Detection Of Single DNA Molecules Research Articles

An aptamer-triggered hybridization chain reaction strategy for ultra-sensitive biological nanopore detection of aflatoxin B1

Biological nanopore refers to a type of pore-forming membrane proteins, providing a unique platform for single molecule detection of DNA, RNA, peptide, etc. The direct detection of small molecules, especially of food chemical contaminants by biological nanopore, remains challenging due to their fast translocation dynamics, trace amount, and the interference from the complex food matrix. The effective recognition and signal conversion methods need to be developed. Herein, we employ an aptamer-triggered hybridization chain reaction (AtHCR) strategy for the detection of a mycotoxin, aflatoxin B1 (AFB1) by an α-hemolysin nanopore. In the absence of AFB1, the formed dsDNA polymers in HCR show a low frequency of long-time blockage signals. The presence of AFB1 can inhibit the hybridization chain reaction through the interaction between AFB1 and its aptamer, so the remaining hairpins can easily block the nanopore and induce frequent long-time blockage signals. The results show that the event frequency of long-blockage has a linear relationship with the AFB1 concentration in the range of 6.6 × 10−1–6.6 × 102 pmol/L. The recognition and amplification effect of AtHCR allows nanopore to detect AFB1 at a detection limit of 0.54 pmol/L. Furthermore, the developed AtHCR strategy can help biological nanopore to detect AFB1 in corn sample with satisfactory accuracy. The recognition and amplification effect achieved by coupling AtHCR to biological nanopore offers an enzyme-free single-molecule technology for the rapid detection of food chemical contaminants.

Deciphering the Structural Variants in Acute Myeloid Leukemia and Myelodysplastic Neoplasms By Optical Genome Mapping

Introduction Acute myeloid leukemia (AML) and myelodysplastic neoplasm (MDS) are common hematological malignancies, and the majority of patients carry genetic mutations, structural variants (SVs) and copy number variants (CNVs). Identifying these variants is important for diagnosis and prognosis, and in some cases can alter treatment management and outcomes. Methods for analyzing SVs and CNVs include karyotype analysis, fluorescence in situ hybridization, gene chips, and high-throughput gene sequencing technologies. Optical genome mapping (OGM) is a novel ultra-long single-molecule DNA detection based high throughput SVs analysis technology, which has the characteristics of whole genome coverage, high detection incidence and sensitivity. There were few publications presenting technical concordance of SVs detection between karyotyping and OGM, and there are no systematic studies comparing the concordance of OGM versus transcriptome sequencing (RNA-seq) in detecting gene fusion. This study aimed to compare the concordance of OGM and RNA-seq in the detection of fusion genes in AML and MDS cases, and the additional findings using OGM technology. Methods Bone marrow aspirates of 52 newly diagnosed or relapsed/refractory AML/MDS patients were recruited for OGM, RNA-seq, and leukemia-related gene DNA panel sequencing. Among these cases, 36 males and 16 were females, 15 pediatric (< 18 years) and 37 adults (≥18 years), with a median age of 31 years. Results Based on the RNA-seq results, 29/52 (55.8%) cases were detected with pathogenic gene fusions, including RUNX1::RUNX1T1, CBFB::MYH11, KMT2Aretc. (Figure 1). All those fusions were detected by OGM. For SVs with VAF>5%, the concordance was 98.1% (51/52 cases) when comparing OGM with karyotyping. OGM missed one case whose translocation happened close to the centromere. OGM reported more pathological variants with clinically significant in 5/52 cases (9.62%) and helped revise karyotype results in 1 case with clarifying marker chromosome. According to the ELN-2022 risk stratification mainly based on karyotype results, OGM's report results helped revise the risk stratification of 3 patients from intermediate to high risk. Fifty of 52 (96.2%) patients carried the gene mutation detected by the gene sequencing panel (Figure 1). In the samples of 4 patients who relapsed after receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) with donors and patients of different genders, we can infer the proportion of male and female DNA based on the copy number of chr Y detected by OGM, and then determine the proportion of donor cells. Conclusion This is the first AML cohort study to systematically compare the performance of OGM and RNA-seq, and the reported results of fusion genes are completely consistent. This is also the first report of applying the OGM technology in patients who relapsed after allo-HSCT and for chimerism analysis. Combined using OGM and targeted sequencing to fully characterize the CNV, SV, and mutation can be used for accurate stratification risk of AML/MDS. In conclusion, combining OGM and NGS can significantly improve the detection rate of genetic abnormalities with important pathological/diagnostic significance, and may bring new discoveries on SVs and pathogenic mechanisms.

Deciphering the Structural Variants in B Cell Acute Lymphoblastic Leukemia By Optical Genome Mapping

Introduction Structural variants (SVs) of the genome play a key role in the leukemogenesis and development of B cell lymphoblastic leukemia (B-ALL), including gene deletion, amplification, translocation, and other forms. Methods for analyzing SVs and CNVs include karyotype analysis, fluorescence in situ hybridization, gene chips, and high-throughput gene sequencing technologies. Optical genome mapping (OGM) is a novel ultra-long single-molecule DNA detection based high throughput SVs analysis technology, which has the characteristics of whole genome coverage, high detection incidence and sensitivity. There were few publications presenting technical concordance of SVs detection beween karyotyping and OGM, and there are no systematic studies comparing the concordance of OGM and RNA-seq in detecting gene fusion. This study aimed to compare the concordance of OGM and RNA-seq in the detection of fusion genes in B-ALL cases, and the additional findings using OGM technology. Methods Bone marrow aspirates of 70 patients with newly diagnosed or relapsed/refractory B-ALL were recruited into the study. All samples were conducted with both OGM and transcriptome sequencing (RNA-seq). Among these cases, 42 males and 28 females, 30 pediatric (< 18 years) and 40 adults (≥ 18 years) cases, ages ranged 4-60 years (median 16 years). Results Totally 50 gene fusions were reported in 48 cases, with 2 cases each carrying two fusion genes ( BCR::ABL1/ PAX5::ELK3 and P2RY8::CRLF2/ PAX5::GSE1, respectively) (Figure 1A). OGM and RNA-seq have complete concordance in reporting gene fusions, and OGM provides further insight into the genomic events that lead to gene fusion. A subtype of B-ALL characterized by UBTF microdeletion leading to UBTF::ATXN7L3 fusion and PAN3 downstream deletion-induced CDX2 aberrant expression has been reported recently, and has been accepted by the International Consensus Classification as a novel molecular subtype of UBTF/CDX2 of B-ALL. We performed the OGM investigation on 6 UBTF/CDX2 cases which were identified by RNA-seq. OGM identified both microdeletion events in all 6 cases, and further reported 1q amplification in 3 of them (Figure 1B, C). B-lymphocyte differentiation-associated transcription factor gene defects are common and important genetic abnormalities in B-ALL, often manifested as complete or partial deletion of genes. We focused on a panel including 30 genes in the OGM data and determined IKZF1 deletion in 35 patients, CDKN2A/B deletion in 32 patients, and PAX5 deletion in 25 patients (Figure 1 A). Follow-up data showed that patients with CDKN2A/B deletion had a worse prognosis than those without CDKN2A/B deletion ( P = 0.025). Conclusion This is the first B-ALL cohort to systematically compare OGM and RNA-seq in gene fusion detection, and the result showed complete consistency. RNA-seq has significant advantages in analyzing gene fusion transcripts, while OGM has technical advantages in analyzing SVs at the genome level. Combining the two technologies can accurately analyze gene fusion and other SVs in B-ALL, significantly improve the detection rate of SVs with important pathological/diagnostic classification significance, and may bring new discoveries on SVs and pathogenic mechanisms.

Plasmonic Bowl-Shaped Nanopore for Raman Detection of Single DNA Molecules in Flow-Through.

Plasmonic nanopores combined with Raman spectroscopy are emerging as platforms for single-molecule detection and sequencing in label-free mode. Recently, the ability of identifying single DNA bases or amino acids has been demonstrated for molecules adsorbed on plasmonic particles and then delivered into the plasmonic pores. Here, we report on bowl-shaped plasmonic gold nanopores capable of direct Raman detection of single λ-DNA molecules in a flow-through scheme. The bowl shape enables the incident laser to be focused into the nanopore to generate a single intense hot spot with no cut off in pore size. Therefore, we achieved ultrasmall focusing of NIR light in a spot of 3 nm. This enabled us to detect 7 consecutive bases along the DNA chain in flow-through conditions. Furthermore, we found a novel electrofluidic mechanism to manipulate the molecular trajectory within the pore volume so that the molecule is pushed toward the hot spot, thus improving the detection efficiency.

De novo design of β-barrel nanopore with Gly-kink towards single-molecule detection.

Nanopore sensing is a rapid and label-free method for single-molecule detection of DNA, peptides, and protein. However, it remains challenging to adjust the size and geometry of nanopores for each target molecule using biological nanopores. Therefore, constructing size and geometry controllable nanopore is highly demanded. Recently, de novo design has enabled the creation of specific structures and functions of proteins. We have previously designed a de novo β-hairpin peptide named SV28, which is assembled to form a multidisperse-sized β-barrel nanopore in a bilayer lipid membrane (BLM). A monodisperse-size nanopore is required for the highly sensitive detection of a specific target. Toward constructing a monodisperse-sized nanopore, we focused on Gly-kink. In the computational protein design, introducing the Gly-kink in a β-barrel structure makes the packing of the β-strands relaxed and the β-barrel structure to be more rigid. Then, we redesigned SVG28 with the introduction of Gly-kink into SV28. To evaluate the pore-forming ability of SVG28, we conducted channel current measurement with BLM in microdevices and signal classification analysis. As result, the SVG28 nanopore converged relatively monodisperse size (SVG28: φ = 1.7 nm, SV28: φ = 1.7-6.3 nm). Moreover, we carried out molecular dynamics simulations of peptide nanopores, indicating the stabilization of the β-barrel structure by introducing the Gly-kink. For future nanopore amino acid sequencing, we applied SVG28 nanopore to polypeptide detection. The SVG28 nanopore could detect and discriminate molecular weight difference of poly-l-lysine (PLL: φ = 1.1-1.4 nm). Furthermore, the SVG28 nanopore could identify the single amino acid in the oligo-arginine with several different types of single amino acids (R7X: φ = 1.0-1.5 nm). The above achievements are due to size and geometry matching between the SVG28 nanopore and the targets. Now, we are redesigning SVG28 to construct nanopores that have specific large pore sizes for protein detection.

DNA Origami Nanoantennas for Fluorescence Enhancement.

The possibility to increase fluorescence by plasmonic effects in the near-field of metal nanostructures was recognized more than half a century ago. A major challenge, however, was to use this effect because placing single quantum emitters in the nanoscale plasmonic hotspot remained unsolved for a long time. This not only presents a chemical problem but also requires the nanostructure itself to be coaligned with the polarization of the excitation light. Additional difficulties arise from the complex distance dependence of fluorescence emission: in contrast to other surface-enhanced spectroscopies (such as Raman spectroscopy), the emitter should not be placed as close as possible to the metallic nanostructure but rather needs to be at an optimal distance on the order of a few nanometers to avoid undesired quenching effects.Our group addressed these challenges almost a decade ago by exploiting the unique positioning ability of DNA nanotechnology and reported the first self-assembled DNA origami nanoantennas. This Account summarizes our work spanning from this first proof-of-principle study to recent advances in utilizing DNA origami nanoantennas for single DNA molecule detection on a portable smartphone microscope.We summarize different aspects of DNA origami nanoantennas that are essential for achieving strong fluorescence enhancement and discuss how single-molecule fluorescence studies helped us to gain a better understanding of the interplay between fluorophores and plasmonic hotspots. Practical aspects of preparing the DNA origami nanoantennas and extending their utility are also discussed.Fluorescence enhancement in DNA origami nanoantennas is especially exciting for signal amplification in molecular diagnostic assays or in single-molecule biophysics, which could strongly benefit from higher time resolution. Additionally, biophysics can greatly profit from the ultrasmall effective detection volumes provided by DNA nanoantennas that allow single-molecule detection at drastically elevated concentrations as is required, e.g., in single-molecule DNA sequencing approaches.Finally, we describe our most recent progress in developing DNA NanoAntennas with Cleared HOtSpots (NACHOS) that are fully compatible with biomolecular assays. The developed DNA origami nanoantennas have proven robustness and remain functional after months of storage. As an example, we demonstrated for the first time the single-molecule detection of DNA specific to antibiotic-resistant bacteria on a portable and battery-driven smartphone microscope enabled by DNA origami nanoantennas. These recent developments mark a perfect moment to summarize the principles and the synthesis of DNA origami nanoantennas and give an outlook of new exciting directions toward using different nanomaterials for the construction of nanoantennas as well as for their emerging applications.

Quantum scale organic semiconductors for SERS detection of DNA methylation and gene expression

Cancer stem cells (CSC) can be identified by modifications in their genomic DNA. Here, we report a concept of precisely shrinking an organic semiconductor surface-enhanced Raman scattering (SERS) probe to quantum size, for investigating the epigenetic profile of CSC. The probe is used for tag-free genomic DNA detection, an approach towards the advancement of single-molecule DNA detection. The sensor detected structural, molecular and gene expression aberrations of genomic DNA in femtomolar concentration simultaneously in a single test. In addition to pointing out the divergences in genomic DNA of cancerous and non-cancerous cells, the quantum scale organic semiconductor was able to trace the expression of two genes which are frequently used as CSC markers. The quantum scale organic semiconductor holds the potential to be a new tool for label-free, ultra-sensitive multiplexed genomic analysis.

Toward the highly sensitive SERS detection of bio-molecules: the formation of a 3D self-assembled structure with a uniform GO mesh between Ag nanoparticles and Au nanoparticles.

We report a structure to form a hybrid system in which a mesh is sandwiched between Au nanoparticles (AuNPs) and Ag nanoparticles (AgNPs). This self-assembly method uses smaller and denser AgNPs "hot spots" that are spin-coated on a AuNPs@GO mesh nanostructure formed by the reaction of GO@MoS2 and HAuCl4 to form AuNPs@GO mesh@AgNPs SERS substrates. Sub-40-nm mesh and 10-nm gaps ensure the landing sites and spacing of the AgNPs. Consequently, the design integrates the strong plasmonic effects of AgNPs and AuNPs with the biological compatibility of the GO mesh. Crystal violet (CV) as low as 10-15 M can be detected, which confirms the ultrahigh sensitivity of AuNPs@GO mesh@AgNPs. Furthermore, the reproducibility, stability, and finite-difference time-domain (FDTD) simulations confirm the value of this SERS substrate. This material can be used for label-free DNA detection, and the AuNPs@GO mesh@AgNPs substrate facilitated single-molecule DNA detection limits.

Single-Molecule Detection of DNA in a Nanochannel by High-Field Strength-Assisted Electrical Impedance Spectroscopy.

Many researchers have fabricated micro and nanofluidic devices incorporating optical, chemical, and electrical detection systems with the aim of achieving on-chip analysis of macromolecules. The present study demonstrates a label-free detection of DNA using a nanofluidic device based on impedance measurements that is both sensitive and simple to operate. Using this device, the electrophoresis and dielectrophoresis effect on DNA conformation and the length dependence were examined. A low alternating voltage was applied to the nanogap electrodes to generate a high intensity field (>0.5 MV/m) under non-faradaic conditions. In addition, a 100 nm thick gold electrode was completely embedded in the substrate to allow direct measurements of a solution containing the sample passing through the gap, without any surface modification required. The high intensity field in this device produced a dielectrophoretic force that stretched the DNA molecule across the electrode gap at a specific frequency, based on back and forth movements between the electrodes with the DNA in a random coil conformation. The characteristics of 100 bp, 500 bp, 1 kbp, 5 kbp, 10 kbp, and 48 kbp λ DNA associated with various conformations were quantitatively analyzed with high resolution (on the femtomolar level). The sensitivity of this system was found to be more than about 10 orders of magnitude higher than that obtained from conventional linear alternating current (AC) impedance for the analysis of bio-polymers. This new high-sensitivity process is expected to be advantageous with regard to the study of complex macromolecules and nanoparticles.

TAMRA-polypyrrole for A/T sequence visualization on DNA molecules.

Fluorophore-linked, sequence-specific DNA binding reagents can visualize sequence information on a large DNA molecule. In this paper, we synthesized newly designed TAMRA-linked polypyrrole to visualize adenine and thymine base pairs. A fluorescent image of the stained DNA molecule generates an intensity profile based on A/T frequency, revealing a characteristic sequence composition pattern. Computer-aided comparison of this intensity pattern with the genome sequence allowed us to determine the DNA sequence on a visualized DNA molecule from possible intensity profile pattern candidates for a given genome. Moreover, TAMRA-polypyrrole offers robust advantages for single DNA molecule detection: no fluorophore-mediated photocleavage and no structural deformation, since it exhibits a sequence-specific pattern alone without the use of intercalating dyes such as YOYO-1. Accordingly, we were able to identify genomic DNA fragments from Escherichia coli cells by aligning them to the genomic A/T frequency map based on TAMRA-polypyrrole-generated intensity profiles. Furthermore, we showed band and interband patterns of polytene chromosomal DNA stained with TAMRA-polypyrrole because it prefers to bind AT base pairs.

Approaching single DNA molecule detection with an ultrasensitive electrochemical genosensor based on gold nanoparticles and cobalt-porphyrin DNA conjugates.

We describe an ultrasensitive electrochemical genosensor based on gold nanoparticles and cobalt-porphyrin functionalised ssDNA probes. The sensitivity at the attomolar concentration level arises from an increased density of redox labels on the electrode surface compared to sensors without NP modification. The electrode detects as few as 23 DNA molecules, approaching single molecule detection.

Probing DNA Translocations in Nanopipettes using High-Speed Detection Electronics

Solid-state chip-based nanopores show great promise as biosensors for single-molecule detection of DNA and proteins(1,2). Nanopipettes are a novel subclass of solid-state nanopores and show desirable properties such as their ease of fabrication, easy integration into microfluidic systems and low device capacitance(3). Typically made from quartz, nanopipettes have a sharp tip with a high aspect ratio whereas chip-based nanopores typically have a more cylindrical shape. These different geometries affect the electric field distribution around the nanometre-sized opening and the DNA capture radius.Previous literature using solid-state nanopores has reported a scaling factor, p of DNA translocation times of p ∼ 1.3(2). Using a custom-built high-speed electrical detection system, p was examined using four different lengths of DNA(4,5). To our knowledge this is the first length dependence study of double-stranded DNA translocations in potassium chloride electrolyte using nanopipettes. Having found p = 1 ± 0.03 we hypothesize that the different p values may be due to DNA adsorption prior to translocation and have developed a theoretical model to rationalise the two results.(1) Plesa, C.; Kowalczyk, S. W.; Zinsmeester, R.; Grosberg, A. Y.; Rabin, Y.; Dekker, C. Nano Lett. 2013, 13, 658-663.(2) Storm, A. J.; Storm, C.; Chen, J.; Zandbergen, H.; Joanny, J. F.; Dekker, C. Nano Lett. 2005, 5, 1193-1197.(3) Gibb, T. R.; Ivanov, A. P.; Edel, J. B.; Albrecht, T. Anal. Chem. 2014, 86, 1864-1871.(4) Ciccarella, P.; Carminati, M.; Ferrari, G.; Fraccari, R.; Bahrami, A. In Integrated Low-Noise Current Amplifier for Glass-Based Nanopore Sensing; 10th Conference on Ph.D. Research in Microelectronics and Electronics (PRIME 2014), Grenoble, France, 2014, pp 1-4.(5) Fraccari, R. L.; Ciccarella, P.; Bahrami, A.; Carminati, M.; Ferrari, G.; Albrecht, T. 2015, (submitted).

Single molecule DNA detection with an atomic vapor notch filter

The detection of single molecules has facilitated many advances in life- and material-sciences. Commonly, it founds on the fluorescence detection of single molecules, which are for example attached to the structures under study. For fluorescence microscopy and sensing the crucial parameters are the collection and detection efficiency, such that photons can be discriminated with low background from a labeled sample. Here we show a scheme for filtering the excitation light in the optical detection of single stranded labeled DNA molecules. We use the narrow-band filtering properties of a hot atomic vapor to filter the excitation light from the emitted fluorescence of a single emitter. The choice of atomic sodium allows for the use of fluorescent dyes, which are common in life-science. This scheme enables efficient photon detection, and a statistical analysis proves an enhancement of the optical signal of more than 15% in a confocal and in a wide-field configuration.

Silicon Nanopore Detection of Single DNA Molecules by Ultraviolet Excitation

Silicon Nanopore Detection of Single DNA Molecules by Ultraviolet Excitation

One-by-one single-molecule detection of mutated nucleobases by monitoring tunneling current using a DNA tip.

A DNA molecule was utilized as a probe tip to achieve single-molecule genetic diagnoses. Hybridization of the probe and target DNAs resulted in electron tunneling along the emergent double-stranded DNA. Simple stationary monitoring of the tunneling current leads to single-molecule DNA detection and discovery of base mismatches and methylation.

Plasmonic Imaging and Detection of Single DNA Molecules

The capability of imaging and detecting single DNA molecules is critical in the study, analysis, and applications of DNA. Fluorescence imaging is a widely used method, but it suffers from blinking and photobleaching, and fluorescence tags may block or affect binding sites on DNA. We report on label-free imaging of single DNA molecules with a differential plasmonic imaging technique. The technique produces high contrast images due to the scattering of surface plasmonic waves by the molecules and the removal of background noises and interference patterns, allowing for quantitative analysis of individual DNA molecules. Simulation of the images based on a scattering model shows good agreement with the experiment. We further demonstrate optical mapping of single DNA molecules.

Slowing Down DNA Translocation Through Solid‐State Nanopores by Pressure

The effect of applied pressure on event duration distributions in 3 kb dsDNA translocation is systematically investigated. The effects of pressure magnitude and nanopore size on the length discrimination between 615 bp and 1.14 kbp dsDNA is studied. The pressure-controlled DNA translocation in solid-state nanopores makes a significant contribution to improve the temporal resolution in DNA single-molecule detection.

Imaging of Nonuniform Motion of Single DNA Molecules Reveals the Kinetics of Varying-Field Isotachophoresis

The nonuniform motion of charged species in a varying electric field may provide unique separation and focusing power for chemical, biochemical, and nanoscale studies. We imaged in real time the nonuniform motion of single DNA molecules under varying-field isotachophoresis (ITP) conditions. From the trajectories of single molecules, we obtained the time- and position-dependent electric field strength (E) and revealed the behavior of adaption barriers within electro-osmotic flow (EOF)-driven and EOF-independent ITP. We found that the initial terminating electrolyte zone of constant E is split into two zones: a highly adapted high-E zone and a low-E zone of gradually adapting electric field. The formation of the two unique zones is associated with the rate-limiting mass transfer barrier in EOF-driven ITP. As a result of the unique E distribution, DNA molecules first slow to a stop and then rapidly move backward to the leading electrolyte/terminating electrolyte boundary. This provides a novel mechanism for selective focusing of target molecules in dilute solutions of large volume. We show that the ITP focusing can improve the detection of single DNA molecules (limit of detection = 4 × 10(-17) mol/L), which are stochastically distributed at extremely low concentrations. The ITP strategy focuses individual molecules into a small volume that is matched with the focal point of single-molecule imaging.

Single-molecule DNA detection using a novel SP1 protein nanopore

SP1 protein as a new type of biological nanopore is described and is utilized to distinguish single-stranded DNA at the single-molecule level. Using the SP1 nanopore to investigate single molecule detection broadens the existing research areas of pore-forming biomaterials from unsymmetrical biological nanopores to symmetrical biological nanopores. This novel nanopore could provide a good candidate for single-molecule detection and characterization of biomaterial applications.

Direct electrical single-molecule detection of DNA through electron transfer induced by hybridization

Single-stranded DNA was utilized as a probe tip for single-molecule DNA detection. Hybridization of the DNA tip and target DNA induces electron tunneling through the resulting DNA duplex. It is demonstrated that the DNA tip allows not only genetic detection but also discovery of single-nucleotide polymorphisms at the single-molecule level.