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/químicaRESUMO
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 TempoRESUMO
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 PolimeraseRESUMO
Studying the earliest stages of precipitation at the nanoscale is technically challenging but quite valuable as such phenomena reflect important processes such as crystallization and biomineralization. Using a quartz nanopipette as a nanoreactor, we induced precipitation of an insoluble salt to generate oscillating current blockades. The reversible process can be used to measure both kinetics of precipitation and relative size of the resulting nanoparticles. Counter ions for the highly water-insoluble salt zinc phosphate were separated by the pore of a nanopipette and a potential applied to cause ion migration to the interface. By analyzing the kinetics of pore blockage, two distinct mechanisms were identified: a slower process due to precipitation from solution, and a faster process attributed to voltage-driven migration of a trapped precipitate. We discuss the potential of these techniques in studying precipitation dynamics, trapping particles within a nanoreactor, and electrical sensors based on nanoprecipitation.
Assuntos
Nanotecnologia , Cinética , Fosfatos/química , Solubilidade , Compostos de Zinco/químicaRESUMO
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.