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Probing molecular pathways for DNA orientational trapping, unzipping and translocation in nanopores by using a tunable overhang sensor

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NANOSCALE 2014年第19期6卷 11372-11379页

作者： Wang, Yong Tian, Kai Hunter, Lehr L. Ritzo, Brandon Gu, Li-Qun Univ Missouri Dept Bioengn Columbia MO 65211 USA Univ Missouri Dalton Cardiovasc Res Ctr Columbia MO 65211 USA

Nanopores provide a unique single-molecule platform for genetic and epigenetic detection. The target nucleic acids can be accurately analyzed by characterizing their specific electric fingerprints or signatures in the nanopore. Here we report a series of novel nanopore signatures generated by target nucleic acids that are hybridized with a probe. A length-tunable overhang appended to the probe functions as a sensor to specifically modulate the nanopore current profile. The resulting signatures can reveal multiple mechanisms for the orientational trapping, unzipping, escaping and translocation of nucleic acids in the nanopore. This universal approach can be used to program various molecular movement pathways, elucidate their kinetics, and enhance the sensitivity and specificity of the nanopore sensor for nucleic acid detection.

关键词： Computer Simulation Computer-Aided Design DNA Probes/chemistry DNA Probes/chemistry DNA Probes/genetics DNA Probes/genetics DNA Probes/ultrastructure DNA Probes/ultrastructure Equipment Design Equipment Failure analysis Materials Testing Models, Chemical Models, Molecular Motion Nucleic Acid Conformation oligonucleotide array sequence analysis/instrumentation

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Electrochemical Patterning and Detection of DNA arrays on a Two-Electrode Platform

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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 2013年第51期135卷 19099-19102页

作者： Furst, Ariel Landefeld, Sally Hill, Michael G. Barton, Jacqueline K. CALTECH Div Chem & Chem Engn Pasadena CA 91125 USA Occidental Coll Dept Chem & Chem Biol Los Angeles CA 90041 USA

We report a novel method of DNA array formation that is electrochemically formed and addressed with a two-electrode platform. Electrochemical activation of a copper catalyst, patterned with one electrode, enables precise placement of multiple sequences of DNA onto a second electrode surface. The two-electrode patterning and detection platform allows for both spatial resolution of the patterned DNA array and optimization of detection through DNA-mediated charge transport with electrocatalysis. This two-electrode platform has been used to form arrays that enable differentiation between well-matched and mismatched sequences, the detection of TATA-binding protein, and sequence-selective DNA hybridization.

关键词： Catalysis Copper/chemistry DNA/chemistry Electrochemistry/instrumentation Electrodes Hybridization, Genetic oligonucleotide array sequence analysis/instrumentation

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A Multistage Volumetric Bar Chart Chip for Visualized Quantification of DNA

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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 2013年第45期135卷 16785-16788页

作者： Song, Yujun Wang, Yuanchen Qin, Lidong Houston Methodist Res Inst Dept Nanomed Houston TX 77030 USA Cornell Univ Dept Cell & Dev Biol Weill Med Coll New York NY 10065 USA

Nucleic acid detection is critical in disease diagnosis as well as in the environmental assays of harmful bacteria or viruses and forensic applications. Current methods for visualized quantification of DNA require costly and sophisticated instruments. Here, we report a multistage propelled volumetric bar chart chip (MV-Chip) for multiplexing and quantitative detection of DNA. Because of its "rocket-like" propelling reaction, the predeposited platinum films could perform cascade amplification and detect as low as 20 pM DNA targets after three stages of platinum-catalyzed propulsion. The resulting ink bar charts can be directly read out by the naked eye, and the signal shows little interference from serum. Single-nucleotide polymorphism and multiplex DNA detection were carried out to demonstrate this powerful application.

关键词： Biosensing Techniques/instrumentation Catalysis DNA/analysis DNA/genetics Ebolavirus/genetics Equipment Design oligonucleotide array sequence analysis/instrumentation Platinum/chemistry Polymorphism, Single Nucleotide

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On-Chip Synthesis of Protein Microarrays from DNA Microarrays via Coupled In Vitro Transcription and Translation for Surface Plasmon Resonance Imaging Biosensor Applications

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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 2012年第30期134卷 12358-12361页

作者： Seefeld, Ting H. Halpern, Aaron R. Corn, Robert M. Univ Calif Irvine Dept Chem Irvine CA 92697 USA

Protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). A microarray of dsDNA elements (denoted as generator elements) that encode either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein is utilized to create multiple copies of mRNA (mRNA) in a surface RNA polymerase reaction;the mRNA transcripts are then translated into proteins by cell-free protein synthesis in a microfluidic format. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements (denoted as detector elements) and are specifically adsorbed. The net result is the on-chip, cell-free synthesis of a protein microarray that can be used immediately for SPRI protein biosensing. The dual element format greatly reduces any interference from the nonspecific adsorption of enzyme or proteins. SPRI measurements for the detection of the antibodies anti-GFP and antiluciferase were used to verify the formation of the protein microarray. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRI biosensing measurements in both clinical and research applications.

关键词： Adsorption Bacteriophage T7/enzymology Copper/chemistry DNA/genetics DNA-Directed RNA Polymerases/metabolism Escherichia coli/genetics Green Fluorescent Proteins/analysis Green Fluorescent Proteins/genetics Histidine/analysis Luciferases/analysis Luciferases/genetics oligonucleotide array sequence analysis/instrumentation Oligopeptides/analysis Protein array analysis/instrumentation Protein Biosynthesis RNA, Messenger/genetics Surface Plasmon Resonance/instrumentation Transcription, Genetic

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Rapid Microarray Detection of DNA and Proteins in Microliter Volumes with Surface Plasmon Resonance Imaging Measurements

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LANGMUIR 2011年第10期27卷 6534-6540页

作者： Seefeld, Ting Hu Zhou, Wen-Juan Corn, Robert M. Univ Calif Irvine Dept Chem Irvine CA 92697 USA

A four-chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm x 18 mm biochip consists of four 3 mu L microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The 12-element (4 x 3) SPRI microarray consists of gold thin film spots (1 mm(2) area;45 nm thickness), each in individually addressable 0.5 mu L volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA, respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels;the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (ssDNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements are also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss is proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18 000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 s in the microliter volume format.

关键词： Base sequence Biological Markers/analysis Dimethylpolysiloxanes/chemistry DNA/analysis DNA/genetics Equipment Design Glass/chemistry Humans Molecular Imaging/instrumentation oligonucleotide array sequence analysis/instrumentation Protein array analysis/instrumentation Proteins/analysis Ribonuclease H/metabolism RNA/analysis RNA/genetics Surface Plasmon Resonance/instrumentation Surface Properties

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DNA Concentration Modulation on Supported Lipid Bilayers Switched by Surface Acoustic Waves

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LANGMUIR 2011年第24期27卷 14721-14725页

作者： Hennig, Martin Wolff, Manuel Neumann, Juergen Wixforth, Achim Schneider, Matthias F. Raedler, Joachim O. Univ Munich Ctr NanoSci Fak Phys D-80539 Munich Germany Biozentrum LMU Munchen Ctr NanoSci D-82152 Planegg Martinsried Germany Univ Augsburg Ctr NanoSci Inst Phys D-86159 Augsburg Germany Boston Univ Boston MA 02215 USA

Spatially addressable arrays of molecules embedded in or anchored to supported lipid bilayers are important for on-chip screening and binding assays;however, methods to sort or accumulate components in a fluid membrane on demand are still limited. Here we apply in-plane surface acoustic shear waves (SAWs) to laterally accumulate double-stranded DNA segments electrostatically bound to a cationic supported lipid bilayer. The fluorescently labeled DNA segments are found to segregate into stripe patterns with a spatial frequency corresponding to the periodicity of the standing SAW wave (similar to 10 mu m). The DNA molecules are accumulated 10-fold in the regions of SAW antinodes. The superposition of two orthogonal sets of SAW sources creates checkerboard like arrays of DNA demonstrating the potential to generate arrayed fields dynamically. The pattern relaxation time of 0.58 s, which is independent of the segment length, indicates a sorting and relaxation mechanism dominated by lipid diffusion rather than DNA self-diffusion.

关键词： Diffusion DNA/chemistry DNA/metabolism Fluorescence Fluorescence Lab-On-A-Chip Devices Lipid Bilayers/chemistry Lipid Bilayers/metabolism oligonucleotide array sequence analysis/instrumentation oligonucleotide array sequence analysis/methods Sound Sound Static Electricity Static Electricity Xanthenes/analysis

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

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ANALYST 2011年第19期136卷 3950-3955页

作者： Sui, Benhui Li, Li Li, Lu Jin, Wenrui Shandong Univ Sch Chem & Chem Engn Jinan 250100 Peoples R China Shandong Normal Univ Key Lab Mol & Nano Probes Coll Chem Chem Engn & Mat Sci Jinan 250014 Peoples R China

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 x 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.

关键词： Animals beta 2-Microglobulin/genetics Breast Neoplasms/genetics Cattle DNA/analysis Ethanolamine Female Humans In Situ Hybridization, Fluorescence/instrumentation In Situ Hybridization, Fluorescence/instrumentation In Situ Hybridization, Fluorescence/methods In Situ Hybridization, Fluorescence/methods oligonucleotide array sequence analysis/instrumentation oligonucleotide array sequence analysis/methods Quantum Dots Quantum Dots RNA, Messenger/analysis RNA, Messenger/genetics Sensitivity and Specificity Serum Albumin, Bovine

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Electric detection of DNA with PDMS microgap electrodes and silver nanoparticles

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ANALYST 2011年第3期136卷 540-544页

作者： Sun, Ziyin Qiang, Weibing Li, Hui Hao, Nan Xu, Danke Chen, Hong-Yuan Nanjing Univ Key Lab Analyt Chem Life Sci Minist Educ Sch Chem & Chem Engn Nanjing 210093 Jiangsu Peoples R China

In this work a novel microdevice sensor has been developed by plating gold on the PDMS surface to generate a sandwich-type gap electrode for DNA detection. The microdevice utilizes a gold band electrode-PDMS-gold band electrode configuration and the minimum detectable volume could be as low as 5 mu L. The 20 mu m PDMS-based gap was chemically modified with DNA capture probes and DNA sandwich hybrids were formed with the addition of DNA target and silver nanoparticle probes. To increase detection sensitivity, parallel detection zones have been developed in which the relevant resistances decrease substantially upon hybridyzation. By measuring the change in electrical conductivity, the DNA target in the concentration range of 1000-0.1 nM can be assayed and the limit of lowest detectable concentration was achieved at 0.01 nM.

关键词： Dimethylpolysiloxanes/chemistry DNA/analysis Electrochemical Techniques/instrumentation Electrochemical Techniques/methods Limit of Detection Microelectrodes Microscopy, Electron, Scanning Nanoparticles/chemistry oligonucleotide array sequence analysis/instrumentation oligonucleotide array sequence analysis/methods oligonucleotides/chemistry Silver/chemistry Surface Properties

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A nanobiosensor to detect single hybridization events

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ANALYST 2009年第12期134卷 2458-2461页

作者： Maruccio, Giuseppe Primiceri, Elisabetta Marzo, Pasquale Arima, Valentina Della Torre, Antonio Rinaldi, Ross Pellegrino, Teresa Krahne, Roman Cingolani, Roberto Univ Salento Natl Nanotechnol Lab CNR Scuola Super ISUFIIIT Res UnitINFM I-73100 Lecce Italy

An economical nanoarray method to electrically detect hybridization events is demonstrated. As a proof of concept, we fabricated a sensor for DNA sequencing, in which targets are oligonucleotides conjugated to gold nanoparticles. As a consequence of target-probe binding events, a conductive bridge forms between two electrodes, resulting in a quantized change in conductivity. This enables a robust detection of a few ( down to single) hybridization events and can be potentially applied also to other binding events ( like specific interactions between proteins, antibodies, ligands and receptors). Moreover, target amplification techniques ( such as PCR) are no longer necessary.

关键词： Base sequence Biosensing Techniques/instrumentation DNA/chemistry Gold Metal Nanoparticles Nanotechnology/instrumentation Nucleic Acid Hybridization oligonucleotide array sequence analysis/instrumentation oligonucleotide array sequence analysis/methods

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A simple gold nanoparticle-mediated immobilization method to fabricate highly homogeneous DNA microarrays having higher capacities than those prepared by using conventional techniques

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NANOTECHNOLOGY 2009年第3期20卷 035607 (7p-035607 (7p页

作者： Jung, Cheulhee Mun, Hyo Young Li, Taihua Park, Hyun Gyu Korea Adv Inst Sci & Technol Dept Chem & Biomol Engn Taejon 305701 South Korea

A simple, highly efficient immobilization method to fabricate DNA microarrays, that utilizes gold nanoparticles as the mediator, has been developed. The fabrication method begins with electrostatic attachment of amine-modified DNA to gold nanoparticles. The resulting gold-DNA complexes are immobilized on conventional amine or aldehyde functionalized glass slides. By employing gold nanoparticles as the immobilization mediator, implementation of this procedure yields highly homogeneous microarrays that have higher binding capacities than those produced by conventional methods. This outcome is due to the increased three-dimensional immobilization surface provided by the gold nanoparticles as well as the intrinsic effects of gold on emission properties. This novel immobilization strategy gives microarrays that produce more intense hybridization signals for the complementary DNA. Furthermore, the silver enhancement technique, made possible only in the case of immobilized gold nanoparticles on the microarrays, enables simple monitoring of the integrity of the immobilized DNA probe.

关键词： Aldehydes/chemistry Amines/chemistry DNA, Complementary Fluorescence Fluorescence Gold/chemistry Metal Nanoparticles/chemistry oligonucleotide array sequence analysis/instrumentation oligonucleotide array sequence analysis/methods Poly A Reproducibility of Results

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