nanoelectrodes whose radius can be precisely tuned within the range 5-200 nm. The functionalization of the nanoelectrode with platinum allowed the monitoring of oxygen consumption outside and inside a brain slice. Furthermore, we show that nanoelectrodes of this type can be used to impale individual cells to perform electrochemical measurements within the cell with minimal disruption to cell function. These nanoelectrodes can be fabricated combined with scanning ion conductance microscopy probes, which should allow high resolution electrochemical mapping of species on or in living cells.


Assuntos
Técnicas Eletroquímicas/instrumentação , Eletrodos , Nanoestruturas , Peróxido de Hidrogênio/análise , Microscopia Eletrônica de Varredura , Oxirredução , Oxigênio/análise , Análise de Célula Única
12.
ACS Nano ; 8(1): 546-53, 2014 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-24279711

RESUMO

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.


Assuntos
Biópsia/métodos , Genômica , Nanotecnologia , Análise de Célula Única , Sequência de Bases , Células Cultivadas , Primers do DNA , Humanos , Microscopia/métodos , Reação em Cadeia da Polimerase
13.
Anal Chem ; 85(19): 9333-42, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24004146

RESUMO

Using nanopipettes to locally deliver molecules to the surface of living cells could potentially open up studies of biological processes down to the level of single molecules. However, in order to achieve precise and quantitative local delivery it is essential to be able to determine the amount and distribution of the molecules being delivered. In this work, we investigate how the size of the nanopipette, the magnitude of the applied pressure or voltage, which drives the delivery, and the distance to the underlying surface influences the number and spatial distribution of the delivered molecules. Analytical expressions describing the delivery are derived and compared with the results from finite element simulations and experiments on delivery from a 100 nm nanopipette in bulk solution and to the surface of sensory neurons. We then developed a setup for rapid and quantitative delivery to multiple subcellular areas, delivering the molecule capsaicin to stimulate opening of Transient Receptor Potential Vanilloid subfamily member 1 (TRPV1) channels, membrane receptors involved in pain sensation. Overall, precise and quantitative delivery of molecules from nanopipettes has been demonstrated, opening up many applications in biology such as locally stimulating and mapping receptors on the surface of live cells.


Assuntos
Capsaicina/metabolismo , Gânglios Espinais/química , Nanotecnologia/instrumentação , Canais de Cátion TRPV/metabolismo , Animais , Capsaicina/química , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Tamanho da Partícula , Ratos , Ratos Sprague-Dawley , Propriedades de Superfície , Canais de Cátion TRPV/química
14.
Nanoscale ; 5(19): 9214-21, 2013 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-23934399

RESUMO

Nanofluidic structures share many properties with ligand-gated ion channels. However, actuating ion conductance in artificial systems is a challenge. We have designed a system that uses a carbohydrate-responsive polymer to modulate ion conductance in a quartz nanopipette. The cationic polymer, a poly(vinylpyridine) quaternized with benzylboronic acid groups, undergoes a transition from swollen to collapsed upon binding to monosaccharides. As a result, the current rectification in nanopipettes can be reversibly switched depending on the concentration of monosaccharides. Such molecular actuation of nanofluidic conductance may be used in novel sensors and drug delivery systems.

15.
Anal Chem ; 85(15): 7519-26, 2013 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-23795948

RESUMO

Dual carbon electrodes (DCEs) are quickly, easily, and cheaply fabricated by depositing pyrolytic carbon into a quartz theta nanopipet. The size of DCEs can be controlled by adjusting the pulling parameters used to make the nanopipet. When operated in generation/collection (G/C) mode, the small separation between the electrodes leads to reasonable collection efficiencies of ca. 30%. A three-dimensional finite element method (FEM) simulation is developed to predict the current response of these electrodes as a means of estimating the probe geometry. Voltammetric measurements at individual electrodes combined with generation/collection measurements provide a reasonable guide to the electrode size. DCEs are employed in a scanning electrochemical microscopy (SECM) configuration, and their use for both approach curves and imaging is considered. G/C approach curve measurements are shown to be particularly sensitive to the nature of the substrate, with insulating surfaces leading to enhanced collection efficiencies, whereas conducting surfaces lead to a decrease of collection efficiency. As a proof-of-concept, DCEs are further used to locally generate an artificial electron acceptor and to follow the flux of this species and its reduced form during photosynthesis at isolated thylakoid membranes. In addition, 2-dimensional images of a single thylakoid membrane are reported and analyzed to demonstrate the high sensitivity of G/C measurements to localized surface processes. It is finally shown that individual nanometer-size electrodes can be functionalized through the selective deposition of platinum on one of the two electrodes in a DCE while leaving the other one unmodified. This provides an indication of the future versatility of this type of probe for nanoscale measurements and imaging.


Assuntos
Carbono/química , Microscopia Eletroquímica de Varredura/instrumentação , Eletrodos , Imagem Molecular , Nanotecnologia , Quartzo/química , Tilacoides/metabolismo
16.
RSC Adv ; 2(31): 11638-11640, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23243499

RESUMO

Protein-metal interactions determine and regulate many biological functions. Nanopipettes functionalized with peptide moieties can be used as sensors for metal ions in solution.

17.
Nanoscale ; 4(19): 5843-6, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22899383

RESUMO

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.


Assuntos
Eletricidade , Nanotecnologia/instrumentação , Linhagem Celular , Membrana Celular/metabolismo , Fibroblastos/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Microscopia Eletrônica de Varredura
18.
Anal Chem ; 83(16): 6121-6, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21761859

RESUMO

The calcium ion response of a quartz nanopipette was enhanced by immobilization of calmodulin to the nanopore surface. Binding to the analyte is rapidly reversible in neutral buffer and requires no change in media or conditions to regenerate the receptor. The signal remained reproducible over numerous measurements. The modified nanopipette was used to measure binding affinity to calcium ions, with a K(d) of 6.3 ± 0.8 × 10(-5) M. This affinity is in good agreement with reported values of the solution-state protein. The behavior of such reversible nanopore-based sensors can be used to study proteins in a confined environment and may lead to new devices for continuous monitoring.


Assuntos
Técnicas Biossensoriais/instrumentação , Cálcio/análise , Calmodulina/metabolismo , Proteínas Imobilizadas/metabolismo , Nanoestruturas/química , Nanotecnologia , Cálcio/metabolismo , Calmodulina/química , Cátions/análise , Cátions/metabolismo , Proteínas Imobilizadas/química , Cinética , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Ligação Proteica , Quartzo/química
19.
Biosens Bioelectron ; 26(11): 4503-7, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21636261

RESUMO

Signal Transduction by Ion NanoGating (STING) is a label-free technology based on functionalized quartz nanopipettes. The nanopipette pore can be decorated with a variety of recognition elements and the molecular interaction is transduced via a simple electrochemical system. A STING sensor can be easily and reproducibly fabricated and tailored at the bench starting from inexpensive quartz capillaries. The analytical application of this new biosensing platform, however, was limited due to the difficult correlation between the measured ionic current and the analyte concentration in solution. Here we show that STING sensors functionalized with aptamers allow the quantitative detection of thrombin. The binding of thrombin generates a signal that can be directly correlated to its concentration in the bulk solution.


Assuntos
Técnicas Biossensoriais/métodos , Trombina/análise , Aptâmeros de Nucleotídeos , Técnicas Biossensoriais/instrumentação , Análise Química do Sangue/instrumentação , Análise Química do Sangue/métodos , Técnicas Eletroquímicas , Desenho de Equipamento , Humanos , Nanoporos , Nanotecnologia , Transdução de Sinais
20.
Langmuir ; 27(10): 6528-33, 2011 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-21510657

RESUMO

Most of the research in the field of nanopore-based platforms is focused on monitoring ion currents and forces as individual molecules translocate through the nanopore. Molecular gating, however, can occur when target analytes interact with receptors appended to the nanopore surface. Here we show that a solid state nanopore functionalized with polyelectrolytes can reversibly bind metal ions, resulting in a reversible, real-time signal that is concentration dependent. Functionalization of the sensor is based on electrostatic interactions, requires no covalent bond formation, and can be monitored in real time. Furthermore, we demonstrate how the applied voltage can be employed to tune the binding properties of the sensor. The sensor has wide-ranging applications and, its simplest incarnation can be used to study binding thermodynamics using purely electrical measurements with no need for labeling.


Assuntos
Eletricidade , Eletrólitos/química , Metais/análise , Metais/química , Nanoporos , Polímeros/química , Técnicas Biossensoriais , Quitosana/química , Concentração de Íons de Hidrogênio , Reprodutibilidade dos Testes , Fatores de Tempo
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