Amit Meller

• Doctor of Philosophy
• Professor (Full) at Technion – Israel Institute of Technology

Mitochondrial DNA (mtDNA) quantification is crucial in understanding mitochondrial dysfunction, which is linked to a variety of diseases, including cancer and neuro-degenerative disorders. Traditional methods often rely on amplification-based techniques, which can introduce bias and lack the precision needed for clinical diagnostics. Solid-state na...

Emerging single‐molecule protein sensing techniques are ushering in a transformative era in biomedical research. Nevertheless, challenges persist in realizing ultra‐fast full‐length protein sensing, including loss of molecular integrity due to protein fragmentation, biases introduced by antibodies affinity, identification of proteoforms, and low th...

Emerging single-molecule protein sensing techniques are ushering in a transformative era in biomedical research. Nevertheless, challenges persist in realizing ultra-fast full-size protein sensing, including loss of molecular integrity due to protein fragmentation, biases introduced by antibodies affinity, identification of proteoforms and low throu...

Solid-state nanopores (ssNPs) are single-molecule sensors capable of label-free quantification of different biomole-cules, which have become highly versatile with the introduction of different surface treatments. By modulating the surface charges of the ssNP, the electro-osmotic flow (EOF) can be controlled in turn affecting the in-pore hydrodynami...

Inspired by the biological processes of molecular recognition and transportation across membranes, nanopore techniques have evolved in recent decades as ultrasensitive analytical tools for individual molecules. In particular, nanopore-based single-molecule DNA/RNA sequencing has advanced genomic and transcriptomic research due to the portability, l...

The ability to routinely identify and quantify the complete proteome from single cells will greatly advance medicine and basic biology research. To meet this challenge of single-cell proteomics, single-molecule technologies are being developed and improved. Most approaches, to date, rely on the analysis of polypeptides, resulting from digested prot...

The majority of RNA based COVID-19 diagnostics employ enzymatic amplification to achieve high sensitivity, but this relies on arbitrary thresholding, which complicates the comparison of test results and may lead to false outcomes. Here we introduce solid-state nanopore sensing for label-free quantification of SARS-CoV-2 RNA in clinical nasal swab s...

Novel techniques for single-protein molecule sequencing are rapidly becoming the focus of contemporary biomedical research. Here, Brinkerhoff et al. (2021) report a significant progress in nanopore-based rereading of DNA-peptide conjugates.

Single biomolecule sensing often requires the quantification of multiple fluorescent species. Here, we theoretically and experimentally use time-resolved fluorescence via Time Correlated Single Photon Counting (TCSPC) to accurately quantify fluorescent species with similar chromatic signatures. A modified maximum likelihood estimator (MLE) is intro...

Nanopores are single-molecule sensors capable of detecting and quantifying a broad range of unlabeled biomolecules including DNA and proteins. Nanopores' generic sensing principle has permitted the development of a vast range of biomolecular applications in genomics and proteomics, including single-molecule DNA sequencing and protein fingerprinting...

Background: Circulating Tumor DNAs (ctDNAs) are a highly promising cancer biomarker, potentially applicable for non-invasive 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) in the circulation derived...

Accurate identification of both abundant and rare proteins hinges on the development of single-protein sensing methods. Given the immense variation in protein expression levels in a cell, separation of proteins by weight would improve protein classification strategies. Upstream separation facilitates sample binning into smaller groups while also pr...

Accurate identification of both abundant and rare proteins hinges on the development of single- protein sensing methods. Given the immense variation in protein expression levels in a cell, separation of proteins by weight would improve protein classification strategies. Upstream separation facilitates sample binning into smaller groups while also p...

RNA quantification methods are broadly used in life science research and in clinical diagnostics. Currently, real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most common analytical tool for RNA quantification. However, in cases of rare transcripts or inhibiting contaminants in the sample, an extensive amplification could b...

Solid-state nanopores (NPs) are label-free single-molecule sensors, capable of performing highly sensitive assays from a small number of biomolecule translocation events. However, single-molecule sensing is challenging at extremly low analyte concentrations due to the limited flux of analytes to the sensing volume. This leads to a low event rate an...

We present a parallel stimulated emission depletion (STED) nanoscope with no mechanical moving parts and sub-millisecond pixel dwell times, relying on electro-optical (EO) phase modulators. The nanoscope offers 1225-fold parallelization over single-doughnut-scanning STED and achieves a spatial resolution of 35 nm. We imaged immunostained nuclear po...

Solid-state nanopore sensing of ultra-long genomic DNA molecules has remained challenging, as the DNA must be controllably delivered by its leading end for efficient entry into the nanopore. Herein we introduce a nanopore sensor device designed for electro-optical detection and sorting of ultra-long (300+ kilobase pair) genomic DNA. The fluidic dev...

Single-molecule techniques for protein sequencing are making headway towards single-cell proteomics and are projected to propel our understanding of cellular biology and disease. Yet, single cell proteomics presents a substantial unmet challenge due to the unavailability of protein amplification techniques, and the vast dynamic-range of protein exp...

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...

The ability to quickly and reliably fabricate nanoscale pore arrays in ultra‐thin membranes such as silicon nitride (SixN) is extremely important for the growing field of nanopore biosensing. Laser‐based etching of thin SixN membranes immersed in aqueous solutions has recently been demonstrated as a method to produce stable functional pores. Herein...

Solid‐state nanopores are an emerging biosensor for nucleic acid and protein characterization. For use in a clinical setting, solid‐state nanopore sensing requires sample preparation and purification, fluid handling, a heating element, electrical noise insulators, and an electrical readout detector, all of which hamper its translation to a point‐of...

Multicolor fluorescence substantially expands the sensing capabilities of nanopores by complementing or substituting the resistive pulsing signals. However, to date single-fluorophore detection in multiple color channels has proven to be challenging primarily due to high photo- luminescence (PL) emanating from the silicon nitride (SiNx) membrane. W...

Proteins are the structural elements and machinery of cells responsible for a functioning biological architecture and homeostasis. Advances in nanotechnology are catalyzing key breakthroughs in many areas, including the analysis and study of proteins at the single-molecule level. Nanopore sensing is at the forefront of this revolution. This tutoria...

Solid-state nanopores (ssNPs) are extremely versatile single-molecule sensors and their potential have been established in numerous biomedical applications. However, the fabrication of ssNPs remains the main bottleneck to their widespread use. Herein, we introduce a rapid and localizable ssNPs fabrication method based on feedback-controlled optical...

Monitoring individual proteins in solution while simultaneously obtaining tertiary and quaternary structural information is challenging. In this study, translocation of the vascular endothelial growth factor (VEGF) protein through a solid-state nanopore (ssNP) produces distinct ion-current blockade amplitude levels and durations likely correspondin...

Most functional transcription factor (TF) binding sites deviate from their 'consensus' recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used single-molecule DNA unzipping with optical tweezers to study how Egr-1, a TF harboring three zinc fingers (ZF1, ZF2 and ZF3), is modulated by the sequ...

Most functional transcription factor (TF) binding sites deviate from their “consensus” recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used single-molecule DNA unzipping with optical tweezers to study how Egr-1, a TF harbouring 3 zinc fingers (ZF1,ZF2 and ZF3), is modulated by the sequence...

Herein, we introduce synchronous, real-time, electro-optical monitoring of nanopore formation by DB. Using the same principle as sub-diffraction microscopy, our nanopore localization platform based on wide-field microscopy and calcium indicators provides nanoscale sensitivity. This enables us to establish critical limitations of the fabrication pro...

Detection and characterization of nucleic acid–protein interactions, particularly those involving DNA and proteins such as transcription factors, enzymes, and DNA packaging proteins, remain significant barriers to our understanding of genetic regulation. Nanopores are an extremely sensitive and versatile sensing platform for label-free detection of...

Detection of epigenetic markers, including 5-methylcytosine, is crucial due to their role in gene expression regulation, and due to the mounting evidence of aberrant DNA methylation patterns in cancer biogenesis. Single-molecule methods to date have primarily been focused on hypermethylation detection; however, many oncogenes are hypomethylated dur...

In clinical settings, rapid and accurate characterization of pathogens is essential for effective treatment of patients; however, subtle genetic changes in pathogens which elude traditional phenotypic typing may confer dangerous pathogenic properties such as toxicity, antibiotic resistance, or virulence. Existing options for molecular typing techni...

3-dimensional substrates with high surface to volume ratios and subsequently large protein binding capacities are of interest for advanced immunosorbent assays utilizing integrated microfluidics and nanosensing elements. A library of bioactive and antifouling electrospun nanofiber substrates are described composed of high molecular weight poly(oxan...

Transcription factor (TF)-DNA interactions are the primary control point in regulation of gene expression. Characterization of these interactions is essential for understanding genetic regulation of biological systems and developing novel therapies to treat cellular malfunctions. Solid-state nanopores are a highly versatile class of single-molecule...

Advances in biosensor sensitivity, specificity, and accessibility are required for the development of next generation diagnostic tools. Solid-state nanopores consisting of sub-10 nm in diameter pores drilled into an insulating material show promise as single-molecule biosensors for detecting both nucleic acid and protein targets. However, this tech...

Nanopore sensing involves an electrophoretic transport of analytes through a nanoscale pore, permitting label-free sensing at the single-molecule level. However, to date, the detection of individual small proteins has been challenging, primarily due to the poor signal/noise ratio that these molecules produce during passage through the pore. Here, w...

The invention relates to a method for characterizing a target polynucleic acid by providing a surface containing a channel of a dimension sufficient to allow sequential monomer-by-monomer passage of a single-stranded polynucleic acid, but not of a double-stranded polynucleic acid; providing a source of hybridized target polynucleic acid at the surf...

The field of nanopore sensing has been gaining increasing attention. Much progress has been made towards biotechnological applications that involve electrical measurements of temporal changes in the ionic current flowing through the pore. But in many cases the electrical signal is restricted by the non-ideal noise components, limited throughput, an...

Optical sensing of solid-state nanopores is a relatively new approach that can enable high-throughput, multicolor readout from a collection of nanopores. It is therefore highly attractive for applications such as nanopore-based DNA sequencing and genotyping using DNA barcodes. However, to date optical readout has been plagued by the need to achieve...

Nanopore sensing has enabled label-free single-molecule measurements on a wide variety of analytes, including DNA, RNA, and protein complexes. Much progress has been made towards biotechnological applications; however, electrically probing the ion current introduces non-ideal noise components. Here we further develop a method to couple an ionic cur...

Significance There is a substantial need for single-cell platforms in which each cell is chemically isolated in its own microenvironment and a lack of such platforms that support adherent cells. We present here a method that generates stationary nanoliter droplet arrays on a substrate of choice and supports long-term incubation and interrogation of...

The eukaryotic translation initiation factor 4AI (eIF4AI) is the prototypical DEAD-box RNA helicase. It has a "dumbbell" structure consisting of two domains connected by a flexible linker. Previous studies demonstrated that eIF4AI, in conjunction with eIF4H, bind to loop structures and repetitively unwind RNA hairpins. Here, we probe the conformati...

Nanopores can be used to detect and analyse biomolecules. However, controlling the translocation speed of molecules through a pore is difficult, which limits the wider application of these sensors. Here, we show that low-power visible light can be used to control surface charge in solid-state nanopores and can influence the translocation dynamics o...

Solid-state nanopores show promise as single-molecule sensors for biomedical applications, but to increase their resolution and efficiency, analyte molecules must remain longer in the nanopore sensing volume. Here we demonstrate a novel, facile, and customizable nanopore sensor modification that reduces the double-stranded DNA translocation velocit...

The optipore approach to nanopore-based sequencing enables direct optical readout of many DNA sequences in parallel using an array of nanopores. Fluorescent beacons hybridized to template strands of DNA threaded through each nanopore are sequentially excited via a 4-color total internal reflection (TIR) system. Here we present fabrication of a chip...

Next generation DNA sequencing methods that utilize solid-state nanopores have been intensively studies in past years. Some of the compelling features of solid-state nanopores are their physical and electrical robustness, the ability to form dense arrays of pores on a small membrane footprint for simultaneous analysis of many DNA molecules[1] and t...

Following epical completion of human genome project, further effort in sequencing arena was geared towards a steep reduction in cost to enable personalized genomic-based diagnostics. Nanopore devices provide an appealing platform for DNA sequencing at the single-molecule level, which enables direct recognition of minute amount of genome fragments w...

The Nanopore method is an emerging technique, which extends gel-electrophoresis to the single-molecule level and allows the analysis of DNAs, RNAs and DNA-protein complexes. The strength of the technique stems from two fundamental facts: First, nanopores due to their nanoscale size can be used to uncoil biopolymers, such as DNA or RNA and slide the...

The translation initiation factor eIF4A is the prototypical DEAD-box RNA helicase and it has a "dumbbell" structure consisting of two domains connected by a linker. Our previous studies have shown that eIF4A/eIF4H complex can bind directly to loop structures and repetitively unwind the RNA hairpin. To further illuminate the conformation change of e...

Controlling DNA translocation speed is of critical importance for nanopore sequencing as free electrophoretic threading is far too rapid to resolve individual bases. A number of promising strategies have been explored in recent years, largely driven by the demands of next-generation sequencing. Engineering DNA-nanopore interactions (known to domina...

Nanopores have emerged as a prominent single-molecule analytic tool with particular promise for genomic applications. In this review, we discuss two potential applications of the nanopore sensors: First, we present a nanopore-based single-molecule DNA sequencing method that utilizes optical detection for massively parallel throughput. Second, we de...

We report the fabrication and characterization of uniformly sized nanopore arrays, integrated into an optical detection system for high-throughput DNA sequencing applications. Nanopore arrays were fabricated using focused ion beam milling, followed by TiO(2) coating using atomic layer deposition. The TiO(2) layer decreases the initial pore diameter...

Eukaryotic translation initiation is a highly regulated process in protein synthesis. The principal translation initiation factor eIF4AI displays helicase activity, unwinding secondary structures in the mRNAs 5′-UTR. Single molecule fluorescence resonance energy transfer (sm-FRET) is applied here to directly observe and quantify the helicase activi...

The ubiquitous and abundant cytoplasmic poly(A) binding protein (PABP) is a highly conserved multifunctional protein, many copies of which bind to the poly(A) tail of eukaryotic mRNAs to promote translation initiation. The N-terminus of PABP is responsible for the high binding specificity and affinity to poly(A), whereas the C-terminus is known to...

Nanopore-based DNA analysis is an extremely attractive area of research due to the simplicity of the method, and the ability to not only probe individual molecules, but also to detect very small amounts of genomic material. Here, we describe the materials and methods of a novel, nanopore-based, single-molecule DNA sequencing system that utilizes op...

A new single-molecule approach for rapid and purely electronic discrimination among similar genes is presented. Combining solid-state nanopores and γ-modified synthetic peptide nucleic acid probes, we accurately barcode genes by counting the number of probes attached to each gene and measuring their relative spacing. We illustrate our method by sen...

IntroductionDNA Capture and Translocation ProcessesProbing DNA/Small Molecule InteractionsNanopore-Based Genomic Profiling Using Sequence-Specific ProbesSummaryAcknowledgmentsReferences

Nanopores have emerged as single-molecule analytic tools for fundamental biophysical characterization of nucleic acids as well as for future genomic applications. The enormous interest in single-molecule analysis has spurred the development of many different approaches to nanopore fabrication. Of these, ultrathin solid-state membranes are the most...

The eukaryotic translation initiation factor 4AI (eIF4AI) is the prototypical DEAD-box RNA helicase. It has a ''dumbbell'' structure consisting of two domains connected by a flexible linker. Previous studies demonstrated that eIF4AI, in conjunction with eIF4H, bind to loop structures and repetitively unwind RNA hairpins. Here, we probe the conforma...

Monitoring the real-time behavior of spatial arrays of single living bacteria cells is only achieved with much experimental difficulty due to the small size and mobility of the cells. To address this problem, we have designed and constructed a simple microfluidic device capable of trapping single bacteria cells in spatially well-defined locations w...

The time-dependent response of individual biomolecular complexes to an applied force can reveal their mechanical properties, interactions with other biomolecules, and self-interactions. In the past decade, a number of single-molecule methods have been developed and applied to a broad range of biological systems, such as nucleic acid complexes, enzy...

The ability to detect and measure dsDNA thermal fluctuations is of immense importance in understanding the underlying mechanisms responsible for transcription and replication regulation. We describe here the ability of solid-state nanopores to detect sub-nanometer changes in DNA structure as a result of chemically enhanced thermal fluctuations. In...

We demonstrate the feasibility of a nanopore based single-molecule DNA sequencing method, which employs multicolor readout. Target DNA is converted according to a binary code, which is recognized by molecular beacons with two types of fluorophores. Solid-state nanopores are then used to sequentially strip off the beacons, leading to a series of det...

The translation of cellular mRNA to protein is a tightly controlled process often deregulated in diseases such as cancer. Furthering our understanding of mRNA structural elements and the intracellular proteins and signaling pathways that affect protein expression is crucial in the development of new therapies. In this review, we discuss the current...

We study the effect of dye-dye interactions in labeled double-stranded DNA molecules on the Förster resonance energy transfer (FRET) efficiency at the single-molecule level. An extensive analysis of internally labeled double-stranded DNA molecules in bulk and at the single-molecule level reveals that donor-acceptor absolute distances can be reliabl...

We provide an experimental demonstration that the focusing of ionic currents in a micron size hole connecting two chambers can produce local temperature increases of up to $100^\circ$ C with gradients as large as $1^\circ$ K$\mu m^{-1}$. We find a good agreement between the measured temperature profiles and a finite elements-based numerical calcula...

Helix-coil transition kinetics of polyadenylic acid [poly(A)] inside a small protein channel is investigated for the first time, at the single molecule level. The confinement of a RNA molecule inside the channel slows its kinetics by nearly 3 orders of magnitude as compared to bulk measurements of free poly(A). These findings are related to the int...

Nanopores have emerged as a prominent single-molecule analytic tool, holding particular promise both for genomic applications and for the fundamental biophysical characterization of biopolymers. The interest in single-molecule analysis has spurred the development of numerous approaches to solid-state nanopore fabrication, which offer exceptional ro...

We demonstrate a purely electrical method for the single-molecule detection of specific DNA sequences, achieved by hybridizing double-stranded DNA (dsDNA) with peptide nucleic acid (PNA) probes and electrophoretically threading the DNA through sub-5 nm silicon nitride pores. Bis-PNAs were used as the tagging probes in order to achieve high affinity...

Dramatically reducing the cost of DNA sequencing will revolutionize the healthcare system by enabling patient genomes to be determined in routine procedures. This belief has resulted in large scale investments in alternative sequencing methodologies. One of the most promising techniques to emerge is nanopore sequencing, where individual biomolecule...

Eukaryotic initiation factor eIF4A is a prototype protein of the DEAD box family of RNA helicases, and is part of the translation initiation complex eIF4F. eIF4A binds to the 5′ cap of mRNA and unwinds structures in the 5′-untranslated regions of mRNAs in ATP dependent manner. Our long-term goal in this project is to decipher the role of the initia...

Polyadenylic acid (poly(A)) forms helical configuration in aqueous solution at neutral and alkaline pH. The transition between its helical and random coil structures has been studied using bulk spectroscopic or calorimetric methods, revealing its thermodynamic properties. Recently, optical tweezers pulling experiments provided further support for t...

We present a novel method for integrating two single-molecule measurement modalities, namely, total internal reflection microscopy and electrical detection of biomolecules using nanopores. Demonstrated here is the electrical measurement of nanopore based biosensing performed simultaneously and in-sync with optical detection of analytes. This method...

This chapter presents biophysical studies of single biopolymers using nanopores. Starting from the fundamental process of voltage-driven biopolymer translocation, the understanding of which is a prerequisite for virtually all nanopore applications, the chapter describes recent experiments that resolve nucleic acid structure and its interaction with...

Solid-state nanopores are sensors capable of analysing individual unlabelled DNA molecules in solution. Although the critical information obtained from nanopores (for example, DNA sequence) comes from the signal collected during DNA translocation, the throughput of the method is determined by the rate at which molecules arrive and thread into the p...

The ability to apply a force on individual biomolecular complexes and measure their time-dependent response have begun to reveal their mechanical properties, interactions with other biomolecules, and self-interactions. A number of single-molecule methods have been developed and applied in the past decade to broad range of biological system, includi...

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in...

Bacteria have a complex internal organization with specific localization of many proteins and DNA, which dynamically move during the cell cycle and in response to changing environmental stimuli. Much less is known, however, about the localization and movements of RNA molecules. By modifying our previous RNA labeling system, we monitor the expressio...