RESUMO
Immunosensing is a bioanalytical technique capable of selective detections of pathogens by utilizing highly specific and strong intermolecular interactions between recognition probes and antigens. Here, we exploited the molecular mechanism in artificial nanopores for selective single-virus identifications. We designed hemagglutinin antibody mimicking oligopeptides with a weak affinity to influenza A virus. By functionalizing the pore wall surface with the synthetic peptides, we rendered specificity to virion-nanopore interactions. The ligand binding thereof was found to perturb translocation dynamics of specific viruses in the nanochannel, which facilitated digital typing of influenza by the resistive pulse bluntness. As amino acid sequence degrees of freedom can potentially offer variety of recognition ability to the molecular probes, this peptide nanopore approach can be used as a versatile immunosensor with single-particle sensitivity that promises wide applications in bioanalysis including bacterial and viral screening to infectious disease diagnosis.
Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Nanoporos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Galinhas , Ouro/química , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Compostos de Silício/química , Carga Viral/métodosRESUMO
Bioinspired pore sensing for selective detection of flagellated bacteria was investigated. The Au micropore wall surface was modified with a synthetic peptide designed from toll-like receptor 5 (TLR5) to mimic the pathogen-recognition capability. We found that intermolecular interactions between the TLR5-derived recognition peptides and flagella induce ligand-specific perturbations in the translocation dynamics of Escherichia coli, which facilitated the discrimination between the wild-type and flagellin-deletion mutant (ΔfliC) by the resistive pulse patterns thereby demonstrating the sensing of bacteria at a single-cell level. These results provide a novel concept of utilizing weak intermolecular interactions as a recognition probes for single-cell microbial identification.
Assuntos
Escherichia coli/citologia , Peptídeos/química , Receptor 5 Toll-Like/química , Flagelina/química , Flagelina/genética , Humanos , MutaçãoRESUMO
Skeletal myoblast (SkMB) transplantation has been conducted as a therapeutic strategy for severe heart failure. However, arrhythmogenicity following transplantation remains unsolved. We developed an in vitro model of myoblast transplantation with "patterned" or "randomly-mixed" co-culture of SkMBs and cardiomyocytes enabling subsequent electrophysiological, and arrhythmogenic evaluation. SkMBs were magnetically labeled with magnetite nanoparticles and co-cultured with neonatal rat ventricular myocytes (NRVMs) on multi-electrode arrays. SkMBs were patterned by a magnet beneath the arrays. Excitation synchronicity was evaluated by Ca(2+) imaging using a gene-encoded Ca(2+) indicator, G-CaMP2. In the monoculture of NRVMs (control), conduction was well-organized. In the randomly-mixed co-culture of NRVMs and SkMBs (random group), there was inhomogeneous conduction from multiple origins. In the "patterned" co-culture where an en bloc SKMB-layer was inserted into the NRVM-layer, excitation homogenously propagated although conduction was distorted by the SkMB-area. The 4-mm distance conduction time (CT) in the random group was significantly longer (197 ± 126 ms) than in control (17 ± 3 ms). In the patterned group, CT through NRVM-area did not change (25 ± 3 ms), although CT through the SkMB-area was significantly longer (132 ± 77 ms). The intervals between spontaneous excitation varied beat-to-beat in the random group, while regular beating was recorded in the control and patterned groups. Synchronized Ca(2+) transients of NRVMs were observed in the patterned group, whereas those in the random group were asynchronous. Patterned alignment of SkMBs is feasible with magnetic nanoparticles. Using the novel in vitro model mimicking cell transplantation, it may become possible to predict arrhythmogenicity due to heterogenous cell transplantation. J. Cell. Physiol. 231: 2249-2256, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Técnicas de Cocultura , Ventrículos do Coração/citologia , Nanopartículas de Magnetita/administração & dosagem , Mioblastos Esqueléticos/citologia , Miócitos Cardíacos/citologia , Animais , Arritmias Cardíacas/fisiopatologia , Células Cultivadas , Infarto do Miocárdio/fisiopatologia , Nanotecnologia/métodos , Ratos WistarRESUMO
This paper proposes a capture device to manipulate and transport a cellular aggregate in a micro-well. A cellular aggregate (a few hundreds µm in diameter) is currently manipulated by a pipette. The manual manipulation by a pipette has problems; low reliability, low throughput, and difficulty in confirmation of task completion. We took into account of compatibility with existing methods such as a micro-well-plate and designed for the capture device of a cellular aggregate in a micro-well. A newly developed capture device flows and carries a cellular aggregate from a bottom of a well to a trap of the capture device. We designed a curved surface at the bottom of the capture device to form a space to act as a channel between the inner wall of the micro-well. This paper presents concept, design, fabrication, and of the proposed cellular aggregate capture, followed by successful experimental results.
Assuntos
Separação Celular/instrumentação , Citometria de Fluxo/instrumentação , Dispositivos Lab-On-A-Chip , Células-Tronco Mesenquimais/citologia , Micromanipulação/instrumentação , Agregação Celular/fisiologia , Técnicas de Cultura de Células/instrumentação , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Células-Tronco Mesenquimais/fisiologia , Manejo de Espécimes/instrumentaçãoRESUMO
This paper describes an advanced Micro Channel Array (MCA) for recording electrophysiological signals of neuronal networks at multiple points simultaneously. The developed MCA is designed for neuronal network analysis which has been studied by the co-authors using the Micro Electrode Arrays (MEA) system, and employs the principles of extracellular recordings. A prerequisite for extracellular recordings with good signal-to-noise ratio is a tight contact between cells and electrodes. The MCA described herein has the following advantages. The electrodes integrated around individual micro channels are electrically isolated to enable parallel multipoint recording. Reliable clamping of a targeted cell through micro channels is expected to improve the cellular selectivity and the attachment between the cell and the electrode toward steady electrophysiological recordings. We cultured hippocampal neurons on the developed MCA. As a result, the spontaneous and evoked spike potentials could be recorded by sucking and clamping the cells at multiple points. In this paper, we describe the design and fabrication of the MCA and the successful electrophysiological recordings leading to the development of an effective cellular network analysis device.
Assuntos
Fenômenos Eletrofisiológicos , Análise em Microsséries/métodos , Neurônios/citologia , Potenciais de Ação , Animais , Células Cultivadas , Impedância Elétrica , Eletrodos , Desenho de Equipamento , Análise em Microsséries/instrumentação , Microtecnologia , Polímeros , Ratos , Ratos Wistar , XilenosRESUMO
We report a proof-of-principle demonstration of particle concentration to achieve high-throughput resistive pulse detections of bacteria using a microfluidic-channel-integrated micropore. We fabricated polymeric nanochannels to trap micrometer-sized bioparticles via a simple water pumping mechanism that allowed aggregation-free size-selective particle concentration with negligible loss. Single-bioparticle detections by ionic current measurements were then implemented through releasing and transporting the thus-collected analytes to the micropore. As a result, we attained two orders of magnitude enhancement in the detection throughput by virtue of an accumulation effect via hydrodynamic control. The device concept presented may be useful in developing nanopores and nanochannels for high-throughput single-particle and -molecule analyses.
Assuntos
Dispositivos Lab-On-A-Chip , Escherichia coli/citologia , Hidrodinâmica , PorosidadeRESUMO
Parallel integration of multiple channels is a fundamental strategy for high-throughput particle detection in solid-state nanopores wherein understanding and control of crosstalk is an important issue for the post resistive pulse analysis. Here we report on a prominent effect of cross-channel electric field interference on the ionic current blockade by nanoparticles in nano-spaced pore arrays in a thin Si3N4 membrane. We systematically investigated the variations in resistive pulse profiles in multipore systems of various inter-channel distances. Although each pore acted independently when they were formed at excessively far distances, we observed significant cross-pore electrostatic interactions under close-integration that led the multipores to virtually act as a single-pore of equivalent area. As a result of the interference, the resistive pulse height demonstrated bimodal distributions due to the pronounced particle trajectory-dependence of the ionic blockade effects. Most importantly, the overcrowded multi-channel structure was found to deliver significant crosstalk with serious degradation of the sensor sensitivity to particle sizes. The present results provide a guide to design multipore structures regarding the trade-off between the detection throughput and sensor sensitivity.
RESUMO
We investigated the roles of silicon substrate material compositions in ionic current blockade in solid-state nanopores. When detecting single nanoparticles using an ionic current in a Si3N4 nanopore supported on a doped silicon wafer, resistive pulses were found to be blunted significantly via signal retardation due to predominant contributions of large capacitance at the ultrathin membrane. Unexpectedly, in contrast, changing the substrate material to non-doped silicon led to the sharpening of the spike-like signal feature, suggesting a better temporal resolution of the cross-channel ionic current measurements by virtue of the thick intrinsic semiconductor layer that served to diminish the net chip capacitance. The present results suggest the importance of the choice of Si compositions regarding the capacitance effects to attain better spatiotemporal resolution in solid-state nanopore sensors.
RESUMO
Signal delay is a crucial factor in resistive pulse analyses using low-thickness-to-diameter aspect-ratio pores that aim to detect fine features in the ionic current blockade during the fast translocation of individual analytes to attain single-molecule tomography. Here we report on evaluations of the ionic current response to dynamic motions of nanoparticles in ultrathin solid-state nanopores. We systematically investigated the effects of pore resistance and membrane capacitance on resistive pulse waveforms under different salt concentration conditions and device configurations. The results revealed substantial modifications in the resistive pulse waveforms due to a slow charging/discharging processes at the water-touching thin dielectrics in the solid-state nanopore chips. We also provide a device design to improve the temporal resolution without compromising the spatial sensitivity. The present findings offer a breakthrough toward nanoporescopy to measure the nanoscopic shape of single-bioparticles and -molecules in electrolyte solution.
RESUMO
Utilization of multiple-channel structure is a promising way of accomplishing high-throughput detections of analytes in solid-state pore sensors. Here we report on systematic investigation of particle capture efficiency in Si3N4 multipore systems of various array configurations. We demonstrated enhanced detection throughput with increasing numbers of pore channels in a membrane. Meanwhile, we also observed significant contributions of the interchannel crosstalk in closely integrated multipores that tended to deteriorate throughput performance by causing shrinkage of the absorption zone via the interference-derived weakening of the electric field around the pore orifice. At the same time, the interference-derived electric field distributions were also found to diminish the electroosmotic contributions to the particle capture efficiency. The present findings can be useful in designing pore arrays with optimal throughput performance.
Assuntos
Nanoporos , Compostos de Silício/química , Simulação por Computador , Eletro-Osmose , Poliestirenos/químicaRESUMO
Rapid diagnosis of flu before symptom onsets can revolutionize our health through diminishing a risk for serious complication as well as preventing infectious disease outbreak. Sensor sensitivity and selectivity are key to accomplish this goal as the number of virus is quite small at the early stage of infection. Here we report on label-free electrical diagnostics of influenza based on nanopore analytics that distinguishes individual virions by their distinct physical features. We accomplish selective resistive-pulse sensing of single flu virus having negative surface charges in a physiological media by exploiting electroosmotic flow to filter contaminants at the Si3N4 pore orifice. We demonstrate identifications of allotypes with 68% accuracy at the single-virus level via pattern classifications of the ionic current signatures. We also show that this discriminability becomes >95% under a binomial distribution theorem by ensembling the pulse data of >20 virions. This simple mechanism is versatile for point-of-care tests of a wide range of flu types.
Assuntos
Vírus da Influenza A/isolamento & purificação , Influenza Humana/diagnóstico , Nanotecnologia/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Eletricidade , Humanos , Vírus da Influenza A/química , Influenza Humana/virologia , Transporte de Íons , Aprendizado de Máquina , Nanoporos , Nanotecnologia/instrumentação , Sensibilidade e Especificidade , Compostos de Silício/químicaRESUMO
Conventional concepts of resistive pulse analysis is to discriminate particles in liquid by the difference in their size through comparing the amount of ionic current blockage. In sharp contrast, we herein report a proof-of-concept demonstration of the shape sensing capability of solid-state pore sensors by leveraging the synergy between nanopore technology and machine learning. We found ionic current spikes of similar patterns for two bacteria reflecting the closely resembled morphology and size in an ultra-low thickness-to-diameter aspect-ratio pore. We examined the feasibility of a machine learning strategy to pattern-analyse the sub-nanoampere corrugations in each ionic current waveform and identify characteristic electrical signatures signifying nanoscopic differences in the microbial shape, thereby demonstrating discrimination of single-bacterial cells with accuracy up to 90%. This data-analytics-driven microporescopy capability opens new applications of resistive pulse analyses for screening viruses and bacteria by their unique morphologies at a single-particle level.
Assuntos
Bactérias/isolamento & purificação , Técnicas Eletroquímicas/métodos , Nanoporos , Nanotecnologia/métodos , Estudo de Prova de Conceito , Bactérias/citologia , Técnicas Eletroquímicas/instrumentação , Estudos de Viabilidade , Íons/química , Aprendizado de Máquina , Nanotecnologia/instrumentaçãoRESUMO
Cell-culture microchips mimicking tissue/organ-specific functions are required as alternatives to animal testing for drug discovery and disease models. Although three-dimensional (3D) cell culture microfluidic devices can create more biologically relevant cellular microenvironments and higher throughput analysis platforms of cell behavior than conventional techniques, devices for skeletal muscle cells have not been developed. In the present study, we aimed to develop microfluidic devices for 3D cultures of skeletal muscle cells. Skeletal muscle cells mixed with a collagen type-I solution was introduced into the microchannel for cells (MC-C) and was gelated. Then, the medium was introduced into the microchannel for medium (MC-M). During this process, connecting microchannels (Con-MCs) prevented leakage of the collagen solution mixed with cells from MC-C to MC-M and supplied the nutrients from the medium in MC-M to the cells in MC-C. Skeletal muscle microtissues cultured in the microchannel for a week consisted of myotubes were confirmed by histological analysis and immunofluorescence staining. The skeletal muscle microtissues in the microchannel contracted in response to externally applied electrical stimulation (1 and 50 Hz). These results indicate that the functional skeletal muscle microtissues were constructed in the microchannel. Thus, the microfluidic device for culturing 3D skeletal muscle microtissues presented in this study has a potential to be used for drug discovery and toxicological tests.