Alon Singer

Optical Recognition of Converted DNA Nucleotides for Single-Molecule DNA Sequencing Using Nanopore Arrays

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 detectable photon bursts, at high speed. We show that signals from multiple nanopores can be detected simultaneously, allowing straightforward parallelization to large nanopore arrays.

Nanopore based sequence specific detection of duplex DNA for genomic profiling.

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 and sequence specificity. Sequence detection is performed by reading the ion current traces of individual translocating DNA molecules, which display a characteristic secondary blockade level, absent in untagged molecules. The potential for barcoding DNA is demonstrated through nanopore analysis of once-tagged and twice-tagged DNA at different locations on the same genomic fragment. Our high-throughput, long-read length method can be used to identify key sequences embedded in individual DNA molecules, without the need for amplification or fluorescent/radio labeling. This opens up a wide range of possibilities in human genomics as well as in pathogen detection for fighting infectious diseases.

Synchronous optical and electrical detection of biomolecules traversing through solid-state nanopores

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 makes it possible, for the first time, to visualize DNA and DNA-protein complexes translocating through a nanopore with high temporal resolution (1000 frames/s) and good signal to background. This paper describes a detailed experimental design of custom optics and data acquisition hardware to achieve simultaneous high resolution electrical and optical measurements on labeled biomolecules as they traverse through a approximately 4 nm synthetic pore. In conclusion, we discuss new directions and measurements, which this technique opens up.

Electronic Barcoding of a Viral Gene at the Single-Molecule Level

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 sensing individual genes from two highly similar human immunodeficiency virus subtypes, demonstrating feasibility of a novel, single-molecule diagnostic platform for rapid pathogen classification.

Fabrication and characterization of solid-state nanopore arrays for high-throughput DNA sequencing

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 down to the sub-10 nm range, compatible with the requirements for nanopore-based sequencing using optical readout. We find that the TiO(2) layers produce a lower photoluminescence background as compared with the more widely used Al(2)O(3) coatings. The functionality of the nanopore array was demonstrated by the simultaneous optical detection of DNA-quantum dot conjugates, which were electro-kinetically driven through the nanopores. Our optical scheme employs total internal reflection fluorescence microscopy to illuminate a wide area of the TiO(2)-coated membrane. A highly parallel system for observing DNA capture events in a uniformly sized 6 × 6 nanopore array was experimentally realized.

DNA sequencing and bar-coding using solid-state nanopores

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 describe a method by which nanopores can be used as single‐molecule genotyping tools. For DNA sequencing, the distinction among the four types of DNA nucleobases is achieved by employing a biochemical procedure for DNA expansion. In this approach, each nucleobase in each DNA strand is converted into one of four predefined unique 16‐mers in a process that preserves the nucleobase sequence. The resulting converted strands are then hybridized to a library of four molecular beacons, each carrying a unique fluorophore tag, that are perfect complements to the 16‐mers used for conversion. Solid‐state nanopores are then used to sequentially remove these beacons, one after the other, leading to a series of photon bursts in four colors that can be optically detected. Single‐molecule genotyping is achieved by tagging the DNA fragments with γ‐modified synthetic peptide nucleic acid probes coupled to an electronic characterization of the complexes using solid‐state nanopores. This method can be used to identify and differentiate genes with a high level of sequence similarity at the single‐molecule level, but different pathology or response to treatment. We will illustrate this method by differentiating the pol gene for two highly similar human immunodeficiency virus subtypes, paving the way for a novel diagnostics platform for viral classification.

Duplex DNA-Invading γ-Modified Peptide Nucleic Acids Enable Rapid Identification of Bloodstream Infections in Whole Blood

ABSTRACT Bloodstream infections are a leading cause of morbidity and mortality. Early and targeted antimicrobial intervention is lifesaving, yet current diagnostic approaches fail to provide actionable information within a clinically viable time frame due to their reliance on blood culturing. Here, we present a novel pathogen identification (PID) platform that features the use of duplex DNA-invading γ-modified peptide nucleic acids (γPNAs) for the rapid identification of bacterial and fungal pathogens directly from blood, without culturing. The PID platform provides species-level information in under 2.5 hours while reaching single-CFU-per-milliliter sensitivity across the entire 21-pathogen panel. The clinical utility of the PID platform was demonstrated through assessment of 61 clinical specimens, which showed >95% sensitivity and >90% overall correlation to blood culture findings. This rapid γPNA-based platform promises to improve patient care by enabling the administration of a targeted first-line antimicrobial intervention. IMPORTANCE Bloodstream infections continue to be a major cause of death for hospitalized patients, despite significant improvements in both the availability of treatment options as well their application. Since early and targeted antimicrobial intervention is one of the prime determinants of patient outcome, the rapid identification of the pathogen can be lifesaving. Unfortunately, current diagnostic approaches for identifying these infections all rely on time-consuming blood culture, which precludes immediate intervention with a targeted antimicrobial. To address this, we have developed and characterized a new and comprehensive methodology, from patient specimen to result, for the rapid identification of both bacterial and fungal pathogens without the need for culturing. We anticipate broad interest in our work, given the novelty of our technical approach combined with an immense unmet need. Bloodstream infections continue to be a major cause of death for hospitalized patients, despite significant improvements in both the availability of treatment options as well their application. Since early and targeted antimicrobial intervention is one of the prime determinants of patient outcome, the rapid identification of the pathogen can be lifesaving. Unfortunately, current diagnostic approaches for identifying these infections all rely on time-consuming blood culture, which precludes immediate intervention with a targeted antimicrobial. To address this, we have developed and characterized a new and comprehensive methodology, from patient specimen to result, for the rapid identification of both bacterial and fungal pathogens without the need for culturing. We anticipate broad interest in our work, given the novelty of our technical approach combined with an immense unmet need.

DNA Sequencing by Nanopore-Induced Photon Emission

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 optical detection. We convert target DNA 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 photon bursts that can be detected with a custom-made microscope. We do not use any enzymes in the readout stage; thus, our system is not limited by the highly variable processivity, lifetime, and inaccuracy of individual enzymes that can hinder throughput and reliability. Furthermore, because our system uses purely optical readout, we can take advantage of high-end, wide-field imaging devices to record from multiple nanopores simultaneously. This allows an extremely straightforward parallelization of our system to nanopore arrays.

Nanopore-based Sensing of Individual Nucleic Acid Complexes

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 robustness to both physical and chemical stresses, as well as control over pore size/shape/location and facilitates parallel detection with nanopore arrays. Nanopores in the 1–5 nm diameter range represent an important size regime for studying nucleic acids, as these pores can translocate nucleic acid molecules only in a linear or unfolded fashion, enabling readout of local nucleic acid structural alterations. In this review, we focus on the fundamental aspects of nanopore-based nucleic acid analysis, namely the DNA capture process and the subsequent translocation process. We compile here a multi-parametric study of DNA molecules spanning a large length scale, and discuss the influence of electrolyte concentrations on the capture and translocation processes. We further discuss the ability of nanopores to identify structural changes in the DNA due to non-specific binding of small molecules or specific hybridization with peptide nucleic acids probes. Through our continuing efforts at understanding the underlying processes which govern the capture and translocation process, we will be better positioned to harness the inherent abilities of nanopores to interrogate the internal structure of nucleic acids, enabling improved sensing applications at the single-molecule level.