The first step toward an accurate, early diagnosis of a disease or for monitoring the efficacy of treatment for it is to identify biomarkers in a clinical specimen. To that end, a tool that identifies a peptide by directly "reading" the sequence of amino acid (AA) residues that constitute its primary structure with extreme sensitivity is badly needed. A nanofluidic assay that uses a sub-nanometer-diameter pore spanning an amorphous silicon (a-Si) membrane a few nanometers thick (i.e., a sub-nanopore) fits the bill. When a denatured peptide isoform (i.e., a peptidoform) is impelled by a high electric field through a sub-nanopore immersed in electrolyte, the ionic current through the pore is partially blocked. The AA sequence can be read by measuring the fluctuations in the blockade current. To call the acid sequence, molecular dynamics (MD) simulations of the pore current, including predictors such as the acid volume, mobility, charge, and nearby water molecules, are used to decipher the amplitudes and timing of the fluctuations. To prove out the prospects for sequencing peptidoforms, a fragment of amyloid-beta (A beta 1-42), along with site-specific post-translational modifications of it and point mutations in it, which are biomarkers for Alzheimer's disease (AD), are assayed this way.

Metamaterials, which started off focused on optical and electromagnetic applications such as invisibility and “hyperlenses” with resolution beyond the diffraction limit, has now broadened in scope to include pretty much everything from wireless communications (antennas), lasers, computing, solar power, sports equipment, medicine; acoustics, structural mechanics and even air conditioning. Yet, the promises derived from these applications have not been exploited in earnest and the market for them has not grown much so far, likely because a facile and economical method for fabricating them without defect has not emerged. This review scrutinizes the methods used to manufacture metamaterials with the aim to remedy this shortcoming.

This is a report on a pilot study that tests the feasibility of assembling photonic metamaterials (PMs) using light gradient forces. Following a strategy that works like modular construction, light gradient forces, produced by a tightly focused, 1D standing wave optical trap, time-multiplexed across a 2D lattice are used to assemble voxels consisting of prefabricated, monodispersed nanoparticles (NPs) with radii ranging from 30 to 500 nm into 3D structures on a hydrogel scaffold. Hundreds of NPs can be manipulated concurrently into a complex heterogeneous voxel this way, and then the process can be repeated by stitching together voxels to form a metamaterial of any size, shape, and constituency although imperfectly. Imperfections introduce random phase shifts and amplitude variations that can have an adverse effect on the band structure. Regardless, PMs are created this way using two different dielectric NPs, polystyrene and rutile, and then the near-infrared performance for each is analyzed with angle-, wavelength-, and polarization-dependent reflection spectroscopy. The cross-polarized spectra show evidence of a resonance peak. Interestingly, whereas the line shape from the polystyrene array is symmetric, the rutile array is not, which may be indicative of Fano resonance. So, even with the structural defects, reflection spectroscopy reveals a resonance.

Metamaterials were assembled using the force of a light gradient in a one-dimensional Standing Wave Optical Trap (SWOT) that was time-shared across the 2-D lattice to create a three-dimensional (3D) array of traps, which was then populated with monodispersed dielectric or metallic nanoparticles (NPs). The NP structure was anchored to a hydrogel scaffold, and then the process was repeated to create macroscopic metamaterials. The error in particle position within a voxel (σ=55 nm) was limited by dark time Brownian motion, whereas the error between voxels, (σ=88 nm) was limited by the microscope stage repeatability. Also, compared to a Gaussian beam SWOT, a non-diffractive, pseudo-Bessel beam SWOT produced a longer array due to greater focus-depth and self-healing distance.

The blockade current that develops when a protein translocates across a thin membrane through a sub-nanometer diameter pore informs with extreme sensitivity on the sequence of amino acids that constitute the protein. The current blockade signals measured during the translocation are called a nanospectrum of the protein. Whereas mass spectrometry (MS) is still the dominant technology for protein identification, it suffers limitations. In proteome-wide studies, MS identifies proteins by database search but often fails to provide high protein sequence coverage. It is also not very sensitive requiring about a femtomole for protein identification. Compared with MS, a sub-nanometer diameter pore (i.e. a sub-nanopore) directly reads the amino acids constituting a single protein molecule, but efficient computational tools are still required for processing and interpreting nanospectra. Here, we delineate computational methods for processing sub-nanopore nanospectra and predicting theoretical nanospectra from protein sequences, which are essential for protein identification.

When a denatured protein isoform (i.e., a proteoform) immersed in electrolyte is impelled by an electric field through a sub-nanometer-diameter pore (i.e., a sub-nanopore) spanning a thin membrane, the sequence of amino acid (AA) residues constituting the proteoform can be directly "read" one at a time by measuring fluctuations in the electrolytic current. Corroborating this assertion, an analysis of the pore current with molecular dynamic (MD) simulations reveals that the fluctuations are correlated to the sequence of AA volumes, the water in the pore and acid mobility. After alignment to account for variations in the acid mobility, the simulated pore current is nearly perfectly correlated to the pattern of empirical fluctuations. To prove out the prospects for decoding proteoforms this way, site-specific post-translational modifications (PTMs) and point mutations in amyloid-beta (Aβ 1-42 ) were analyzed with a sub-nanopore. The results show that single acids can be resolved in proteoforms with a dynamic range limited by the size of phenylalanine and glycine. With this sensitivity and single acid resolution, the sequence of a scrambled variant of Aβ 1-42 was discriminated with a p-value < 10-5.

Proteins can be the root cause of a disease, and they can be used to cure it. The need to identify these critical actors was recognized early (1951) by Sanger; the first biopolymer sequenced was a peptide, insulin. With the advent of scalable, single-molecule DNA sequencing, genomics and transcriptomics have since propelled medicine through improved sensitivity and lower costs, but proteomics has lagged behind. Currently, proteomics relies mainly on mass spectrometry (MS), but instead of truly sequencing, it classifies a protein and typically requires about a billion copies of a protein to do it. Here, we offer a survey that illuminates a few alternatives with the brightest prospects for identifying whole proteins and displacing MS for sequencing them. These alternatives all boast sensitivity superior to MS and promise to be scalable and seem to be adaptable to bioinformatics tools for calling the sequence of amino acids that constitute a protein.

The size of an ion affects everything from the structure of water to life itself. In this report, to gauge their size, ions dissolved in water are forced electrically through a sub-nanometer-diameter pore spanning a thin membrane and the current is measured. The measurements reveal an ion-selective conductance that vanishes in pores <0.24 nm in diameter—the size of a water molecule—indicating that permeating ions have a grossly distorted hydration shell. Analysis of the current noise power spectral density exposes a threshold, below which the noise is independent of current, and beyond which it increases quadratically. This dependence proves that the spectral density, which is uncorrelated below threshold, becomes correlated above it. The onset of correlations for Li + , Mg 2+ , Na + and K + -ions extrapolates to pore diameters of 0.13 ± 0.11 nm, 0.16 ± 0.11 nm, 0.22 ± 0.11 nm and 0.25 ± 0.11 nm, respectively—consonant with diameters at which the conductance vanishes and consistent with ions moving through the sub-nanopore with distorted hydration shells in a correlated way.

Secreted proteins mediate cell-to-cell communications. Thus, eavesdropping on the secretome could reveal the cellular phenotype, but it is challenging to detect the proteins because they are secreted only in minute amounts and then diluted in blood plasma or contaminated by cell culture medium or the lysate. In this pilot study, it is demonstrated that secretions from single cancer cells can be detected and dynamically analyzed through measurements of blockades in the electrolytic current due to single molecules translocating through a nanopore in a thin inorganic membrane. It is established that the distribution of blockades can be used to differentiate three different cancer cell lines (U937, MDA-MB-231, and MCF-7) in real time and quickly (<20 s). Importantly, the distinctive blockades associated with the chemokine CCL5, a prognostic factor for disease progression in breast cancer, along with other low-mass biomarkers of breast cancer (PI3, TIMP1, and MMP1) were identified in the context of the secretome of these three cell types, tracked with time, and used to provide information on the cellular phenotype.

The size and charge of an ion affects everything from the structure of water to life itself. To gauge their size, metal alkali and alkali earth ions, and protons dissolved in water were forced through a sub-nanometer-diameter pore spanning an ultra-thin silicon nitride membrane and the current was measured. Except for protons, the measurements revealed a conductance selective to positive ions that vanished when extrapolated to pores smaller than about 0.25 nm in diameter, which was comparable to the diameter of a water molecule (0.28 nm). On the other hand, the proton conductance persisted, extrapolating to zero only when the pore diameter was about 0.15 nm. Furthermore, an analysis of the low frequency (pink) noise power spectral density exposed a threshold, below which the noise was independent of the current, and beyond which it increased quadratically. This dependence on current proved that the spectral density components of the noise, which were uncorrelated below threshold, became nearly perfectly correlated above it. Coincidently, the onset of correlations in the noise current for Li+, Mg2+, Na+and K+ ions extrapolates to pore diameters of 0.12 ± 0.11 nm, 0.12 ± 0.11 nm, 0.21 ± 0.11 nm and 0.23 ± 0.11 nm, respectively. Altogether, these data were consistent with the correlated motion of (at least partially) unscreened metal ions with a grossly distorted hydration shell permeating the smallest pores at high current.

The information gleaned from precise, comprehensive whole-protein analysis will elucidate disease phenotypes. However, conventional tools like top-down mass spectrometry (TD-MS) lack the sensitivity to interrogate proteome and discriminate proteoforms without a priori knowledge of the target protein required for enrichment (including all the different molecular forms for a protein product of a single gene, genetic variations, post-translational modifications and alternative splicing). Recently, it was discovered that denatured, charge linearized single proteins translocating a sub-nanopore exhibit current blockade fluctuations that correlate with the amino acid sequence of the protein. Small volume differences (<0.1 nm3), associated with post-translation modifications or single residue substitutions can be detected even in a single molecule. Here, we describe how proteins can be discriminated with high accuracy (p < 10−5) with supervised machine learning using random forest regression, based on improvements that include a revised model of the volume that accounts for hydrophilicity. This accuracy can be achieved for small blockade ensembles (n < 10), indicating that the current technology is already capable of identification at E-value = 0.01 for small (<104 constituents) proteomes.

It is now possible to create, in a thin inorganic membrane, a single, sub-nanometer-diameter pore (i.e., a sub-nanopore) about the size of an amino acid residue. To explore the prospects for sequencing protein with it, measurements of the force and current were performed as two denatured histones, which differed by four amino acid residue substitutions, were impelled systematically through the sub-nanopore one at a time using an atomic force microscope. The force measurements revealed that once the denatured protein, stabilized by sodium dodecyl sulfate (SDS), translocated through the sub-nanopore, a disproportionately large force was required to pull it back. This was interpreted to mean that the SDS was cleaved from the protein during the translocation. The force measurements also exposed a dichotomy in the translocation kinetics: either the molecule slid nearly frictionlessly through the pore or it slipped-and-stuck. When it slid frictionlessly, regardless of whether the molecule was pulled N-terminus or C-terminus first through the pore, regular patterns were observed intermittently in the force and blockade current fluctuations that corresponded to the distance between stretched residues. Furthermore, the amplitude of the fluctuations in the current blockade were correlated with the occluded volume associated with the amino acid residues in the pore. Finally, a comparison of the patterns in the current fluctuations associated with the two practically identical histones supported the conclusion that a sub-nanopore was sensitive enough to discriminate amino acid substitutions in the sequence of a single protein molecule by measuring volumes of 0.1 nm3 per read.

ADVERTISEMENT RETURN TO ISSUEPREVLetter to the EditorNEXTGene Expression in Electron-Beam-Irradiated Bacteria in Reply to "Live Cell Electron Microscopy Is Probably Impossible"Eamonn Kennedy†, Edward M. Nelson†, John Damiano‡, and Gregory Timp*§View Author Information† Department of Electrical Engineering, University of Notre Dame, Notre Dame, Indiana 46556, United States‡ Protochips, Inc., Morrisville, North Carolina 27560, United States§ Departments of Electrical Engineering and Biological Science, University of Notre Dame, Notre Dame, Indiana 46556, United States*E-mail: [email protected]Cite this: ACS Nano 2017, 11, 1, 3–7Publication Date (Web):January 24, 2017Publication History Received30 September 2016Published online24 January 2017Published inissue 24 January 2017https://pubs.acs.org/doi/10.1021/acsnano.6b06616https://doi.org/10.1021/acsnano.6b06616letterACS PublicationsCopyright © 2017 American Chemical Society. This publication is available under these Terms of Use. Request reuse permissions This publication is free to access through this site. Learn MoreArticle Views1763Altmetric-Citations18LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail PDF (1 MB) Get e-AlertscloseSUBJECTS:Assays,Fluorescence,Genetics,Irradiation,Membranes Get e-Alerts

Recent advances in top-down mass spectrometry enabled identification of intact proteins, but this technology still faces challenges. For example, top-down mass spectrometry suffers from a lack of sensitivity since the ion counts for a single fragmentation event are often low. In contrast, nanopore technology is exquisitely sensitive to single intact molecules, but it has only been successfully applied to DNA sequencing, so far. Here, we explore the potential of sub-nanopores for single-molecule protein identification (SMPI) and describe an algorithm for identification of the electrical current blockade signal (nanospectrum) resulting from the translocation of a denaturated, linearly charged protein through a sub-nanopore. The analysis of identification p-values suggests that the current technology is already sufficient for matching nanospectra against small protein databases, e.g., protein identification in bacterial proteomes.

The promise of adapting biology to information processing will not be realized until engineered gene circuits, operating in different cell populations, can be wired together to express a predictable function. Here, elementary biological integrated circuits (BICs), consisting of two sets of transmitter and receiver gene circuit modules with embedded memory placed in separate cell populations, were meticulously assembled using live cell lithography and wired together by the mass transport of quorum-sensing (QS) signal molecules to form two isolated communication links (comlinks). The comlink dynamics were tested by broadcasting "clock" pulses of inducers into the networks and measuring the responses of functionally linked fluorescent reporters, and then modeled through simulations that realistically captured the protein production and molecular transport. These results show that the comlinks were isolated and each mimicked aspects of the synchronous, sequential networks used in digital computing. The observations about the flow conditions, derived from numerical simulations, and the biofilm architectures that foster or silence cell-to-cell communications have implications for everything from decontamination of drinking water to bacterial virulence.

Capillaries pervade human physiology. The mean intercapillary distance is only about 100 μm in human tissue, which indicates the extent of nutrient diffusion. In engineered tissue the lack of capillaries, along with the associated perfusion, is problematic because it leads to hypoxic stress and necrosis. However, a capillary is not easy to engineer due to its complex cytoarchitecture. Here, it is shown that it is possible to create in vitro, in about 30 min, a tubular microenvironment with an elastic modulus and porosity consistent with human tissue that functionally mimicks a bona fide capillary using “live cell lithography”(LCL) to control the type and position of cells on a composite hydrogel scaffold. Furthermore, it is established that these constructs support the forces associated with blood flow and produce nutrient gradients similar to those measured in vivo . With LCL, capillaries can be constructed with single cell precision—no other method for tissue engineering offers such precision. Since the time required for assembly scales with the number of cells, this method is likely to be adapted first to create minimal functional units of human tissue that constitute organs, consisting of a heterogeneous population of 100–1000 cells, organized hierarchically to express a predictable function.

The primary structure of a protein consists of a sequence of amino acids (AAs) that essentially dictates how it folds and functions. Thus, sequencing a protein is essential to proteomics, the next step beyond genomics, in the analysis of biology. It is shown here that the sequence of AA quadromers in a denatured protein can be determined using a sub-nanometer diameter pore through a thin inorganic membrane. When a sub-nanopore was immersed in electrolyte and denaturants, and a voltage was applied across it, measurements of a blockade in the current associated with the translocation of a molecule revealed nearly regular fluctuations, the number of which coincided with the number of residues in the protein. Furthermore, the fluctuation amplitudes were highly correlated with the volumes occluded by quadromers in the protein sequence. Scrutiny of the fluctuations revealed that a sub-nanopore was sensitive enough to detect the occluded volume of post-translational modifications at a single residue in a quadromer. Thus, each fluctuation represents a read of a quadromer. To validate these results, the force and blockade characterizing the translocation of a single protein, tethered to the tip of an atomic force microscope cantilever, were also measured as the molecule was impelled systematically through a sub-nanopore. The force measurements revealed a dichotomy in the translocation kinetics: either the molecule slid frictionlessly through the pore or it slipped-and-stuck. When the molecule slid frictionlessly, periodic fluctuations were observed in force and current with lags that corresponded to the AA separation, and the amplitudes of the current fluctuations were correlated with the quadromer volumes, corroborating results obtained without systematic control of the translocation.

It is now possible to visualize at nanometer resolution the infection of a living biological cell with virus without compromising cell viability using scanning transmission electron microscopy (STEM). To provide contrast while preserving viability, Escherichia coli and P1 bacteriophages were first positively stained with a very low concentration of uranyl acetate in minimal phosphate medium and then imaged with low-dose STEM in a microfluidic liquid flow cell. Under these conditions, it was established that the median lethal dose of electrons required to kill half the tested population was LD50 = 30 e(-)/nm(2), which coincides with the disruption of a wet biological membrane, according to prior reports. Consistent with the lateral resolution and high-contrast signal-to-noise ratio (SNR) inferred from Monte Carlo simulations, images of the E. coli membrane, flagella, and the bacteriophages were acquired with 5 nm resolution, but the cumulative dose exceeded LD50. On the other hand, with a cumulative dose below LD50 (and lower SNR), it was still possible to visualize the infection of E. coli by P1, showing the insertion of viral DNA within 3 s, with 5 nm resolution.

The primary structure of a protein consists of a sequence of amino acids and is a key factor in determining how a protein folds and functions. However, conventional methods for sequencing proteins, such as mass spectrometry and Edman degradation, suffer from short reads and lack sensitivity, so alternative approaches are sought. Here, we show that a subnanometre-diameter pore, sputtered through a thin silicon nitride membrane, can be used to detect the primary structure of a denatured protein molecule. When a denatured protein immersed in electrolyte is driven through the pore by an electric field, measurements of a blockade in the current reveal nearly regular fluctuations, the number of which coincides with the number of residues in the protein. Furthermore, the amplitudes of the fluctuations are highly correlated with the volumes that are occluded by quadromers (four residues) in the primary structure. Each fluctuation, therefore, represents a read of a quadromer. Scrutiny of the fluctuations reveals that the subnanometre pore is sensitive enough to read the occluded volume that is related to post-translational modifications of a single residue, measuring volume differences of ∼0.07 nm 3 , but it is not sensitive enough to discriminate between the volumes of all twenty amino acids.

The secretome of a single living cell contains the totality of its secreted proteins,(1) and therefore can act as a fingerprint by which to identify cell type. Although between 10 % and 20 % of the human genome encodes proteins that are secreted, measuring the secretome from an individual living cell is challenging as the secreted proteins are present in vanishingly small concentrations due to the very large dilutions involved. However, a nanopore is able to detect single proteins through the distinctive blockage profile that develops in the ionic conductance current when a protein passes through the nanopore.(2,3) Using a synthetic nanopore in a silicon membrane we investigate the distinctive blockage patterns, in essence the fingerprint, that arise from a single living cell. The cell is placed in proximity to the nanopore using optical tweezers and held stationary. The ionic conductance current is measured across the nanopore, and translocation events (distinct blockage currents) are observed and measured. When the events are plotted in scatter plots (as dwell time versus average blockage current) the distinct fingerprint of individual cells can be observed. For instance, lymphoma cells (U937) and breast cancer cells (MCF7) produce distinct event patterns that enable them to be distinguished. This shows for the first time cell identification based entirely on the secretome, measured using a simple, non-invasive, non-destructive nanopore. (1) Skalnikova, H.; Motlik, J.; Gadher, S.; Kovarova, H. Proteomics, 2011, 11 691-708. (2) Nelson, E. M.; Kurz, V.; Shim, J.; Timp, W.; Timp, G. Analyst, 2012, 137, 3020. (3) Kurz, V.; Nelson, E. M.; Shim, J.; Timp G. ACS Nano, 2013, 7(5), 4057-4069