DNA microspot assay using single-molecule detection and requiring 1.8 nL samples only

An ultra-sensitive DNA microspot assay was developed that required 1.8?nL samples and was based on single-molecule detection. The solution of the target DNA (tDNA) was spotted onto the coverslip modified with capture DNA (DNA1) and blocked with ethanolamine and bovine serum albumin using a pintool type microspoting robot. The microspot had a diameter of ~300???m. The tDNA was captured by the DNA1, and the tDNA was then labeled with a detection DNA that previously was labeled with a quantum dot. Next, a fluorescence microscopic image of the microspot was acquired using a single-molecule microspot reader during total internal reflection fluorescence excitation. As little as 4?×?10^?22 mole (240 molecules) of tDNA can be detected by this method. The response is linear in the range from 6.0?×?10^?22 to 1.2?×?10^?19 mole of tDNA. All operations (including the acquisition of microspot images and single-molecule counting) were performed using the MetaMorph software. The assay was applied to the determination of osteopontin messenger RNA in single decidual stromal cells without the need for PCR amplification.

Simple lab-on-chip approach with time-based detection

A simple lab-on-chip approach with time-based detection is proposed. A platform is made from a piece of acrylic differently shaped channels for introducing sample and reagent(s) using flow manipulation. Time-based changes involving migration of the reaction zone are monitored. The changes can be visually monitored by using a stop-watch with naked eyes observation. Some applications for the determination of ascorbic acid, acetic acid and iron in real samples with different chemistries were demonstrated.     

Assay of chondroitin sulfate using time-based detection in a simple lab-on-chip

Amount of chondroitin sulfate in supplementary tablets is determined using the reaction with methylene blue in a proposed simple lab-on-chip platform. An easy approach of detection by timing the migration of the reaction zone, which depends upon concentration of chondroitin sulfate, is described. This simple assay system does not require any complicated or expensive instrumentation and has a potential of further development for on-site analysis.     

Integration of exonuclease III-powered three-dimensional DNA walker with single-molecule detection for multiple initiator caspases assay

Initiator caspases are important components of cellular apoptotic signaling and they can activate effector caspases in extrinsic and intrinsic apoptotic pathways. The simultaneous detection of multiple initiator caspases is essential for apoptosis mechanism studies and disease therapy. Herein, we develop a sensitive nanosensor based on the integration of exonuclease III (Exo III)-powered three-dimensional (3D) DNA walker with single-molecule detection for the simultaneous measurement of initiator caspase-8 and caspase-9. This assay involves two peptide–DNA detection probe-conjugated magnetic beads and two signal probe-conjugated gold nanoparticles (signal probes@AuNPs). The presence of caspase-8 and caspase-9 can induce the cleavage of peptides in two peptide–DNA detection probes, releasing two trigger DNAs from the magnetic beads, respectively. The two trigger DNAs can serve as the walker DNA to walk on the surface of the signal probes@AuNPs powered by Exo III digestion, liberating numerous Cy5 and Texas Red fluorophores which can be quantified by single-molecule detection, with Cy5 indicating caspase-8 and Texas Red indicating caspase-9. Notably, the introduction of the AuNP-based 3D DNA walker greatly reduces the background signal and amplifies the output signals, and the introduction of single-molecule detection further improves the detection sensitivity. This nanosensor is very sensitive with a detection limit of 2.08 × 10⁻⁶ U μL⁻¹ for caspase-8 and 1.71 × 10⁻⁶ U μL⁻¹ for caspase-9, and it can be used for the simultaneous screening of caspase inhibitors and the measurement of endogenous caspase activity in various cell lines at the single-cell level. Moreover, this nanosensor can be extended to detect various proteases by simply changing the peptide sequences of the detection probes.

We demonstrate the simultaneous detection of multiple initiator caspases by integrating exonuclease III-powered three-dimensional DNA walker with single-molecule detection.     

Comparative quantification of nucleic acids using single-molecule detection and molecular beacons

This paper reports a highly sensitive homogenous method for comparative quantification of nucleic acids based on single-molecule detection (SMD) and molecular beacons (MBs). Two different color MBs were used to perform a separation-free comparative hybridization assay for simultaneous quantification of both target and control strands. A fluorescent burst, emitted from a single hybrid when it passes through a minuscule laser-focused region, is detected with high signal-to-noise ratio (SNR) by using single-molecule fluorescence spectroscopy. Targets are quantified via counting of discrete fluorescent bursts. The high SNR achieved in both detection channels overcame the complications of fluorescent variability usually observed in dual-color ensemble measurements. In comparison with the conventional ensemble methods, this method improved the detection limit by 3 orders of magnitude and reduced the probe consumption by 6 orders of magnitude, facilitating a highly sensitive approach for comparative quantification of nucleic acids and offering great promise for genomic quantification without amplification.     

Highly-integrated lab-on-chip system for point-of-care multiparameter analysis

A novel innovative approach towards a marketable lab-on-chip system for point-of-care in vitro diagnostics is reported. In a consortium of seven Fraunhofer Institutes a lab-on-chip system called "Fraunhofer ivD-platform" has been established which opens up the possibility for an on-site analysis at low costs. The system features a high degree of modularity and integration. Modularity allows the adaption of common and established assay types of various formats. Integration lets the system move from the laboratory to the point-of-need. By making use of the microarray format the lab-on-chip system also addresses new trends in biomedicine. Research topics such as personalized medicine or companion diagnostics show that multiparameter analyses are an added value for diagnostics, therapy as well as therapy control. These goals are addressed with a low-cost and self-contained cartridge, since reagents, microfluidic actuators and various sensors are integrated within the cartridge. In combination with a fully automated instrumentation (read-out and processing unit) a diagnostic assay can be performed in about 15 min. Via a user-friendly interface the read-out unit itself performs the assay protocol, data acquisition and data analysis. So far, example assays for nucleic acids (detection of different pathogens) and protein markers (such as CRP and PSA) have been established using an electrochemical read-out based on redoxcycling or an optical read-out based on total internal reflectance fluorescence (TIRF). It could be shown that the assay performance within the cartridge is similar to that found for the same assay in a microtiter plate. Furthermore, recent developments are the integration of sample preparation and polymerase chain reaction (PCR) on-chip. Hence, the instrument is capable of providing heating-and-cooling cycles necessary for DNA-amplification. In addition to scientific aspects also the production of such a lab-on-chip system was part of the development since this heavily affects the success of a later market launch. In summary, the Fraunhofer ivD-platform covers the whole value chain ranging from microfluidics, material and polymer sciences, assay and sensor development to the production and assembly design. In this consortium the gap between diagnostic needs and available technologies can be closed.     

A one-step highly sensitive method for DNA detection using dynamic light scattering

A one-step homogeneous DNA detection method with high sensitivity was developed using gold nanoparticles (AuNPs) coupled with dynamic light scattering (DLS) measurement. Citrate-protected AuNPs with a diameter of 30 nm were first functionalized with two sets of single-stranded DNA probes and then used as optical probes for DNA detection. In the presence of target DNA, the hybridization between target DNA and the two nanoparticle probes caused the formation of nanoparticle dimers, trimers, and oligomers. As a result, the nanoparticle aggregation increased the average diameter of the whole nanoparticle population, which can be monitored simply by DLS measurement. A quantitative correlation can be established between the average diameter of the nanoparticles and the target DNA concentration. This DLS-based assay is extremely easy to conduct and requires no additional separation and amplification steps. The detection limit is around 1 pM, which is 4 orders of magnitude better than that of light-absorption-based methods. Single base pair mismatched DNAs can be readily discriminated from perfectly matched target DNAs using this assay.     

Label-free "digital detection" of single-molecule DNA hybridization with a single electron transistor

Here we present a novel assay that eliminates fluorescent labels and enables "digital detection" of single-molecule DNA hybridization in complex matrixes with greatly simplified protocols. Electronic coupling of the binding state of a single oligonucleotide to the quantum dot (QD) of a single electron transistor (SET) affords direct observation of binding events in real-time via "molecular gating". The change of electrostatic charge associated with the molecular capture is used in lieu of a gate electrode to modulate the SET conductivity. Target oligos containing base mismatches do not elicit SET response under 0.1X SSC at room temperature nor do changes in ionic strength or pH. Furthermore, hybridization is detected even in optically inaccessible matrixes such as serum or quanidinium thiocyanate lysis buffer.     

Time-of-flight thermal flowrate sensor for lab-on-chip applications

We describe a thermal microflowrate sensor for measuring liquid flow velocity in microfluidic channels, which is capable of providing a highly accurate response independent of the thermal and physical properties of the working liquid. The sensor consists of a rectangular channel containing a heater and several temperature detectors microfabricated on suspended silicon bridges. Heat pulses created by the heater are advected downstream by the flow and are detected using the temperature detector bridges. By injecting a pseudo-stochastic thermal signal at the heater and performing a cross correlation between the detected and the injected signals, we can measure the single-pulse response of the system with excellent signal-to-noise ratio and hence deduce the thermal signal time-of-flight from heater to detector. Combining results from several detector bridges allows us to eliminate diffusion effects, and thus calculate the flow velocity with excellent accuracy and linearity over more than two orders of magnitude. The experimental results obtained with several test fluids closely agree with data from finite element analysis. We developed a phenomenological model which supports and explains the observed sensor response. Several fully functional sensor prototypes were built and characterized, proving the feasibility and providing a critical component to microfluidic lab-on-chip applications where accurate flow measurements are of importance.     

Lipid bilayer membrane technologies: A review on single-molecule studies of DNA sequencing by using membrane nanopores

Nanopores based on α-hemolysin and MspA represent attractive sensing platforms due to easy production and operation with relatively low background noise. Such characteristics make them highly favorable for sequencing nucleic acids. Artificial lipid bilayer membranes, also referred to as black lipid membranes, in conjunction with membrane nanopores, can be applied to both the detection and highly efficient sequencing of DNA on a single-molecule level. However, the inherently weak physical properties of the membrane have impeded progress in these areas. Current issues impeding the ultimate recognition of the artificial lipid bilayer as a viable platform for detection and sequencing of DNA include membrane stability, lifespan, and automation. This review (with 105 references) highlights attempts to improve the attributes of the artificial lipid bilayer membrane starting with an overview on the present state and limitations. The first main section covers lipid bilayer membranes (BLM) in general. The following section reviews the various kinds of lipid bilayer membrane platforms with subsections on polymer membranes, solid-supported membranes, hydrogel-encapsulated membranes, shippable and storable membrane platforms, and droplet interface bilayers. A further section covers engineered biological nanopore sensor applications using BLMs with subsections offering a comparative view of different DNA sequencing methods, a detailed look at DNA Sequencing by synthesis using alpha-hemolysin nanopores, sequencing by synthesis using the MspA nanopore and quadromer map, and on limitations of sequencing based on synthesis technology. We present an outlook at the end that discusses current research trends on single-molecule sequencing to highlight the significance of this technology and its potential in the medical and environmental fields.

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Graphical abstract Sequencing by Synthesis, a novel method of sequencing DNA, uses the αHL biological nanopore and the artificial lipid bilayer membrane platform. Polymer tags attached to nucleotides bind to the polymerase-primer–template complex and are characterized by the nanopore upon release.

     

Detection of single-molecule DNA hybridization by using dual-color total internal reflection fluorescence microscopy

We examined the use of prism-type simultaneous dual-color total internal reflection fluorescence microscopy (TIRFM) to probe DNA molecules at the single-molecule level. The system allowed the direct detection of the complementary interactions between single-stranded probe DNA molecules (16-mer) and various lengths of single-stranded target DNA molecules (16-mer and 55-mer) that had been labeled with different fluorescent dyes (Cy3, Cy5, and fluorescein). The polymer-modified glass substrate and the extent of DNA probe immobilization were easily characterized either with standard TIRFM or with atomic force microscopy. However, only dual-color TIRFM could provide unambiguous images of individual single-stranded target DNA molecules hybridized with the correct sequence in the range of fM–aM. Succinic anhydride showed low RMS roughness and was found to be an optimal blocking reagent against non-specific adsorption, with an efficiency of 92%. This study provides a benchmark for directly monitoring the interactions and the detection of co-localization of two different DNA molecules and can be applied to the development of a nanoarray biochip at the single-molecule level.     

A new method for the covalent attachment of DNA to a surface for single-molecule studies

Attachments between DNA and a surface or bead are often necessary for single-molecule studies of DNA and DNA-protein interactions. In single-molecule mechanical studies using optical or magnetic tweezers, such attachments must be able to withstand the applied forces. Here we present a new method for covalently attaching DNA to a glass surface, which uses N-hydroxysuccinimide (NHS) modified PEG that is suitable for high-force single-molecule mechanical studies. A glass surface is coated with silane-PEG-NHS and DNA is covalently linked through a reaction between the NHS group and an amine modified nucleotide that has been incorporated into the DNA. After DNA attachment, non-reacted NHS groups are hydrolyzed leaving a PEG-covered surface which has the added benefit of reducing non-specific surface interactions. This method permits specific binding of the DNA to the surface through a covalent bond. At the DNA end not attached to the surface, we attach a streptavidin-coated polystyrene bead and measure force-versus-extension using an optical trap. We show that our method allows a tethered DNA molecule to be pulled through its overstretching transition (> 60pN) multiple times. We anticipate this simple yet powerful method will be useful for many researchers.     

An ultra-sensitive DNA assay based on single-molecule detection coupled with hybridization accumulation and its application

An ultra-sensitive assay for quantification of DNA based on single-molecule detection coupled with hybridization accumulation was developed. In this assay, target DNA (tDNA) in solution was accumulated on a silanized substrate blocked with ethanolamine and bovine serum albumin (BSA) through a hybridization reaction between tDNA and capture DNA immobilized on the substrate. The tDNA on the substrate was labeled with quantum dots which had been modified with detection DNA and blocked with BSA. The fluorescence image of single QD-labeled tDNA molecules on the substrate was acquired using total internal reflection fluorescence microscopy. The tDNA was quantified by counting the bright dots on the image from the QDs. The limit of detection of the DNA assay was as low as 6.4 × 10(-18) mol L(-1). Due to the ultra-high sensitivity, the DNA assay was applied to measure the beta-2-microglobulin messenger RNA level in single human breast cancer cells without a need for PCR amplification.     

Continuous-flow single-molecule CE with high detection efficiency

This paper describes the use of two-beam line-confocal detection geometry for measuring the total mobility of individual molecules undergoing continuous-flow CE separation. High-sensitivity single-molecule confocal detection is usually performed with a diffraction limited focal spot (approximately 500 nm in diameter), which necessitates the use of nanometer-sized channels to ensure all molecules flow through the detection volume. To allow for the use of larger channels that are a few micrometers in width, we employed cylindrical optics to define a rectangular illumination area that is diffraction-limited (approximately 500 nm) in width, but a few micrometers in length to match the width of the microchannel. We present detailed studies that compare the performance of this line-confocal detection geometry with the more widely used point-confocal geometry. Overall, we found line-confocal detection to provide the highest combination of signal-to-background ratio and spatial detection efficiency when used with micrometer-sized channels. For example, in a 2 microm wide channel we achieved a 94% overall detection efficiency for single Alexa488 dye molecules when a 2 microm x 0.5 microm illumination area was used, but only 34% detection efficiency with a 0.5 microm-diameter detection spot. To carry out continuous-flow CE, we used two-beam fluorescent cross-correlation spectroscopy where the transit time of each molecule is determined by cross-correlating the fluorescence registered by two spatially offset line-confocal detectors. We successfully separated single molecules of FITC, FITC-tagged glutamate, and FITC-tagged glycine.     

A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution

The ability to monitor DNA polymerase activity with single-nucleotide resolution has been the cornerstone of a number of advanced single-molecule DNA sequencing concepts. Toward this goal, we report the first observation of the base-by-base DNA polymerase activity with single-base resolution at the single-molecule level. We describe the design and characterization of a supramolecular nanopore device capable of detecting up to nine consecutive DNA polymerase-catalyzed single-nucleotide primer extensions with high sensitivity and spatial resolution (相似文献   

Automated single-molecule imaging to track DNA shape

We describe a straightforward, automated line tracking method to visualize linear macromolecules as they rearrange shape by brownian diffusion and under external fields such as electrophoresis. The analysis, implemented here with 30 ms time resolution, identifies contour lines from one end of the molecule to the other without attention to structure smaller than the optical resolution. There are three sequential stages of analysis: first, "feature finding" to discriminate signal from noise; second, "line tracking" to approximate those shapes as lines; and third, "temporal consistency check" to discriminate reasonable from unreasonable fitted conformations in the time domain. Automation makes it straightforward to accumulate vast quantities of data while excluding the unreliable parts of it. We implement this analysis on fluorescence images of λ-DNA molecules in agarose gel to demonstrate its capability to produce large data sets for subsequent statistical analysis.     

Injection molded nanofluidic chips: fabrication method and functional tests using single-molecule DNA experiments

We demonstrate that fabrication of well-defined nanofluidic systems can be greatly simplified by injection molding of thermoplastic polymers. Chips featuring nanochannel arrays, microchannels and integrated interconnects are produced in a single processing step by injection molding. The resulting open channel structures are subsequently sealed by facile plasma-enhanced thermal bonding of a polymer film. This fast, inexpensive and industry-compatible method thus provides a single-use all-polymer platform for nanofluidic lab-on-a-chip applications. Its applicability for nanofluidics is demonstrated by DNA stretching experiments performed on individual double-stranded DNA molecules confined in the injection molded nanochannels. The obtained results are consistent with measurements performed in costly state-of-the-art silica nanochannels, for both straight and tapered channel geometries.     

A label-free fluorescent turn-on enzymatic amplification assay for DNA detection using ligand-responsive G-quadruplex formation

A facile and general label-free assay for sensitive and selective DNA detection has been developed based on enzyme amplification and ligand-responsive quadruplex formation.