Nanopore Biosensor Research Articles

Detection of Intrinsically Disordered Peptides by Biological Nanopore.

Intrinsically disordered protein regions (IDPRs) are pivotal in regulation of transcription and facilitation of signal transduction. Because of their multiple conformational states of structure, characterizing the highly flexible structures of IDPRs becomes challenging. Herein, we employed the wild-type (WT) aerolysin nanopore as a real-time biosensor for identification and monitoring of long peptides containing IDPRs. This sensor successfully identified three intrinsically disordered peptides, with the lengths up to 43 amino acids, by distinguishing the unique signatures of blockade current and duration time. The analysis of the binding constant revealed that interactions between the nanopore and peptides are critical for peptide translocation, which suggests that mechanisms beyond mere volume exclusion. Furthermore, we were able to compare the conformational stabilities of various IDPRs by examining the detailed current traces of blockade events. Our approach can detect the conformational changes of IDPR in a confined nanopore space. These insights broaden the understanding of peptide structural changes. The nanopore biosensor showed the potential to study the conformations change of IDPRs, IDPRs transmembrane interactions, and protein drug discovery.

Direct detection of C9orf72 hexanucleotide repeat expansions by nanopore biosensor

Diagnostic C9orf72 hexanucleotide repeat expansions (C9-HRE) is essential for the early and accurate diagnosis of amyotrophic lateral sclerosis (ALS) and will provide support for the prognosis and gene therapy of ALS. In the present study, by combining catalytic hairpin assembly (CHA) with Mycobacterium smegmatis porin A (MspA) nanopore, a new nanopore-based strategy for the detection of C9-HRE was reported. Less than 30 repeats of C9-HRE could be detected via this method, and the results have the potential to help distinguish between patients and healthy individuals. Moreover, the method demonstrated its great specificity for C9-HRE by identifying other repeat expansions. Given the high selectivity, this approach had been successfully used to detect C9-HRE in cell and blood samples with high accuracy. This detection strategy is user-friendly and has a strong anti-interference ability, thus providing a powerful tool for clinical diagnosis.

E. coli outer membrane protein T (OmpT) nanopore for peptide sensing

Peptide detection methods with facility and high sensitivity are essential for diagnosing disease associated with peptide biomarkers. Nanopore sensing technology had emerged as a low cost, high-throughput, and scalable tool for peptide detection. The omptins family proteins which can form β-barrel pores have great potentials to be developed as nanopore biosensor. However, there are no study about the channel properties of E. coli OmpT and the development of OmpT as a nanopore biosensor. In this study, the OmpT biological nanopore channel was constructed with a conductance of 1.49 nS in 500 mM NaCl buffer and a three-step gating phenomenon under negative voltage higher than 100 mV and then was developed as a peptide biosensor which can detect peptide without the interfere of ssDNA and dNTPs. The OmpT constructed in this study has potential application in peptide detection, and also provides a new idea for the detection of peptides using the specific binding ability of protease.

Real-Time Monitoring of Exosomes Secretion from Single Cell Using Dual-Nanopore Biosensors.

Exosomes secreted from cells carry rich information from their parent cells, representing a promising biomarker for investigation of diseases. We develop a dual-nanopore biosensor using DNA aptamers to specifically recognize CD63 protein on the exosome's surface, which enables label-free exosome detection based on ionic current change. The sensor allows for sensitive detection of exosomes with a detection limit of 3.4 × 106 particles/mL. The dual-nanopore biosensor was able to form an intrapipette electric circuit for ionic current measurement due to its unique structure, which is crucial to achieve detection of exosome secretion from a single cell. We utilized a microwell array chip to entrap a single cell into a confined microwell with small volume, enabling the accumulation of exosomes with high concentration. The dual-nanopore biosensor was positioned into the microwell with a single cell, and monitoring of exosome secretion from a single cell in different cell lines and under different stimulations has been achieved. Our design may provide a useful platform for developing nanopore biosensors for detecting cell secretions from a single living cell.

An Introduction to Nanopore Sequencing: Past, Present, and Future Considerations.

There has been significant progress made in the field of nanopore biosensor development and sequencing applications, which address previous limitations that restricted widespread nanopore use. These innovations, paired with the large-scale commercialization of biological nanopore sequencing by Oxford Nanopore Technologies, are making the platforms a mainstay in contemporary research laboratories. Equipped with the ability to provide long- and short read sequencing information, with quick turn-around times and simple sample preparation, nanopore sequencers are rapidly improving our understanding of unsolved genetic, transcriptomic, and epigenetic problems. However, there remain some key obstacles that have yet to be improved. In this review, we provide a general introduction to nanopore sequencing principles, discussing biological and solid-state nanopore developments, obstacles to single-base detection, and library preparation considerations. We present examples of important clinical applications to give perspective on the potential future of nanopore sequencing in the field of molecular diagnostics.

Nanopore single-molecule biosensor in protein denaturation analysis

Unclear issues in protein studies include but not limited to the stability and denaturation mechanism in the presence of denaturants. Herein, we report a dynamic monitoring approach based on nanopore single-molecule biosensor, which can detect the protein's folding and unfolding transitions by recording a nanopore ionic current. When gradually increasing the concentration of denaturant guanidine hydrochloride (GdmCl), sensitive responses were observed with lysozyme unfolding. The emergence of the featured biphasic-pulse demonstrated the existence of a stable intermediate. It was the first time to experimentally confirm the dynamic equilibrium between the intermediate and the native states at single molecule level, therefore consolidating the standpoint of lysozyme denaturation process following the three-state model. Additionally, we got more insights into the conformation about the intermediate as globular-like structure, larger gyration radius, and enhanced positive charge density. We considered that the manner of denaturant toward lysozyme adopts the “direct” model based on stronger electrostatic and van der Waals forces. Nanopore biosensor exhibited excellent sensitivity with a low detection concentration of 280 pM and reproducibility in analysing the folding intermediate of lysozyme.

Highly sensitive α-hemolysin nanopore detection of MUC1 based on 3D DNA walker

Mucin 1(MUC1) is an effective marker of breast cancer, so it is of great significance to develop a simple, sensitive and highly selective MUC1 detection sensor. Herein, we constructed a label-free nanopore biosensor for rapid and highly sensitive detection of MUC1. The presence of MUC1 triggered the modification of the DNAzyme walking chain on the surface of Fe3O4 nanoparticles and separation from the aptamer. In the presence of Zn2+, DNAzyme catalyzed hydrolytic cleavage of the hairpin substrate at the scissile rA. The DNAzyme was divided into two fragments and ssDNA was released. ssDNA products from the hairpin substrate can generate a current blocking signal during α-hemolysin nanopore testing. The frequency of signature events showed a linear response toward the concentration of MUC1 in the range of 0.01 nM–100 nM. The sensing system also exhibited high selectivity against other protein and can be used for the detection of real sample.

An aptamer-assisted biological nanopore biosensor for ultra-sensitive detection of ochratoxin A with a portable single-molecule measuring instrument

Biological nanopore-based single-molecule detection technology has shown ultrahigh sensitivity to various target analyte. But the detection scope of interesting targets is limited due to the lack of effective signal conversion strategies. In addition, conventional nanopore detection instruments are cumbersome, resulting nanopore detection can only be performed in laboratory. Herein, a customizable nanopore current amplifier is constructed to lower the cost and increase the portability of the nanopore instrument, and then an immobilized aptamer-based signal conversion strategy is proposed for α-hemolysin (α-HL) nanopore to detect small molecules (ochratoxin A, OTA). The presence of OTA in sample would trigger the release of probe single-strand DNA (ssDNA) from magnetic beads, which could subsequently cause current blockage in nanopore. The results show that the signal frequency of probe ssDNA has a linear relationship with the OTA concentration in the range of 2 × 101～2 × 103 pmol/L. Compared to other methods, our sensing system has achieved an ultra-sensitive detection of OTA with the detection limit as low as 1.697 pmol/L. This strategy could broaden the scope of nanopore detection and have the potential for rapid and in-situ detection of other food contaminants in the future.

Numerical Study of Single Molecular Charge Sensing by FET-Integrated Nanopore Biosensor

This report studies the charge-based sensing modality of FET-embedded nanopore biosensors through FEM simulation. PNP equation is solved to analyze the mirror charge introduced by charged biomolecule while threading through the nanopore-FET sensor. Negative and positive charged molecules are analyzed respectively. Obvious local potential change induced by the presenting of charged molecules nearby is observed. In addition, the transport-induced descreening effect is observed under intensive bias, which might explain the capability of charge sensing even under high concentrations such as 1 M for FET-nanopore biosensors.

Spatiotemporally Resolved Protein Detection in Live Cells Using Nanopore Biosensors.

Spatiotemporal detection of proteins in living cells is a persistent challenge but is the key to understanding their cellular biology and developing theranostic technologies. We develop a dual-nanopore biosensor using affinity-tunable peptide probes, which enables label-free and spatiotemporal monitoring of protein abundance and its concentration change in single live cells. We demonstrate that by screening for peptide probes with tunable affinities, the nanopore modified with a medium-affinity peptide allowed reversible and sensitive detection of the protein kinase A (PKA) catalytic subunit with a detection limit of 0.04 nM. The sensor is shown to have the ability to effectively eliminate interferences from cell membrane resistance and coexisting species in live cell detection. Moreover, our sensor is successfully implemented in monitoring of dynamic PKA activity changes (PKA catalytic subunit dynamic concentration changes) under different stimulations in single live cells. Our design may provide a paradigm for developing nanopore biosensors for spatiotemporally resolved protein analysis in live cells.

Ultrasensitive and Label-Free Detection of Multiple DNA Methyltransferases by Asymmetric Nanopore Biosensor.

DNA methylation is catalyzed by a family of DNA methyltransferases that play crucial roles in various biological processes. Therefore, an ultrasensitive methyltransferase assay is highly desirable in biomedical research and clinical diagnosis. However, conventional assays for the detection of DNA methyltransferase activity often involve radioactive labeling, costly equipment, and laborious operation. In this study, an ultrasensitive and label-free method for detecting DNA adenine methyltransferase (Dam) and CpG methyltransferase (M.SssI) was developed using the nanopore technique coupled with DNA cascade signal amplification reactions. A hairpin DNA (HD) comprising of the methylation-responsive sequences was skillfully designed. In the presence of Dam methyltransferase, the corresponding recognition site of hairpin HD was methylated and specifically cleaved by DpnI endonuclease, thus forming a DNA fragment that induces the catalytic hairpin assembly and hybridization chain reaction (CHA-HCR). The generated products could be absorbed onto the Zr4+-coated nanopore, resulting in an ion current rectification signal change. Considering the high sensitivity of the nanopore and excellent specificity toward the recognition of methyltransferase/endonuclease, our developed method could detect both Dam and M.SssI methyltransferases in the same sensing platform. Furthermore, the designed nanopore sensor could realize the multiplex detection of Dam and M.SssI methyltransferases after integration with the cascaded INHIBIT-AND logic gate. This ultrasensitive methyltransferase assay holds great promise in the field of cancer diagnosis.

A molecular dynamics investigation of Taq DNA polymerase and its complex with a DNA substrate using a solid-state nanopore biosensor.

Proteins have a small volume difference by the diversity of amino acids, which make protein detection and identification a great challenge. Solid-state nanopore as label-free biosensors has attracted attention with high sensitivity. In this work, we investigated the Taq DNA polymerase before and after combining it with a DNA substrate on a solid-state nanopore through molecular dynamics. In simulation, we analyzed the contribution source of nanopore current blockage. In addition to considering the traditional physical exclusion volume model, the non-covalent interaction between the protein molecules and the pore wall also showed to affect the current blockage in the nanopore. When choosing pores of comparable size to protein molecules, the two states of Taq DNA polymerase produce differentiated non-covalent interactions with the pore wall, which enhanced the amplitude difference in current blockage. As a result, the two DNA polymerases can be distinguished through the distinct current blockage. However, when applying additional pulling force or increasing the pore size of the nanopore, the differences between the current blockages are not significant enough to distinguish. The introduction of the non-covalent interaction makes it clear to understand the current blockage differences, which guide the mechanism between molecules with similar structures or volumes.

In Vitro Biosensing of β-Amyloid Peptide Aggregation Dynamics using a Biological Nanopore.

Alzheimer's disease and other neurodegenerative disorders are becoming more prevalent as advances in technology and medicine increase living standards and life expectancy. Alzheimer's disease is thought to initiate development early in the patient's life and progresses continuously into old age. This process is characterized molecularly by the amyloid hypothesis, which asserts that self-aggregating amyloid peptides are core to the pathophysiology in Alzheimer's progression. Precise quantification of amyloid peptides in human bodily fluid samples (i.e. cerebrospinal fluid, blood) may inform diagnosis and prognosis, and has been studied using established biosensing technologies like liquid chromatography, mass spectrometry, and immunoassays. However, existing methods are challenged to provide single molecule, quantitative analysis of the disease-causing aggregation process. Ultra-sensitive nanopore biosensors can step in to fill this role as a dynamic mapping tool. The work in this paper establishes characteristic signals of β-amyloid 40 monomers, oligomers, and soluble aggregates, as well as a proof-of-concept foundation where a biological nanopore biosensor is used to monitor the extent of in vitro β-amyloid 40 peptide aggregation at the single molecule level. This foundation allows for future work to expand in drug screening, diagnostics, and aggregation dynamic experiments.

Unbalanced Ion Flushing Effect in MoS2 Nanopore Biosensors

In MoS2 nanopores, in addition to the widely measured ionic current signal to detect DNA nucleotides or epigenetic malformations, recent efforts have focused on the use of the transverse electronic current along the membrane for biomolecules sensing. This approach provides larger current magnitude and allows detection at the observed DNA translocation speed in the solid-state nanopore, which are crucial for accurate biomolecule sensing and faster DNA sequencing. As of today, no existing model is able to relate the variations of the transverse current to the interaction between ions, biomolecules and the MoS2 membrane. In this presentation, we report a comprehensive transport model based on the Poisson-Boltzmann transport formalism that considers the effect of biomolecules, electrolyte, and ion screening on conducting carriers along a MoS2 membrane. By accounting for membrane geometry and material properties used in recent experiment, we are able to extract the different electrical contributions to the transverse current variation during a DNA translocation event. In particular, we show that the major contribution to the electrical signal is caused by an unbalanced-ion-flushing effect that arises when the ion double-layer around the nanopore edge is swept out of the pore by the biomolecule translocation. By optimizing the MoS2 membrane geometry and nanopore size, we predict higher sensitivity to detect the translocation event in the corresponding transverse current signal. Thus,the identification of the unbalanced ion effect on the transverse electronic current foresees a critical new direction to improving the signal-to-noise ratio in solid-state nanopore sensing device.

Multiplexed Molecular Counters using a High-Voltage Transmembrane Pore Platform

Synthetic andBiological nanopores have been used as powerful platforms for label-free detection and identification of a spectrum of biomolecules, which are used in applications that range from biosensing, single-molecule biophysics, and DNA/RNA sequencing. Nonetheless, their relatively high limit of detection (LOD) has inhibited further development of nanopore biosensors. Traditionally, biological nanopores are incorporated into planar lipid membranes painted on PTFE apertures, and thus their LOD is limited by the maximum voltage bias that can be applied across the lipid bilayer and protein nanopore. Here, by using grayscale lithography, we developed a polymer support for lipid bilayers that consists of an aperture with a unique 3D profile. We find that lipid bilayer membranes formed on the support exhibit longer lifetimes and a consistent ability to sustain voltages as high as 350 mV, without compromising the diameter of the aperture. By increasing the applied voltage, in addition to improving the signal-to-noise ratio, the LOD of DNA hairpins in α-hemolysin nanopores was pushed to the nM regime. To harness this platform, we demonstrate the use of different length DNA hairpins as multiplexed reporter molecules for biosensing. As proof-of-concept, we demonstrate DNA hairpin release from magnetic microbeads through a site-specific enzymatic digestion reaction, followed by direct quantification of the hairpins using a nanopore on our platform at high voltage. This convenient approach, which is general for any ELISA-type biomarker assay and does not require further cleanup steps following digestion, paves the way for development of a novel nanopore-based multiplexed biosensing strategies in which small concentrations/volumes are required. Finally, since our polymer support chips are housed on a silicon-based chip, our devices are compatible with optical sensing, as well as integration with silicon-based microfluidic elements.

Nanopore Fabrication and Application as Biosensors in Neurodegenerative Diseases.

Since its conception as an applied biomedical technology nearly 30 years ago, nanopore is emerging as a promising, high-throughput, biomarker-targeted diagnostic tool for clinicians. The attraction of a nanopore-based detection system is its simple, inexpensive, robust, user-friendly, high-throughput blueprint with minimal sample preparation needed prior to analysis. The goal of clinical-based nanopore biosensing is to go from sample acquisition to a meaningful readout quickly. The most extensive work in nanopore applications has been targeted at DNA, RNA, and peptide identification. Although, biosensing of pathological biomarkers, which is covered in this review, is on the rise. This review is broken into two major sections: (i) the current state of existing biological, solid state, and hybrid nanopore systems and (ii) the applications of nanopore biosensors toward detecting neurodegenerative biomarkers.

Nanopore biphasic-pulse biosensor

Nanopores as artificial biomimetic nanodevices are of great importance for their applications in biosensing, nanomedicine and bioelectronics. However, it remains a challenge to detect small biomolecules especially small-sized proteins with high sensitivity and selectivity. In the article, we report a simple and efficient method for small-sized protein detection by constructing biphasic-pulse nanopore biosensor. Unlike the traditional resistive pulse sensing, the biphasic-pulse event can provide unique and abundant fingerprint information. Although the nanopore biphasic-pulse electrical signal is originated from both the molecular exclusion electrical resistance and the surface-charged effect of confined molecule, its frequency and amplitude of the waveform can be adjusted by pH, applied potential and salt concentration. Based on the frequency of the biphasic pulse, nanomolar concentration of proteins could be specifically detected and the limit of detection is 1.2 nM. In addition, the biphasic-pulse nanopore shows well discrimination in similar-sized protein detection and its signal generation is highly reproducible. The nanopore biphasic-pulse biosensor should have broad applications as a new generation of powerful single-molecule device.

Nanopore blockade sensors for ultrasensitive detection of proteins in complex biological samples

Nanopore sensors detect individual species passing through a nanoscale pore. This experimental paradigm suffers from long analysis times at low analyte concentration and non-specific signals in complex media. These limit effectiveness of nanopore sensors for quantitative analysis. Here, we address these challenges using antibody-modified magnetic nanoparticles ((anti-PSA)-MNPs) that diffuse at zero magnetic field to capture the analyte, prostate-specific antigen (PSA). The (anti-PSA)-MNPs are magnetically driven to block an array of nanopores rather than translocate through the nanopore. Specificity is obtained by modifying nanopores with anti-PSA antibodies such that PSA molecules captured by (anti-PSA)-MNPs form an immunosandwich in the nanopore. Reversing the magnetic field removes (anti-PSA)-MNPs that have not captured PSA, limiting non-specific effects. The combined features allow detecting PSA in whole blood with a 0.8 fM detection limit. Our ‘magnetic nanoparticle, nanopore blockade’ concept points towards a strategy to improving nanopore biosensors for quantitative analysis of various protein and nucleic acid species.

Reversing current rectification to improve DNA-sensing sensitivity in conical nanopores.

Herein, we report the ultrasensitive DNA detection through designing an elegant nanopore biosensor as the first case to realize the reversal of current rectification direction for sensing. Attributed to the unique asymmetric structure, the glass conical nanopore exhibits the sensitive response to the surface charge, which can be facilely monitored by ion current rectification curves. In our design, an enzymatic cleavage reaction was employed to alter the surface charge of the nanopore for DNA sensing. The measured ion current rectification was strongly responsive to DNA concentrations, even reaching to the reversed status from the negative ratio (-6.5) to the positive ratio (+16.1). The detectable concentration for DNA was as low as 0.1fM. This is an ultrasensitive and label-free DNA sensing approach, based on the rectification direction-reversed amplification in a single glass conical nanopore.

Plasmonic-Nanopore Biosensors for Superior Single-Molecule Detection.

Plasmonic and nanopore sensors have separately received much attention for achieving single-molecule precision. A plasmonic "hotspot" confines and enhances optical excitation at the nanometer length scale sufficient to optically detect surface-analyte interactions. A nanopore biosensor actively funnels and threads analytes through a molecular-scale aperture, wherein they are interrogated by electrical or optical means. Recently, solid-state plasmonic and nanopore structures have been integrated within monolithic devices that address fundamental challenges in each of the individual sensing methods and offer complimentary improvements in overall single-molecule sensitivity, detection rates, dwell time and scalability. Here, the physical phenomena and sensing principles of plasmonic and nanopore sensing are summarized to highlight the novel complementarity in dovetailing these techniques for vastly improved single-molecule sensing. A literature review of recent plasmonic nanopore devices is then presented to delineate methods for solid-state fabrication of a range of hybrid device formats, evaluate the progress and challenges in the detection of unlabeled and labeled analyte, and assess the impact and utility of localized plasmonic heating. Finally, future directions and applications inspired by the present state of the art are discussed.