Single nucleotide variation detection on 3D DNA microarray by ligation of two-terminal-modified universal probes

Exploiting the advantages of three-dimensional (3D) DNA microarray, we put forward a novel strategy termed "ligation of two-terminal-modified universal probes" for single nucleotide variation (SNV) detection on 3D microarray. By performing specific ligation reaction between the unmodified hybridization primer with 3' hydroxyl terminus and the universal probe with phosphorylated 5' terminus and fluorescently labeled 3' terminus, two point mutations (C3206T and A5301G) in the PCR products immobilized on the 3D polyacrylamide gel DNA microarray were accurately discriminated. This method can not only maintain the predominance of 3D DNA microarray as a platform with high through-put, but also can exert this predominance to the cases of detecting a small quantity of samples and multiple SNVs since four universal probes can be employed to detect all SNVs, therefore, it is more feasible for laboratory research and provide an effective tool for clinical diagnosis.     

Multiple and simultaneous detection of specific bacteria in enriched bacterial communities using a DNA microarray chip with randomly generated genomic DNA probes

A DNA microarray chip for detecting the presence of specific bacterial strains was developed using random genomic probes derived from genomic DNA, i.e., without any sequence information. Thirteen bacteria from different genuses were selected as targets. For the fabrication of the random genomic probes, genomic DNA from pure cultures of each bacterium was fractionated using several pairs of restriction endonucleases. After size fractionation of the genomic DNA fragments, random genomic libraries for each bacterium were constructed. From the library, specific probes were amplified by PCR and the probes were affixed to a slide glass to fabricate the DNA microarray chip. The results from tests with pure and mixed cultures of the bacteria used in the fabrication of the chips showed specific responses and only a small portion of cross-hybridization. This DNA microarray chip was also tested to detect the presence of specific bacteria in mixed populations. In these tests, it was demonstrated that this system provided a fast and specific response to the presence of bacterial species in mixed samples, even in activated sludge samples. This indicates that any DNA microarray chip for the detection of specific bacteria can be fabricated using the same protocols as presented in this study without requiring any genus level sequence information from pure isolates.     

Emerging low-dimensional materials for mid-infrared detection

Nano Research - Mid-infrared (IR) detectors based on the emerging low-dimensional (two-dimensional and quasi one-dimensional) materials offer unique characteristics including large bandgap...     

Development of a microfluidic platform with an optical imaging microarray capable of attomolar target DNA detection

In this paper, DNA hybridization in a microfluidic manifold is performed using fluorescence detection on a fiber-optic microarray. The microfluidic device integrates optics, sample transport, and fluidic interconnects on a single platform. A high-density optical imaging fiber array containing oligonucleotide-labeled microspheres was developed. DNA hybridization was observed at concentrations as low as 10 aM with response times of less than 15 min at a flow rate of 1 microL/min using 50 microL of target DNA samples. The fast response times coupled with the low sample volumes and the use of a high-density, fiber-optic microarray format make this method highly advantageous. This paper describes the initial development, optimization, and integration of the microfluidic platform with imaging fiber arrays.     

A free-labeled method for DNA-binding protein detection using a double-stranded DNA microarray

We have developed a new type of double-stranded DNA microarray to perform detection of sequence-specific DNA-binding proteins. The DNA-binding site of a DNA-binding protein is divided into two fragments. One fragment was immobilized on an aldehyde-coated glass microscope slide surface via chemical bonds. The other fragment was labeled with a fluorescent molecule. When using this kind of double-stranded DNA microarray, the labeled DNA fragment was pre-incubated with detection sample for 5 to 10 minutes and then hybridized with the microarray. In our experiment, six different concentrations of Nuclear Factor kappa-B P50 homodimer in detection samples were tested. The microarray fluorescence intensity was obtained and the relationship between the intensity and the protein concentration was calculated. The detection results suggested that this free-labeled detection system could have the ability to be used in research and medical diagnosis and for high-throughput screening of drugs targeted to DNA-binding proteins.     

Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55?°C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 μg ml(-1) swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.     

Quantum dots based probes conjugated to annexin V for photostable apoptosis detection and imaging

Quantum dots (Qdots) are nanoparticles exhibiting fluorescent properties that can be used for cell staining. We present here the development of quantum dots conjugated to Annexin V for specific targeting of apoptotic cells, for both apoptosis detection and staining of apoptotic "living" cells. For that purpose, Qdots Streptavidin Conjugates are coupled to biotinylated Annexin V, a 35-kDa protein which specifically recognizes and binds to phosphatidylserine (PS) moieties present on the outer membrane of apoptotic cells and not on healthy or necrotic cells. By using Annexin V, our Qdots probes are made specific for apoptotic cells. Staining of apoptotic cells was checked using fluorescence and confocal microscopy techniques and nonfixed cells. It is shown here that Qdots are insensitive to bleaching after prolonged exposure as opposed to organic dyes. This makes Qdots excellent candidates to continuously follow fast changes occurring at the membrane of apoptotic cells and facilitates time-lapse imaging as they alleviate any bleaching issue.     

Room-temperature mid-infrared laser sensor for trace gas detection

Design and operation of a compact, portable, room-temperature mid-infrared gas sensor is reported. The sensor is based on continuous-wave difference-frequency generation (DFG) in bulk periodically poled lithium niobate at 4.6 mum, pumped by a solitary GaAlAs diode laser at 865 nm and a diode-pumped monolithic ring Nd:YAG laser at 1064.5 nm. The instrument was used for detection of CO in air at atmospheric pressure with 1 ppb precision (parts in 10(9), by mole fraction) and 0.6% accuracy for a signal averaging time of 10 s. It employed a compact multipass absorption cell with a 18-m path length and a thermoelectrically cooled HgCdTe detector. Precision was limited by residual interference fringes arising from scattering in the multipass cell. This is the first demonstration of a portable high-precision gas sensor based on diode-pumped DFG at room temperature. The use of an external-cavity diode laser can provide a tuning range of 700 cm(-1) and allow the detection of several trace gases, including N(2) O, CO(2), SO(2), H(2) CO, and CH(4).     

Restoration and spectral recovery of mid-infrared chemical images

Fourier transform infrared (FTIR) microspectroscopy is a powerful technique for label-free chemical imaging that has supplied important chemical information about heterogeneous samples for many problems across a variety of disciplines. State-of-the-art synchrotron based infrared (IR) microspectrometers can yield high-resolution images, but are truly diffraction limited for only a small spectral range. Furthermore, a fundamental trade-off exists between the number of pixels, acquisition time and the signal-to-noise ratio, limiting the applicability of the technique. The recently commissioned infrared synchrotron beamline, infrared environmental imaging (IRENI), overcomes this trade off and delivers 4096-pixel diffraction limited IR images with high signal-to-noise ratio in under a minute. The spatial oversampling for all mid-IR wavelengths makes the IRENI data ideal for spatial image restoration techniques. Here, we measured and fitted wavelength-dependent point-spread-functions (PSFs) at IRENI for a 74× objective between the sample plane and detector. Noise-free wavelength-dependent theoretical PSFs are deconvoluted from images generated from narrow bandwidths (4 cm(-1)) over the entire mid-infrared range (4000-900 cm(-1)). The stack of restored images is used to reconstruct the spectra. Restored images of metallic test samples with features that are 2.5 μm and smaller are clearly improved in comparison to the raw data images for frequencies above 2000 cm(-1). Importantly, these spatial image restoration methods also work for samples with vibrational bands in the recorded mid-IR fingerprint region (900-1800 cm(-1)). Improved signal-to-noise spectra are reconstructed from the restored images as demonstrated for a mixture of spherical polystyrene beads in a polyurethane matrix. Finally, a freshly thawed retina tissue section is used to demonstrate the success of deconvolution achievable with a heterogeneous, irregularly shaped, biologically relevant sample with distinguishing spectroscopic features across the entire mid-IR spectral range.     

Nanoparticle probes with surface enhanced Raman spectroscopic tags for cellular cancer targeting

We have developed biocompatible, photostable, and multiplexing-compatible surface-enhanced Raman spectroscopic tagging material (SERS dots) composed of silver nanoparticle-embedded silica spheres and organic Raman labels for cellular cancer targeting in living cells. SERS dots showed linear dependency of Raman signatures on their different amounts, allowing their possibility for the quantification of targets. In addition, the antibody-conjugated SERS dots were successfully applied to the targeting of HER2 and CD10 on cellular membranes and exhibited good specificity. SERS dots demonstrate the potential for high-throughput screening of biomolecules using vibrational information.     

Molecular detection of bacterial pathogens using microparticle enhanced double-stranded DNA probes

Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.     

Two-dimensional infrared and mid-infrared imaging by single-photon frequency upconversion

Single-photon frequency upconversion is an effective method of infrared single-photon detection and imaging by converting the long-wavelength photons to shorter wavelengths to match the detector’s spectral response. We realized few-photon level 2D infrared imaging with a coincidence frequency upconversion system in a bulk periodically poled lithium niobate crystal. Moreover, the infrared photons carrying orbital angular momentum were converted to the visible regime with high efficiency, while the orbital angular momentum of the photons was well conserved during the frequency upconversion process. The single-photon frequency upconversion method was also used for mid-infrared imaging at 3.39 µm with high efficiency and low noise.     

Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection

A fully integrated biochip device that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and DNA microarray sensors was developed to perform DNA analysis of complex biological sample solutions. Sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis), polymerase chain reaction, DNA hybridization, and electrochemical detection were performed in this fully automated and miniature device. Cavitation microstreaming was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads and accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from approximately milliliters of whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.     

Microfabricated linear hydrogel microarray for single-nucleotide polymorphism detection

A platform is developed for rapid, multiplexed detection of single-nucleotide polymorphisms using gels copolymerized with oligonucleotide capture probes in a linear microchannel array. DNA samples are analyzed by electrophoresis through the linear array of gels, each containing 20-40 μM of a unique oligonucleotide capture probe. Electrophoresis of target DNA through the capture sites and the high concentration of capture probes within the gels enables significantly shorter incubation times than standard surface DNA microarrays. These factors also result in a significant concentration of target within the gels, enabling precise analysis of as little as 0.6 femtomoles of DNA target. Differential melting of perfectly matched and mismatched targets from capture probes as a function of electric field and temperature enables rapid, unambiguous identification of single-nucleotide polymorphisms.     

A microarray immunoassay for simultaneous detection of proteins and bacteria

We report the development and characterization of an antibody microarray biosensor for the rapid detection of both protein and bacterial analytes under flow conditions. Using a noncontact microarray printer, biotinylated capture antibodies were immobilized at discrete locations on the surface of an avidin-coated glass microscope slide. Preservation of capture antibody function during the deposition process was accomplished with the use of a low-salt buffer containing sucrose and bovine serum albumin. The slide was fitted with a six-channel flow module that conducted analyte-containing solutions over the array of capture antibody microspots. Detection of bound analyte was subsequently achieved using fluorescent tracer antibodies. The pattern of fluorescent complexes was interrogated using a scanning confocal microscope equipped with a 635-nm laser. This microarray system was employed to detect protein and bacterial analytes both individually and in samples containing mixtures of analytes. Assays were completed in 15 min, and detection of cholera toxin, staphylococcal enterotoxin B, ricin, and Bacillus globigii was demonstrated at levels as low as 8 ng/mL, 4 ng/mL, 10 ng/mL, and 6.2 x 10(4) cfu/mL, respectively. The assays presented here are very fast, as compared to previously published methods for measuring antibody-antigen interactions using microarrays (minutes versus hours).     

Gadonanotubes as ultrasensitive pH-smart probes for magnetic resonance imaging

With their nanoscalar, superparamagnetic Gd(3+)-ion clusters (1 x 5 nm) confined within ultrashort (20-80 nm) single-walled carbon nanotube capsules, gadonanotubes are high-performance T1-weighted contrast agents for magnetic resonance imaging (MRI). At 1.5 T, 37 degrees C, and pH 6.5, the r1 relaxivity (ca. 180 mM(-1) s(-1) per Gd(3+) ion) of gadonanotubes is 40 times greater than any current Gd(3+) ion-based clinical agent. Herein, we report that gadonanotubes are also ultrasensitive pH-smart probes with their r1/pH response from pH 7.0-7.4 being an order of magnitude greater than for any other MR contrast agent. This result suggests that gadonanotubes might be excellent candidates for the development of clinical agents for the early detection of cancer where the extracellular pH of tumors can drop to pH=7 or below. In the present study, gadonanotubes have also been shown to maintain their integrity when challenged ex vivo by phosphate-buffered saline solution, serum, heat, and pH cycling.     

Coaxial atomic force microscope probes for imaging with dielectrophoresis

We demonstrate atomic force microscope (AFM) imaging using dielectrophoresis (DEP) with coaxial probes. DEP provides force contrast allowing coaxial probes to image with enhanced spatial resolution. We model a coaxial probe as an electric dipole to provide analytic formulas for DEP between a dipole, dielectric spheres, and a dielectric substrate. AFM images taken of dielectric spheres with and without an applied electric field show the disappearance of artifacts when imaging with DEP. Quantitative agreement between our model and experiment shows that we are imaging with DEP.     

Bioconjugated quantum dots as fluorescent probes for biomedical imaging

Luminescent semiconductor quantum dots have become an important class of fluorescent labels for biological and biomedical imaging. In comparison with conventional organic dyes and fluorescent proteins, quantum dots have extraordinary fluorescent properties including high brightness, high resistance to photobleaching and tunable wavelengths. In this review, we briefly discuss the properties and modification of quantum dots. We focus on the applications of quantum dots in biomedical imaging, including molecular detection, live cell imaging and in vivo imaging. The toxicity of the quantum dots to cells and animals is also discussed.