Single-Molecule Electrical Detection with Real-Time Label-Free Capability and Ultrasensitivity

Small Methods ◽  
2017 ◽  
Vol 1 (5) ◽  
pp. 1700071 ◽  
Author(s):  
Chunhui Gu ◽  
Chuancheng Jia ◽  
Xuefeng Guo
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Label Free ◽  
Electrical Detection

An integrated system for optical and electrical detection of single molecules/particles inside a solid-state nanopore

2015 ◽  
Vol 184 ◽  
pp. 85-99 ◽  
Author(s):  
Xin Shi ◽  
Rui Gao ◽  
Yi-Lun Ying ◽  
Wei Si ◽  
Yunfei Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Detection System ◽  
Single Molecules ◽  
Ionic Current ◽  
Dark Field ◽  
Integrated System ◽  
High Temporal Resolution ◽  
Label Free ◽  
Electrical Detection ◽  
Current Detection

Nanopore techniques have proven to be useful tools for single-molecule detection. The combination of optical detection and ionic current measurements enables a new possibility for the parallel readout of multiple nanopores without complex nanofluidics and embedded electrodes. In this study, we developed a new integrated system for the label-free optical and electrical detection of single molecules based on a metal-coated nanopore. The entire system, containing a dark-field microscopy system and an ultralow current detection system with high temporal resolution, was designed and fabricated. An Au-coated nanopore was used to generate the optical signal. Light scattering from a single Au-coated nanopore was measured under a dark-field microscope. A lab-built ultralow current detection system was designed for the correlated optical and electrical readout. This integrated system might provide more direct and detailed information on single analytes inside the nanopore compared with classical ionic current measurements.

scholarly journals A label-free approach to detect ligand binding to cell surface proteins in real time

2018 ◽  
Author(s):  
Verena Burtscher ◽  
Matej Hotka ◽  
Yang Li ◽  
Michael Freissmuth ◽  
Walter Sandtner
Keyword(s):  
Ligand Binding ◽  
Real Time ◽  
Single Molecule ◽  
Membrane Capacitance ◽  
Surface Proteins ◽  
Displacement Current ◽  
High Temporal Resolution ◽  
Label Free ◽  
Single Molecule Level ◽  
Electrophysiological Recordings

AbstractElectrophysiological recordings allow for monitoring the operation of proteins with high temporal resolution down to the single molecule level. This technique has been exploited to track either ion flow arising from channel opening or the synchronized movement of charged residues and/or ions within the membrane electric field. Here, we describe a novel type of current by using the serotonin transporter (SERT) as a model. We examined transient currents elicited on rapid application of specific SERT inhibitors. Our analysis shows that these currents originate from ligand binding and not from a conformational change. The Gouy-Chapman model predicts that a ligand-induced elimination/neutralization of surface charge must produce a displacement current and related apparent changes in membrane capacitance. Here we verified these predictions with SERT. Our observations demonstrate that ligand binding to a protein can be monitored in real time and in a label-free manner by recording the membrane capacitance.

scholarly journals A label-free approach to detect ligand binding to cell surface proteins in real time

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Verena Burtscher ◽  
Matej Hotka ◽  
Yang Li ◽  
Michael Freissmuth ◽  
Walter Sandtner
Keyword(s):  
Ligand Binding ◽  
Real Time ◽  
Single Molecule ◽  
Membrane Capacitance ◽  
Surface Proteins ◽  
High Temporal Resolution ◽  
Label Free ◽  
Single Molecule Level ◽  
Electrophysiological Recordings ◽  
Displacement Currents

Electrophysiological recordings allow for monitoring the operation of proteins with high temporal resolution down to the single molecule level. This technique has been exploited to track either ion flow arising from channel opening or the synchronized movement of charged residues and/or ions within the membrane electric field. Here, we describe a novel type of current by using the serotonin transporter (SERT) as a model. We examined transient currents elicited on rapid application of specific SERT inhibitors. Our analysis shows that these currents originate from ligand binding and not from a long-range conformational change. The Gouy-Chapman model predicts that adsorption of charged ligands to surface proteins must produce displacement currents and related apparent changes in membrane capacitance. Here we verified these predictions with SERT. Our observations demonstrate that ligand binding to a protein can be monitored in real time and in a label-free manner by recording the membrane capacitance.

Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

2020 ◽  
Author(s):  
Nikolas Hundt
Keyword(s):  
Single Molecule ◽  
Cellular Systems ◽  
Single Molecule Imaging ◽  
Label Free ◽  
Measurement Sensitivity ◽  
Mass Distributions ◽  
Quantitative Measurements ◽  
Contrast Mechanism ◽  
Cellular Components ◽  
Scattering Signal

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

scholarly journals Combined Transcriptome Analysis Reveals the Ovule Abortion Regulatory Mechanisms in the Female Sterile Line of Pinus tabuliformis Carr.

2021 ◽  
Vol 22 (6) ◽  
pp. 3138
Author(s):  
Zaixin Gong ◽  
Rui Han ◽  
Li Xu ◽  
Hailin Hu ◽  
Min Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Regulatory Mechanisms ◽  
Cdna Libraries ◽  
Ovule Development ◽  
Ovule Abortion ◽  
Pinus Tabuliformis ◽  
Wild Type Female ◽  
Fertile Line ◽  
Next Generation Sequencing Ngs

Ovule abortion is a common phenomenon in plants that has an impact on seed production. Previous studies of ovule and female gametophyte (FG) development have mainly focused on angiosperms, especially in Arabidopsis thaliana. However, because it is difficult to acquire information about ovule development in gymnosperms, this remains unclear. Here, we investigated the transcriptomic data of natural ovule abortion mutants (female sterile line, STE) and the wild type (female fertile line, FER) of Pinus tabuliformis Carr. to evaluate the mechanism of ovule abortion during the process of free nuclear mitosis (FNM). Using single-molecule real-time (SMRT) sequencing and next-generation sequencing (NGS), 18 cDNA libraries via Illumina and two normalized libraries via PacBio, with a total of almost 400,000 reads, were obtained. Our analysis showed that the numbers of isoforms and alternative splicing (AS) patterns were significantly variable between FER and STE. The functional annotation results demonstrate that genes involved in the auxin response, energy metabolism, signal transduction, cell division, and stress response were differentially expressed in different lines. In particular, AUX/IAA, ARF2, SUS, and CYCB had significantly lower expression in STE, showing that auxin might be insufficient in STE, thus hindering nuclear division and influencing metabolism. Apoptosis in STE might also have affected the expression levels of these genes. To confirm the transcriptomic analysis results, nine pairs were confirmed by quantitative real-time PCR. Taken together, these results provide new insights into ovule abortion in gymnosperms and further reveal the regulatory mechanisms of ovule development.

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