scholarly journals Real-time sensing of neurotransmitters by functionalized nanopores embedded in a single live cell

2021 ◽  
Vol 2 (1) ◽  
Author(s):  
Xialin Zhang ◽  
Linqin Dou ◽  
Ming Zhang ◽  
Yu Wang ◽  
Xin Jiang ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Single Channel ◽  
Drug Carrier ◽  
Hek293t Cell ◽  
Live Cell ◽  
In Situ Monitoring ◽  
Electrophysiological Recording ◽  
Directed Mutation ◽  
Artificial Synapse

AbstractInterface between neuron cells and biomaterials is the key to real-time sensing, transmitting and manipulating of neuron activities, which are the long-term pursue of scientists and gain intense research focus recently. It is of great interest to develop a sensor with exquisite sensitivity and excellent selectivity for real-time monitoring neurotransmitters transport through single live cell. Sensing techniques including electrode-based methods, optogenetics, and nanowire cell penetration systems have been developed to monitor the neuron activities. However, their biocompatibilities remain a challenge. Protein nanopores with membrane compatibility and lumen tunability provide real-time, single-molecule sensitivities for biosensing of DNA, RNA, peptides and small molecules. In this study, an engineered protein nanopore MspA (Mycobacterium smegmatis porin A) through site-directed mutation with histidine selectively bind with Cu2+ in its internal lumen. Chelation of neurotransmitters such as L-glutamate (L-Glu), dopamine (DA) and norepinephrine (NE) with the Cu2+ creates specific current signals, showing different transient current blockade and dwell time in single channel electrophysiological recording. Furthermore, the functionalized M2MspA-N91H nanopores have been embedded in live HEK293T cell membrane for real-time, in situ monitoring of extracellular L-glutamate translocating through the nanopore. This biomimetic neurotransmitter nanopore has provided a new platform for future development of neuron sensors, drug carrier and artificial synapse.

scholarly journals Active DNA unwinding and transport by a membrane-adapted helicase nanopore

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ke Sun ◽  
Changjian Zhao ◽  
Xiaojun Zeng ◽  
Yuejia Chen ◽  
Xin Jiang ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Single Cell Analysis ◽  
Single Channel ◽  
Vital Role ◽  
Live Cell ◽  
Bovine Papillomavirus ◽  
Nanoscale Transport ◽  
Single Molecule Level

Abstract Nanoscale transport through nanopores and live-cell membranes plays a vital role in both key biological processes as well as biosensing and DNA sequencing. Active translocation of DNA through these nanopores usually needs enzyme assistance. Here we present a nanopore derived from truncated helicase E1 of bovine papillomavirus (BPV) with a lumen diameter of c.a. 1.3 nm. Cryogenic electron microscopy (cryo-EM) imaging and single channel recording confirm its insertion into planar lipid bilayer (BLM). The helicase nanopore in BLM allows the passive single-stranded DNA (ssDNA) transport and retains the helicase activity in vitro. Furthermore, we incorporate this helicase nanopore into the live cell membrane of HEK293T cells, and monitor the ssDNA delivery into the cell real-time at single molecule level. This type of nanopore is expected to provide an interesting tool to study the biophysics of biomotors in vitro, with potential applications in biosensing, drug delivery and real-time single cell analysis.

scholarly journals FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins

2020 ◽  
Author(s):  
Matthieu Lagardère ◽  
Ingrid Chamma ◽  
Emmanuel Bouilhol ◽  
Macha Nikolski ◽  
Olivier Thoumine
Keyword(s):  
Real Time ◽  
Protein Dynamics ◽  
Single Molecule ◽  
Imaging Techniques ◽  
Simulated Data ◽  
Super Resolution ◽  
Molecular Complexes ◽  
Live Cell ◽  
Single Molecule Level ◽  
Resolution Imaging

AbstractFluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these limitations, we present here a unified computer program that allows one to model and predict membrane protein dynamics at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments. FluoSim is an interactive real-time simulator of protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. The software, thoroughly validated against experimental data on the canonical neurexin-neuroligin adhesion complex, integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the distribution of thousands of independent molecules in 2D cellular geometries, providing simulated data of protein dynamics and localization directly comparable to actual experiments.

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.

scholarly journals A machine learning approach for accurate and real-time DNA sequence identification

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yiren Wang ◽  
Mashari Alangari ◽  
Joshua Hihath ◽  
Arindam K. Das ◽  
M. P. Anantram
Keyword(s):  
Real Time ◽  
Dna Sequence ◽  
Single Molecule ◽  
Identification Accuracy ◽  
Measuring System ◽  
Identification System ◽  
Genetic Sequencing ◽  
Sequence Identification ◽  
Tree Classifier ◽  
Single Mismatch

Abstract Background The all-electronic Single Molecule Break Junction (SMBJ) method is an emerging alternative to traditional polymerase chain reaction (PCR) techniques for genetic sequencing and identification. Existing work indicates that the current spectra recorded from SMBJ experimentations contain unique signatures to identify known sequences from a dataset. However, the spectra are typically extremely noisy due to the stochastic and complex interactions between the substrate, sample, environment, and the measuring system, necessitating hundreds or thousands of experimentations to obtain reliable and accurate results. Results This article presents a DNA sequence identification system based on the current spectra of ten short strand sequences, including a pair that differs by a single mismatch. By employing a gradient boosted tree classifier model trained on conductance histograms, we demonstrate that extremely high accuracy, ranging from approximately 96 % for molecules differing by a single mismatch to 99.5 % otherwise, is possible. Further, such accuracy metrics are achievable in near real-time with just twenty or thirty SMBJ measurements instead of hundreds or thousands. We also demonstrate that a tandem classifier architecture, where the first stage is a multiclass classifier and the second stage is a binary classifier, can be employed to boost the single mismatched pair’s identification accuracy to 99.5 %. Conclusions A monolithic classifier, or more generally, a multistage classifier with model specific parameters that depend on experimental current spectra can be used to successfully identify DNA strands.

Highly Sensitive Detection of Paraquat with Pillar[5]arene as an aptamer in α-Hemolysin Nanopore

2021 ◽  
Author(s):  
Xiaojia Jiang ◽  
Mingsong Zang ◽  
Fei Li ◽  
Chunxi Hou ◽  
Quan Luo ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Single Molecule Detection ◽  
Sensitive Detection ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
The Real ◽  
Highly Sensitive ◽  
Highly Sensitive Detection

Biological nanopore-based techniques have attracted more and more attention recently in the field of single-molecule detection, because they allow the real-time, sensitive, high-throughput analysis. Herein, we report an engineered biological...

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