Identification of Conformational Variants for Bradykinin Biomarker Peptides from a Biofluid Using a Nanopore and Machine Learning.

There is a current need to develop methods for the sensitive detection of peptide biomarkers in complex mixtures of molecules, such as biofluids, to enable early disease detection. Moreover, to our knowledge, there is currently no detection method capable of identifying the different conformations of a peptide biomarker differing by a single amino acid. Single-molecule nanopore sensing promises to provide this level of resolution. In order to be able to identify these differences in a biofluid such as serum, it is necessary to carefully characterize electrical parameters to obtain specific signatures of each biomarker population observed. We are interested here in a family of peptide biomarkers, kinins such as bradykinin and des-Arg9 bradykinin, that are involved in many disabling pathologies (allergy, asthma, angioedema, sepsis, or cancer). We show the proof of concept for direct identification of these biomarkers in serum at the single-molecule level using a protein nanopore. Each peptide exhibits two unique electrical signatures attributed to specific conformations in bulk. The same signatures are found in serum, allowing their discrimination and identification in a complex mixture such as biofluid. To extend the utility of our experimental results, we developed a principal component analysis approach to define the most relevant electrical parameters for their identification. Finally, we used semisupervised classification to assign each event type to a specific biomarker at physiological serum concentration. In the future, single-molecule scale analysis of peptide biomarkers using a powerful nanopore coupled with machine learning will facilitate the identification and quantification of other clinically relevant biomarkers from biofluids.

Nanopore Discrimination of Coagulation Biomarker Derivatives and Characterization of a Post-Translational Modification.

One of the most important health challenges is the early and ongoing detection of disease for prevention, as well as personalized treatment management. Development of new sensitive analytical point-of-care tests are, therefore, necessary for direct biomarker detection from biofluids as critical tools to address the healthcare needs of an aging global population. Coagulation disorders associated with stroke, heart attack, or cancer are defined by an increased level of the fibrinopeptide A (FPA) biomarker, among others. This biomarker exists in more than one form: it can be post-translationally modified with a phosphate and also cleaved to form shorter peptides. Current assays are long and have difficulties in discriminating between these derivatives; hence, this is an underutilized biomarker for routine clinical practice. We use nanopore sensing to identify FPA, the phosphorylated FPA, and two derivatives. Each of these peptides is characterized by unique electrical signals for both dwell time and blockade level. We also show that the phosphorylated form of FPA can adopt two different conformations, each of which have different values for each electrical parameter. We were able to use these parameters to discriminate these peptides from a mix, thereby opening the way for the potential development of new point-of-care tests.

Circulating Tumor Cells in Cancer Diagnostics and Prognostics by Single-Molecule and Single-Cell Characterization.

Circulating tumor cells (CTCs) represent an interesting source of biomarkers for diagnosis, prognosis, and the prediction of cancer recurrence, yet while they are extensively studied in oncobiology research, their diagnostic utility has not yet been demonstrated and validated. Their scarcity in human biological fluids impedes the identification of dangerous CTC subpopulations that may promote metastatic dissemination. In this Perspective, we discuss promising techniques that could be used for the identification of these metastatic cells. We first describe methods for isolating patient-derived CTCs and then the use of 3D biomimetic matrixes in their amplification and analysis, followed by methods for further CTC analyses at the single-cell and single-molecule levels. Finally, we discuss how the elucidation of mechanical and morphological properties using techniques such as atomic force microscopy and molecular biomarker identification using nanopore-based detection could be combined in the future to provide patients and their healthcare providers with a more accurate diagnosis.

Dynamics of DNA Through Solid-state Nanopores Fabricated by Controlled Dielectric Breakdown.

Controlled dielectric breakdown (CDB) is gaining popularity for fabricating solid-state nanopores in situ with size control in a simple, low-cost, and scalable way. This technique could be used for a broad type of applications in the field of nucleic acid analysis and even for protein studies. In this work, we studied the entry and transport of double-stranded DNAs using a solid-state nanopore fabricated by CDB as a function of applied voltage for two different DNA lengths. We showed that the blockade rate increases exponentially with voltage up to 120 mV. The energy barrier depends on the chain length, and the dwell times decrease with applied voltage up to 120 mV. Moreover, no matter the chain length, it is possible to differentiate two families of blockade amplitudes, high and low ones, due to DNA folding.

Focus on using nanopore technology for societal health, environmental, and energy challenges.

With an increasing global population that is rapidly ageing, our society faces challenges that impact health, environment, and energy demand. With this ageing comes an accumulation of cellular changes that lead to the development of diseases and susceptibility to infections. This impacts not only the health system, but also the global economy. As the population increases, so does the demand for energy and the emission of pollutants, leading to a progressive degradation of our environment. This in turn impacts health through reduced access to arable land, clean water, and breathable air. New monitoring approaches to assist in environmental control and minimize the impact on health are urgently needed, leading to the development of new sensor technologies that are highly sensitive, rapid, and low-cost. Nanopore sensing is a new technology that helps to meet this purpose, with the potential to provide rapid point-of-care medical diagnosis, real-time on-site pollutant monitoring systems to manage environmental health, as well as integrated sensors to increase the efficiency and storage capacity of renewable energy sources. In this review we discuss how the powerful approach of nanopore based single-molecule, or particle, electrical promises to overcome existing and emerging societal challenges, providing new opportunities and tools for personalized medicine, localized environmental monitoring, and improved energy production and storage systems.

Selective target protein detection using a decorated nanopore into a microfluidic device.

Solid-state nanopores provide a powerful tool to electrically analyze nanoparticles and biomolecules at single-molecule resolution. These biosensors need to have a controlled surface to provide information about the analyte. Specific detection remains limited due to nonspecific interactions between the molecules and the nanopore. Here, a polymer surface modification to passivate the membrane is performed. This functionalization improves nanopore stability and ionic conduction. Moreover, one can control the nanopore diameter and the specific interactions between protein and pore surface. The effect of ionic strength and pH are probed. Which enables control of the electroosmotic driving force and dynamics. Furthermore, a study of polymer chain structure and permeability in the pore are carried out. The nanopore chip is integrated into a microfluidic device to ease its handling. Finally, a discussion of an ionic conductance model through a permeable crown along the nanopore surface is elucidated. The proof of concept is demonstrated by the capture of free streptavidin by the biotins grafted into the nanopore. In the future, this approach could be used for virus diagnostic, nanoparticle or biomarker sensing.

Aerolysin, a Powerful Protein Sensor for Fundamental Studies and Development of Upcoming Applications.

The nanopore electrical approach is a breakthrough in single molecular level detection of particles as small as ions, and complex as biomolecules. This technique can be used for molecule analysis and characterization as well as for the understanding of confined medium dynamics in chemical or biological reactions. Altogether, the information obtained from these kinds of experiments will allow us to address challenges in a variety of biological fields. The sensing, design, and manufacture of nanopores is crucial to realize these objectives. For some time now, aerolysin, a pore forming toxin, and its mutants have shown high potential in real time analytical chemistry, size discrimination of neutral polymers, oligosaccharides, oligonucleotides and peptides at monomeric resolution, sequence identification, chemical modification on DNA, potential biomarkers detection, and protein folding analysis. This review focuses on the results obtained with aerolysin nanopores on the fields of chemistry, biology, physics, and biotechnology. We discuss and compare as well the results obtained with other protein channel sensors.

Thermostable virus portal proteins as reprogrammable adapters for solid-state nanopore sensors.

Nanopore-based sensors are advancing the sensitivity and selectivity of single-molecule detection in molecular medicine and biotechnology. Current electrical sensing devices are based on either membrane protein pores supported in planar lipid bilayers or solid-state (SS) pores fabricated in thin metallic membranes. While both types of nanosensors have been used in a variety of applications, each has inherent disadvantages that limit its use. Hybrid nanopores, consisting of a protein pore supported within a SS membrane, combine the robust nature of SS membranes with the precise and simple engineering of protein nanopores. We demonstrate here a novel lipid-free hybrid nanopore comprising a natural DNA pore from a thermostable virus, electrokinetically inserted into a larger nanopore supported in a silicon nitride membrane. The hybrid pore is stable and easy to fabricate, and, most importantly, exhibits low peripheral leakage allowing sensing and discrimination among different types of biomolecules.

Differential Enzyme Flexibility Probed Using Solid-State Nanopores.

Enzymes and motor proteins are dynamic macromolecules that coexist in a number of conformations of similar energies. Protein function is usually accompanied by a change in structure and flexibility, often induced upon binding to ligands. However, while measuring protein flexibility changes between active and resting states is of therapeutic significance, it remains a challenge. Recently, our group has demonstrated that breadth of signal amplitudes in measured electrical signatures as an ensemble of individual protein molecules is driven through solid-state nanopores and correlates with protein conformational dynamics. Here, we extend our study to resolve subtle flexibility variation in dihydrofolate reductase mutants from unlabeled single molecules in solution. We first demonstrate using a canonical protein system, adenylate kinase, that both size and flexibility changes can be observed upon binding to a substrate that locks the protein in a closed conformation. Next, we investigate the influence of voltage bias and pore geometry on the measured electrical pulse statistics during protein transport. Finally, using the optimal experimental conditions, we systematically study a series of wild-type and mutant dihydrofolate reductase proteins, finding a good correlation between nanopore-measured protein conformational dynamics and equilibrium bulk fluorescence probe measurements. Our results unequivocally demonstrate that nanopore-based measurements reliably probe conformational diversity in native protein ensembles.

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization.

Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules, such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics, and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein's insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal's DNA-ratchet function.

Nanopore-Based Measurements of Protein Size, Fluctuations, and Conformational Changes.

Proteins are structurally dynamic macromolecules, and it is challenging to quantify the conformational properties of their native state in solution. Nanopores can be efficient tools to study proteins in a solution environment. In this method, an electric field induces electrophoretic and/or electro-osmotic transport of protein molecules through a nanopore slightly larger than the protein molecule. High-bandwidth ion current measurement is used to detect the transit of each protein molecule. First, our measurements reveal a correlation between the mean current blockade amplitude and the radius of gyration for each protein. Next, we find a correlation between the shape of the current signal amplitude distributions and the protein fluctuation as obtained from molecular dynamics simulations. Further, the magnitude of the structural fluctuations, as probed by experiments and simulations, correlates with the ratio of α-helix to ß-sheet content. We highlight the resolution of our measurements by resolving two states of calmodulin, a canonical protein that undergoes a conformational change in response to calcium binding.

Dynamics and Energy Contributions for Transport of Unfolded Pertactin through a Protein Nanopore.

To evaluate the physical parameters governing translocation of an unfolded protein across a lipid bilayer, we studied protein transport through aerolysin, a passive protein channel, at the single-molecule level. The protein model used was the passenger domain of pertactin, an autotransporter virulence protein. Transport of pertactin through the aerolysin nanopore was detected as transient partial current blockades as the unfolded protein partially occluded the aerolysin channel. We compared the dynamics of entry and transport for unfolded pertactin and a covalent end-to-end dimer of the same protein. For both the monomer and the dimer, the event frequency of current blockades increased exponentially with the applied voltage, while the duration of each event decreased exponentially as a function of the electrical potential. The blockade time was twice as long for the dimer as for the monomer. The calculated activation free energy includes a main enthalpic component that we attribute to electrostatic interactions between pertactin and the aerolysin nanopore (despite the low Debye length), plus an entropic component due to confinement of the unfolded chain within the narrow pore. Comparing our experimental results to previous studies and theory suggests that unfolded proteins cross the membrane by passing through the nanopore in a somewhat compact conformation according to the "blob" model of Daoud and de Gennes.

Protein transport through a narrow solid-state nanopore at high voltage: experiments and theory.

We report experimentally the transport of an unfolded protein through a narrow solid-state nanopore of 3 nm diameter as a function of applied voltage. The random coil polypeptide chain is larger than the nanopore. The event frequency dependency of current blockades from 200 to 750 mV follows a van't Hoff-Arrhenius law due to the confinement of the unfolded chain. The protein is an extended conformation inside the pore at high voltage. We observe that the protein dwell time decreases exponentially at medium voltage and is inversely proportional to voltage for higher values. This is consistent with the translocation mechanism where the protein is confined in the pore, creating an entropic barrier, followed by electrophoretic transport. We compare these results to our previous work with a larger pore of 20 nm diameter. Our data suggest that electro-osmotic flow and protein adsorption on the narrowest nanopore wall are minimized. We discuss the experimental data obtained as compared with recent theory for the polyelectrolyte translocation process. This theory reproduces clearly the experimental crossover between the entropic barrier regime with medium voltage and the electrophoretic regime with higher voltage.

DNA unzipping and protein unfolding using nanopores.

We present here an overview on unfolding of biomolecular structures as DNA double strands or protein folds. After some theoretical considerations giving orders of magnitude about transport timescales through pores, forces involved in unzipping processes … we present our experiments on DNA unzipping or protein unfolding using a nanopore. We point out the difficulties that can be encountered during these experiments, such as the signal analysis problems, noise issues, or experimental limitations of such system.

Wild type, mutant protein unfolding and phase transition detected by single-nanopore recording.

Understanding protein folding remains a challenge. A difficulty is to investigate experimentally all the conformations in the energy landscape. Only single molecule methods, fluorescence and force spectroscopy, allow observing individual molecules along their folding pathway. Here we observe that single-nanopore recording can be used as a new single molecule method to explore the unfolding transition and to examine the conformational space of native or variant proteins. We show that we can distinguish unfolded states from partially folded ones with the aerolysin pore. The unfolding transition curves of the destabilized variant are shifted toward the lower values of the denaturant agent compared to the wild type protein. The dynamics of the partially unfolded wild type protein follows a first-order transition. The denaturation curve obtained with the aerolysin pore is similar to that obtained with the α-hemolysin pore. The nanopore geometry or net charge does not influence the folding transition but changes the dynamics.

Dynamics of completely unfolded and native proteins through solid-state nanopores as a function of electric driving force.

We report experimentally the dynamic properties of the entry and transport of unfolded and native proteins through a solid-state nanopore as a function of applied voltage, and we discuss the experimental data obtained as compared to theory. We show an exponential increase in the event frequency of current blockades and an exponential decrease in transport times as a function of the electric driving force. The normalized current blockage ratio remains constant or decreases for folded or unfolded proteins, respectively, as a function of the transmembrane potential. The unfolded protein is stretched under the electric driving force. The dwell time of native compact proteins in the pore is almost 1 order of magnitude longer than that of unfolded proteins, and the event frequency for both protein conformations is low. We discuss the possible phenomena hindering the transport of proteins through the pores, which could explain these anomalous dynamics, in particular, electro-osmotic counterflow and protein adsorption on the nanopore wall.