Label-free and amplification-free viral RNA quantification from primate biofluids using a trapping-assisted optofluidic nanopore platform.

Viral outbreaks can cause widespread disruption, creating the need for diagnostic tools that provide high performance and sample versatility at the point of use with moderate complexity. Current gold standards such as PCR and rapid antigen tests fall short in one or more of these aspects. Here, we report a label-free and amplification-free nanopore sensor platform that overcomes these challenges via direct detection and quantification of viral RNA in clinical samples from a variety of biological fluids. The assay uses an optofluidic chip that combines optical waveguides with a fluidic channel and integrates a solid-state nanopore for sensing of individual biomolecules upon translocation through the pore. High specificity and low limit of detection are ensured by capturing RNA targets on microbeads and collecting them by optical trapping at the nanopore location where targets are released and rapidly detected. We use this device for longitudinal studies of the viral load progression for Zika and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections in marmoset and baboon animal models, respectively. The up to million-fold trapping-based target concentration enhancement enables amplification-free RNA quantification across the clinically relevant concentration range down to the assay limit of RT-qPCR as well as cases in which PCR failed. The assay operates across all relevant biofluids, including semen, urine, and whole blood for Zika and nasopharyngeal and throat swab, rectal swab, and bronchoalveolar lavage for SARS-CoV-2. The versatility, performance, simplicity, and potential for full microfluidic integration of the amplification-free nanopore assay points toward a unique approach to molecular diagnostics for nucleic acids, proteins, and other targets.

Synthetic heparan sulfate standards and machine learning facilitate the development of solid-state nanopore analysis.

The application of solid-state (SS) nanopore devices to single-molecule nucleic acid sequencing has been challenging. Thus, the early successes in applying SS nanopore devices to the more difficult class of biopolymer, glycosaminoglycans (GAGs), have been surprising, motivating us to examine the potential use of an SS nanopore to analyze synthetic heparan sulfate GAG chains of controlled composition and sequence prepared through a promising, recently developed chemoenzymatic route. A minimal representation of the nanopore data, using only signal magnitude and duration, revealed, by eye and image recognition algorithms, clear differences between the signals generated by four synthetic GAGs. By subsequent machine learning, it was possible to determine disaccharide and even monosaccharide composition of these four synthetic GAGs using as few as 500 events, corresponding to a zeptomole of sample. These data suggest that ultrasensitive GAG analysis may be possible using SS nanopore detection and well-characterized molecular training sets.

Electrical unfolding of cytochrome c during translocation through a nanopore constriction.

Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field-driven translocation behavior of cytochrome c (cyt c) through ultrathin silicon nitride (SiNx) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5-nm-diameter pore, we find that, in a threshold electric-field regime of ∼30 to 100 MV/m, cyt c is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt c is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5- and 2.0-nm-diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (∼ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens avenues for exploring protein folding structures, internal contacts, and electric-field-induced deformability.

Aggregation and Oligomerization Characterization of ß-Lactoglobulin Protein Using a Solid-State Nanopore Sensor.

Protein aggregation is linked to many chronic and devastating neurodegenerative human diseases and is strongly associated with aging. This work demonstrates that protein aggregation and oligomerization can be evaluated by a solid-state nanopore method at the single molecule level. A silicon nitride nanopore sensor was used to characterize both the amyloidogenic and native-state oligomerization of a model protein ß-lactoglobulin variant A (ßLGa). The findings from the nanopore measurements are validated against atomic force microscopy (AFM) and dynamic light scattering (DLS) data, comparing ßLGa aggregation from the same samples at various stages. By calibrating with linear and circular dsDNA, this study estimates the amyloid fibrils' length and diameter, the quantity of the ßLGa aggregates, and their distribution. The nanopore results align with the DLS and AFM data and offer additional insight at the level of individual protein molecular assemblies. As a further demonstration of the nanopore technique, ßLGa self-association and aggregation at pH 4.6 as a function of temperature were measured at high (2 M KCl) and low (0.1 M KCl) ionic strength. This research highlights the advantages and limitations of using solid-state nanopore methods for analyzing protein aggregation.

Probing the Effect of Ubiquitinated Histone on Mononucleosomes by Translocation Dynamics Study through Solid-State Nanopores.

Post-translational modifications (PTMs), such as ubiquitination, are critically important in regulating genetic expressions by adjusting the nucleosome stability. A fast and label-free technology inspecting dynamic nucleosome structures can facilitate the interrogation of PTMs effects. Here we leverage the advantages of mechanically stable solid-state nanopores and detect the effect of a ubiquitinated histone on mononucleosomes at the single-molecule level. By comparing the translocation dynamics of natural and cross-linked mononucleosomes, we verified that the nucleosomal DNA unravelled from histones in natural mononucleosomes. Furthermore, we found that a turning point of voltage corresponds to the onset of nucleosome rupture. More importantly, we reveal that ubH2A stabilizes the nucleosome by shifting the turning point to a larger value and investigated the effect of ubiquitination on different histones (ubH2A and ubH2B). These findings open promising possibilities for developing a miniaturized and portable device for the fast screening of PTMs on nucleosomes.

Registration of Functioning of a Single Horseradish Peroxidase Macromolecule with a Solid-State Nanopore.

Currently, nanopore-based technology for the determination of the functional activity of single enzyme molecules continues its development. The use of natural nanopores for studying single enzyme molecules is known. At that, the approach utilizing artificial solid-state nanopores is also promising but still understudied. Herein, we demonstrate the use of a nanotechnology-based approach for the investigation of the enzymatic activity of a single molecule of horseradish peroxidase with a solid-state nanopore. The artificial 5 nm solid-state nanopore has been formed in a 40 nm thick silicon nitride structure. A single molecule of HRP has been entrapped into the nanopore. The activity of the horseradish peroxidase (HRP) enzyme molecule inserted in the nanopore has been monitored by recording the time dependence of the ion current through the nanopore in the course of the reaction of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) oxidation reaction. We have found that in the process of ABTS oxidation in the presence of 2.5 mM hydrogen peroxide, individual HRP enzyme molecules are able to retain activity for approximately 700 s before a decrease in the ion current through the nanopore, which can be explained by structural changes of the enzyme.

Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation.

Solid-state nanopores have been used extensively in biomolecular studies involving DNA and proteins. However, the interpretation of signals generated by the translocation of proteins or protein-DNA complexes remains challenging. Here, we investigate the behavior of monovalent streptavidin and the complex it forms with short biotinylated DNA over a range of nanopore sizes, salts, and voltages. We describe a simple geometric model that is broadly applicable and employ it to explain observed variations in conductance blockage and dwell time with experimental conditions. The general approach developed here underscores the value of nanopore-based protein analysis and represents progress toward the interpretation of complex translocation signals.

Fabrication of solid-state nanopores.

Nanopores are valuable single-molecule sensing tools that have been widely applied to the detection of DNA, RNA, proteins, viruses, glycans, etc. The prominent sensing platform is helping to improve our health-related quality of life and accelerate the rapid realization of precision medicine. Solid-state nanopores have made rapid progress in the past decades due to their flexible size, structure and compatibility with semiconductor fabrication processes. With the development of semiconductor fabrication techniques, materials science and surface chemistry, nanopore preparation and modification technologies have made great breakthroughs. To date, various solid-state nanopore materials, processing technologies, and modification methods are available to us. In the review, we outline the recent advances in nanopores fabrication and analyze the virtues and limitations of various membrane materials and nanopores drilling techniques.

Nanopore Identification of Single Nucleotide Mutations in Circulating Tumor DNA by Multiplexed Ligation.

BACKGROUND: Circulating tumor DNAs (ctDNAs) are highly promising cancer biomarkers, potentially applicable for noninvasive liquid biopsy and disease monitoring. However, to date, sequencing of ctDNAs has proven to be challenging primarily due to small sample size and high background of fragmented cell-free DNAs (cfDNAs) derived from normal cells in the circulation, specifically in early stage cancer. METHODS: Solid-state nanopores (ssNPs) have recently emerged as a highly efficient tool for single-DNA sensing and analysis. Herein, we present a rapid nanopore genotyping strategy to enable an amplification-free identification and classification of ctDNA mutations. A biochemical ligation detection assay was used for the creation of specific fluorescently-labelled short DNA reporter molecules. Color conjugation with multiple fluorophores enabled a unique multi-color signature for different mutations, offering multiplexing potency. Single-molecule readout of the fluorescent labels was carried out by electro-optical sensing via solid-state nanopores drilled in titanium oxide membranes. RESULTS: As proof of concept, we utilized our method to detect the presence of low-quantity ERBB2 F310S and PIK3Ca H1047R breast cancer mutations from both plasmids and xenograft mice blood samples. We demonstrated an ability to distinguish between a wild type and a mutated sample, and between the different mutations in the same sample. CONCLUSIONS: Our method can potentially enable rapid and low cost ctDNA analysis that completely circumvents PCR amplification and library preparation. This approach will thus meet a currently unmet demand in terms of sensitivity, multiplexing and cost, opening new avenues for early diagnosis of cancer.

Detection of nucleotides in hydrated ssDNA via 2D h-BN nanopore with ionic-liquid/salt-water interface.

Accomplishing slow translocation speed with high sensitivity has been the most critical mission for solid-state nanopore (SSN) device to electrically detect nucleobases in ssDNA. In this study, a method to detect nucleobases of ssDNA using a 2D SSN is introduced by considerably reducing the translocation speed and effectively increasing its sensitivity. The ultra-thin titanium dioxide coated hexagonal boron nitride nanopore was fabricated, along with an ionic-liquid 1-butyl-3-methylimidazolium hexafluorophosphate/2.0 M KCl aqueous (cis/trans) interface, for increasing both the spatial and the temporal resolutions. As the ssDNA molecules entered the nanopore, a brief surge of electrical conductivity occurred, which was followed by multiple resistive pulses from nucleobases during the translocation of ssDNA and another brief current surge flagging the exit of the molecule. The continuous detection of nucleobases using a 2D SSN device is a novel achievement: the water molecules bound to ssDNA increased the molecular conductivity and amplified electrical signals during the translocation. Along with the experiment, computational simulations using COMSOL Multiphysics are presented to explain the pivotal role of water molecules bound to ssDNA to detect nucleobases using a 2D SSN.

The effect of laser irradiation on reducing the noise of solid-state nanopore.

The performance of solid-state nanopore is affected by the noise level. This study aimed to investigate the effect of laser irradiation on the noise performance of solid-state nanoporein situ. Laser irradiation is applied to fresh and contaminated nanopores. The measurement results show that the noise of fresh and contaminated nanopores decreases with the laser power and there is a threshold of laser power in reducing the noise of contaminated nanopores. The possible reasons for reducing noise in the laser irradiation process are discussed. The laser treatment is proven to provide a convenient method for reducing the noise of solid-state nanopore.

Solid-state nanopore analysis of human genomic DNA shows unaltered global 5-hydroxymethylcytosine content associated with early-stage breast cancer.

5-Hydroxymethylcytosine (5hmC), the first oxidized form of the well-known epigenetic modification 5-methylcytosine, is an independent regulator of gene expression and therefore a potential marker for disease. Here, we report on methods developed for a selective solid-state nanopore assay that enable direct analysis of global 5hmC content in human tissue. We first describe protocols for preparing genomic DNA derived from both healthy breast tissue and stage 1 breast tumor tissue and then use our approach to probe the net abundance of the modified base in each cohort. Then, we employ empirical data to adjust for the impact of nanopore diameter on the quantification. Correcting for variations in nanopore diameter among the devices used for analysis reveals no detectable difference in global 5hmC content between healthy and tumor tissue. These results suggest that 5hmC changes may not be associated with early-stage breast cancer and instead are a downstream consequence of the disease.

Monitoring nanobubble nucleation at early-stage within a sub-9 nm solid-state nanopore.

Nanobubble nucleation study is important for understanding the dynamic behavior of nanobubble growth, which is instructive for the nanobubble applications. Benefiting from nanopore fabrication, herein, we fabricated a sub-9 nm SiNX nanopore with the comparable size to nanobubbles at early-stage. The confined nanopore interface serves as a generator for producing nanobubbles by the chemical reaction between NaBH4 and H2 O and as an ultra-sensitive sensor for monitoring the H2 nanobubble nucleation process. By carrying out the NaBH4 concentration-dependent experiments, we found the life-time of nanobubbles decreased 250 times and the frequency of nanobubble generation increased 38 times with the NaBH4 concentration increasing from 6 to 100 mM. The long-time equilibrium between gas molecules inward flux and outward flux could prolong the life-time of nanobubbles to hundreds of milliseconds at low NaBH4 concentration. The raw current trace depicted that the transient accumulation and dissolution of cavity occurred during all the life-time of nanobubbles. Therefore, the sub-9 nm SiNX nanopore shows a strong ability for real-time monitoring the nanobubble nucleation at early-stage with high temporal and spatial resolution. This work provides a guide to study the dynamic and stochastic characteristics of nanobubbles.

Application of Solid-State Nanopore in Protein Detection.

A protein is a kind of major biomacromolecule of life. Its sequence, structure, and content in organisms contains quite important information for normal or pathological physiological process. However, research of proteomics is facing certain obstacles. Only a few technologies are available for protein analysis, and their application is limited by chemical modification or the need for a large amount of sample. Solid-state nanopore overcomes some shortcomings of the existing technology, and has the ability to detect proteins at a single-molecule level, with its high sensitivity and robustness of device. Many works on detection of protein molecules and discriminating structure have been carried out in recent years. Single-molecule protein sequencing techniques based on solid-state nanopore are also been proposed and developed. Here, we categorize and describe these efforts and progress, as well as discuss their advantages and drawbacks.

Detection of structured single-strand DNA via solid-state nanopore.

Nanopore is a single-molecule analysis method which also employed electrophoresis has achieved promising single-molecule detections. In this study, we designed two kinds of confined spaces by fabricating solid-state nanopores with desirable diameters to study the structured single-strand DNA of C-rich quadruplex. For the nanopore whose diameter is larger than the quadruplex size, the DNA molecule could directly translocate through the nanopore with extremely high speed. For the nanopore whose diameter is smaller than the quadruplex size, DNA molecule which is captured by nanopore could return to the solution without translocation or unzip the quadruplex structure into single-strand and then pass the nanopore. This study certifies that choosing a suitable sensing interface is the vital importance of observing detailed single-molecule information. The solid-state nanopores hold the great potential to study the structural dynamics of quadruplex DNA molecule.

Stiffness measurement of nanosized liposomes using solid-state nanopore sensor with automated recapturing platform.

This paper describes a method to gauge the stiffness of nanosized liposomes - a nanoscale vesicle - using a custom-made recapture platform coupled to a solid-state nanopore sensor. The recapture platform electrically profiles a given liposome vesicle multiple times through automated reversal of the voltage polarity immediately following a translocation instance to re-translocate the same analyte through the nanopore - provides better statistical insight at the molecular level by analyzing the same particle multiple times compared to conventional nanopore platforms. The capture frequency depends on the applied voltage with lower voltages (i.e., 100 mV) permitting higher recapture instances than at higher voltages (>200 mV) since the probability of particles exiting the nanopore capture radius increases with voltage. The shape deformation was inferred by comparing the normalized relative current blockade ( ΔI/I0̂) at the two voltage polarities to that of a rigid particle, i.e., polystyrene beads. We found that liposomes deform to adopt a prolate shape at higher voltages. This platform can be further applied to investigate the stiffness of other types of soft matters, e.g., virus, exosomes, endosomes, and accelerate the potential studies in pharmaceutics for increasing the drug packing and unpacking mechanism by controlling the stiffness of the drug vesicles.

Mechanical characterization of HIV-1 with a solid-state nanopore sensor.

Enveloped viruses fuse with cells to transfer their genetic materials and infect the host cell. Fusion requires deformation of both viral and cellular membranes. Since the rigidity of viral membrane is a key factor in their infectivity, studying the rigidity of viral particles is of great significance in understating viral infection. In this paper, a nanopore is used as a single molecule sensor to characterize the deformation of pseudo-type human immunodeficiency virus type 1 at sub-micron scale. Non-infective immature viruses were found to be more rigid than infective mature viruses. In addition, the effects of cholesterol and membrane proteins on the mechanical properties of mature viruses were investigated by chemically modifying the membranes. Furthermore, the deformability of single virus particles was analyzed through a recapturing technique, where the same virus was analyzed twice. The findings demonstrate the ability of nanopore resistive pulse sensing to characterize the deformation of a single virus as opposed to average ensemble measurements.

Increased dwell time and occurrence of dsDNA translocation events through solid state nanopores by LiCl concentration gradients.

Solid-state nanopore based biosensors are cost effective, high-throughput engines for single molecule detection of biomolecules with the added benefit of size modification. Progress in the translation of the science into a viable diagnostic tool is impeded by inadequate sensitivity of data acquisition systems in detection of fast DNA translocations through the pore. To combat this, slowing the transport of DNA through the nanopore by use of various media or by altering experimental parameters is common. Applying a concentration gradient of KCl in the experimental ionic solution has been shown to effectively prolong dwell times as well as increase the capture rate of DNA by the nanopore. Our previous work has corroborated the ability of LiCl ionic solution to slow down the transport of dsDNA through the nanopore by up to 10-fold through cation-DNA interactions. However, this drastically reduced the event occurrence frequency, thus hindering the efficacy of this system as a reliable biosensor downstream. Here, we present the use of a concentration gradient of lithium chloride ionic solution to increase the event frequency of single molecule dsDNA translocation through a solid state nanopore. By using 0.5 M/3 M LiCl on the cis/trans chambers respectively, average dwell times experienced up to a 3-fold increase when compared to experiments run in symmetric 1 M LiCl. Additionally, experiments using the 0.5 M/3 M displayed a greater than 10-fold increase in event frequency, confirming the capture propensity of the asymmetric conditions.

Integrating Sub-3 nm Plasmonic Gaps into Solid-State Nanopores.

Plasmonic nanopores combine the advantages of nanopore sensing and surface plasmon resonances by introducing confined electromagnetic fields to a solid-state nanopore. Ultrasmall nanogaps between metallic nanoantennas can generate the extremely enhanced localized electromagnetic fields necessary for single-molecule optical sensing and manipulation. Challenges in fabrication, however, hamper the integration of such nanogaps into nanopores. Here, a top-down approach for integrating a plasmonic antenna with an ultrasmall nanogap into a solid-state nanopore is reported. Employing a two-step e-beam lithography process, the reproducible fabrication of nanogaps down to a sub-1 nm scale is demonstrated. Subsequently, nanopores are drilled through the 20 nm SiN membrane at the center of the nanogap using focused-electron-beam sculpting with a transmission electron microscope, at the expense of a slight gap expansion for the smallest gaps. Using this approach, sub-3 nm nanogaps can be readily fabricated on solid-state nanopores. The functionality of these plasmonic nanopores for single-molecule detection is shown by performing DNA translocations. These integrated devices can generate intense electromagnetic fields at the entrance of the nanopore and can be expected to find applications in nanopore-based single-molecule trapping and optical sensing.

Single Molecule DNA Resensing Using a Two-Pore Device.

A nanofluidic device is presented that, enables independent sensing and resensing of a single DNA molecule translocating through two nanopores with sub-micrometer spacing. The device concept is based upon integrating a thin nitride membrane with microchannels etched in borosilicate glass. Pores, coupled to each microchannel, are connected via a fluid-filled half-space on the device backside, enabling translocation of molecules across each pore in sequence. Critically, this approach allows for independent application of control voltage and measurement of trans-pore ionic current at each of the two pores, leading to 1) controlled assessment of molecular time of flight, 2) voltage-tuned selective molecule recapture, and 3) ability to acquire two correlated translocation signatures for each molecule analyzed. Finally, the rare cocapture of a single chain threading simultaneously through each of the two pores is reported.