MicroRNA profiling by simultaneous selective isotachophoresis and hybridization with molecular beacons

We present and demonstrate a novel assay for the detection and quantification of microRNA (miRNA) that leverages isotachophoresis (ITP) and molecular beacon (MB) hybridization. We use ITP to selectively preconcentrate miRNA from total RNA. We simultaneously focus MBs and use the ITP zone as a 10 pL reactor with active mixing where MBs fluoresce upon hybridization to target miRNA. To increase both sensitivity and selectivity, we leverage a multistage ITP strategy composed of three discrete regions of different concentrations of denaturant, sieving matrix, and magnesium chloride. We show that ITP hybridization is specific and selective to the miRNA target. We demonstrate ITP hybridization of miRNA in a biologically relevant case by detecting and quantifying miR-122 in human kidney and liver. ITP hybridization is a cheap, simple, high-speed, and amplification-free miRNA profiling method which requires small amounts (order 100 ng) of sample. The technique therefore represents an attractive alternative to PCR or Northern blot for miRNAs.     

Rapid identification of the bacterial pathogens responsible for urinary tract infections using direct injection CE

The use of high-performance capillary electrokinetic techniques for the separation, identification, and quantitation of intact microbes represents a new frontier for separation science. In this work, it is demonstrated that pathogens most responsible for urinary tract infections can be distinguished from one another after direct injection of untreated urine. High efficiencies (often exceeding 1000000 plates/m) and short analysis times (< 10 min) are characteristics of this approach. The concentration of the urine matrix appears to be able to cause a small, but definite, change in the electroosmotic flow velocity. This high-efficiency separation-based approach could prove to be invaluable for the diagnosis and tracking of certain diseases. It also could form the basis for a variety of rapid microbial assays.     

Enzyme-free and amplified fluorescence DNA detection using bimolecular beacons

In this work, we propose a simple and enzyme-free strategy for sensitive and selective DNA detection by using two different types of molecular beacons (MBs), MB1 and MB2. In this method, the target DNA binds with and restores the fluorescence of MB1 first. Then, MB2 hybridizes with MB1 and free the target, which is used to trigger another reaction cycle. The cycling use of the target and the employment of bi-MBs amplify the fluorescence intensity for sensitive DNA detection. The detection limit of this method was obtained as 10 pM, which is about 2 orders of magnitude sensitive than the conventional MB-based approaches.     

Rapid and sensitive detection of respiratory virus molecular signatures using a silver nanorod array SERS substrate

A spectroscopic assay based on surface enhanced Raman scattering (SERS) using silver nanorod array substrates has been developed that allows for rapid detection of trace levels of viruses with a high degree of sensitivity and specificity. This novel SERS assay can detect spectral differences between viruses, viral strains, and viruses with gene deletions in biological media. The method provides rapid diagnostics for detection and characterization of viruses generating reproducible spectra without viral manipulation.     

Unimolecular beacons for the detection of DNA-binding proteins

A new methodology for detecting sequence-specific DNA-binding proteins has been recently developed (Heyduk, T.; Heyduk, E. Nat. Biotechnol. 2002, 20, 171). The core feature of this methodology is protein-dependent association of two fluorochrome-labeled DNA fragments, which allows generation of a fluorescence signal reporting the presence of the target protein. Previous kinetic experiments identified the association of the two DNA fragments as the rate-limiting step of the assay. Here we report on a variant of the assay, in which components of the assay--fluorescent DNA fragments--were covalently tethered by a non-DNA linker with the goal of increasing the rate of association of the two fragments. We investigated the effect of the tether on the performance of the assay under a variety of conditions using a model DNA-binding protein. Quantitative titrations and rapid kinetic stopped-flow experiments were conducted to validate the molecular model that describes the two linked equilibria: oscillation of the tethered construct between the open and closed states and the exclusive association of the protein with the closed state. Experiments were also performed to demonstrate the ability of these tethered constructs to signal when attached to a solid surface. The major advantage of this new assay format is the faster response time for the detection allowing the higher throughput of the analysis. Additionally, it will be possible to attach tethered beacons to other solid surfaces, thus allowing the preparation of arrays containing molecular beacons for many different DNA-binding proteins.     

Using molecular beacons to probe molecular interactions between lactate dehydrogenase and single-stranded DNA

The interactions between two key macromolecular species, nucleic acids and proteins, control many important biological processes. There have been limited effective methodologies to study these interactions in real time. In this work, we have applied a newly developed molecular beacon (MB) DNA probe for the analysis of an enzyme, lactate dehydrogenase (LDH), and for the investigation of its properties of binding with single-stranded DNA. Molecular beacons are single-stranded oligonucleotide probes designed to report the presence of specific complementary nucleic acids by fluorescence detection. The interaction between LDH and MB has resulted in a significant fluorescence signal enhancement, which is used for the elucidation of MB/LDH binding properties. The processes of binding between MB and different isoenzymes of LDH have been studied. The results show that the stoichiometry of LDH-5/MB binding is 1:1, and the binding constant is 1.9 x 10(-7) M(-1). We have also studied salt effects, binding sites, temperature effects, pH effects, and the binding specificities for different isoenzymes. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNA interaction studies. MB has a signal transduction mechanism built within the molecule and can thus be used for the development of rapid protein assays and for real-time measurements.     

Immobilization of aptamer-based molecular beacons onto optically-encoded micro-sized beads

This paper presents a method for the novel immobilization of aptamer-based molecular beacons (apta-beacons) onto optically-encoded micro-sized beads (apta-beacon beads). To immobilize apta-beacons onto flourescently-encoded micro-sized beads, core-shell type beads containing a fluorescent dye-encoded core and apta beacon-coupled shell were prepared. The fluorescent dye-encoded beads were prepared from TentaGel resins by coupling RITC to the amino groups of the core region, after partial protection of amino groups with Fmoc-OSu in a diffusion-controlled manner. After deprotection of the Fmoc-amino groups, FITC-coupled molecular beacons (MBs) were immobilized to the beads together with a quencher by covelent bonding. Briefly, aspartic acid (Asp) was coupled to the shell part of the beads. Then, the quencher was coupled to the N-terminal amino group of Asp and the MBs were coupled to the side chain carboxyl group. In a model study, thrombin was directly detected using this apta-beacon bead method. The thrombin-bound apta-beacon beads were easily recognized by the appearance of fluorescence without any further labeling step.     

Separation and analysis of nanomole quantities of heparin oligosaccharides using on-line capillary isotachophoresis coupled with NMR detection

Glycosaminoglycans (GAGs) are important in a number of biological processes and are structurally altered in many pathological conditions. The complete determination of GAG primary structures has been hampered by the lack of sensitive and specific analytical techniques. Nuclear magnetic resonance spectroscopy (NMR) is a powerful tool for GAG structure elucidation despite its relatively poor limits of detection. Solenoidal microcoils have greatly enhanced the mass limits of detection of NMR, enabling the on-line coupling of microseparation and concentration techniques such as capillary isotachophoresis (cITP), which can separate and concentrate analytes by 2-3 orders of magnitude. We have successfully used cITP coupled with on-line NMR detection to separate and concentrate nanomole quantities of heparin oligosaccharides. This sensitive on-line measurement approach has the potential to provide new insights into the relationships between biological function and GAG microstructures.     

Catheters coated with Zn-doped CuO nanoparticles delay the onset of catheter-associated urinary tract infections

Catheter-associated urinary tract infections (CAUTIs) are among the most common bacterial infections associated with medical devices.In the current study,the synthesis,coating,antibiofilm properties,and biocompatibility of urinary catheters coated with Zn-doped CuO (Zn0.12Cu0.~O) nanoparticles (NPs) were examined.The doped NPs were synthesized and subsequently deposited on the catheter by the sonochemical method.The coated catheters displayed high antibiofilm activity and promising biocompatibility,as indicated by low in vitro cytotoxicity,negligible associated cytokine secretion,and absence of detectable irritation.The biocompatibility and ability of the Zn-doped CuO coating to inhibit biofilm formation were also evaluated in vivo using a rabbit model.Rabbits catheterized with uncoated catheters scored positive for CAUTI by day 4 of the experiment.In contrast,rabbits catheterized with Zn-doped CuO-coated catheters did not exhibit CAUTI until day 7 or remained completely uninfected for the whole duration of the 7-day experiment.Furthermore,.the in vivo biocompatibility assays and examinations supported the biosafety of Zn-doped CuO-coated catheters.Taken together,these data highlight the potential of Zn-doped CuO nanocomposite as effective antibiofilm compound.     

Ligase detection reaction generation of reverse molecular beacons for near real-time analysis of bacterial pathogens using single-pair fluorescence resonance energy transfer and a cyclic olefin copolymer microfluidic chip

Detection of pathogenic bacteria and viruses require strategies that can signal the presence of these targets in near real-time due to the potential threats created by rapid dissemination into water and/or food supplies. In this paper, we report an innovative strategy that can rapidly detect bacterial pathogens using reporter sequences found in their genome without requiring polymerase chain reaction (PCR). A pair of strain-specific primers was designed based on the 16S rRNA gene and were end-labeled with a donor (Cy5) or acceptor (Cy5.5) dye. In the presence of the target bacterium, the primers were joined using a ligase detection reaction (LDR) only when the primers were completely complementary to the target sequence to form a reverse molecular beacon (rMB), thus bringing Cy5 (donor) and Cy5.5 (acceptor) into close proximity to allow fluorescence resonance energy transfer (FRET) to occur. These rMBs were subsequently analyzed using single-molecule detection of the FRET pairs (single-pair FRET; spFRET). The LDR was performed using a continuous flow thermal cycling process configured in a cyclic olefin copolymer (COC) microfluidic device using either 2 or 20 thermal cycles. Single-molecule photon bursts from the resulting rMBs were detected on-chip and registered using a simple laser-induced fluorescence (LIF) instrument. The spFRET signatures from the target pathogens were reported in as little as 2.6 min using spFRET.     

Extraction of DNA from malaria-infected erythrocytes using isotachophoresis

We demonstrate a technique for purification of nucleic acids from malaria parasites infecting human erythrocytes using isotachophoresis (ITP). We release nucleic acids from malaria-infected erythrocytes by lysing with heat and proteinase K for 10 min and immediately, thereafter, load sample onto a capillary device. We study the effect of temperature on lysis efficiency. We also implement pressure-driven counterflow during ITP extraction to extend focusing time and increase nucleic acid yield. We show that the purified genomic DNA samples are compatible with polymerase chain reaction (PCR) and demonstrate a clinically relevant limit of detection of 0.5 parasites per nanoliter using quantitative PCR.     

Coupled isotachophoretic preconcentration and electrophoretic separation using bidirectional isotachophoresis

We present a novel technique for coupling isotachophoretic preconcentration and electrophoretic separation using bidirectional isotachophoresis (ITP). Bidirectional ITP simultaneously sets up sharp ITP interfaces between relatively high- and low-mobility cations and high- and low-mobility anions. These two interfaces can migrate toward each other and be described as ion concentration shock waves. We here demonstrate a bidirectional ITP process in which we use the interaction of these anionic and cationic ITP shock waves to trigger a transformation from ITP preconcentration to electrophoretic separation. We use anionic ITP to focus anionic sample species prior to shock interaction. The interaction of the counter-propagating anionic and cationic ITP shocks then changes the local pH (and ionic strength) of the focused analyte zones. Under this new condition, the analytes no longer focus and begin to separate electrophoretically. The method provides faster and much less dispersive transition from ITP preconcentration to electrophoretic separation compared with traditional (unidirectional) transient ITP. It eliminates the need for intermediate steps between focusing and separation, such as manual buffer exchanges. We illustrate the technique with numerical simulations of species transport equations. We have validated our simulations with experimental visualization of bidirectional ITP zones. We then show the effectiveness of the technique by coupling ITP preconcentration and high-resolution separation of a 1 kbp DNA ladder via shock interaction in bidirectional ITP.     

Spectroscopic features of dual fluorescence/luminescence resonance energy-transfer molecular beacons

Molecular beacons have the potential to become a powerful tool in gene detection and quantification in living cells. Here we report a novel dual molecular beacons approach to reduce false-positive signals in detecting target nucleic acids in homogeneous assays. A pair of molecular beacons, each containing a fluorescence quencher and a reporter fluorophore, one with a donor and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that seen in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of possible false-positive events. Further, when a lanthanide chelate is used as a donor in a dual-probe assay, extremely high signal-to-background ratios can be achieved owing to the long lifetime and sharp emission peaks of the donor and the time-gated detection of acceptor fluorescence emission. These new approaches allow for the ultrasensitive detection of target molecules in a way that could be readily applied to real-time imaging of gene expression in living cells.     

Rapid detection of food pathogens using RNA aptamers-immobilized slide

The purpose of this study was to develop a simple and rapid detection system for foodborne bacteria, which consisted of an optical microscope and its slide chip with artificial antibodies, or RNA aptamers. From an RNA pool, three each RNA aptamers were built by the method of SELEX (systematic evolution of ligands by exponential enrichment) for components of cell wall, LPS (lipopolysaccharide) from E. coli O157:H7, teichoic acid from Staphylococcus aureus and a cell membrane protein of OmpC from Salmonella typhimurium, respectively. These aptamers were hybridized with thiol-conjugated 16 dT-linker molecules in order to be immobilized on silver surface which was, in advance, fabricated on glass slide, using a spin-coating method. To confirm that each aptamers retained its specific binding activities to their antigenic live bacteria, microscopic view of bound cells immobilized on silver film were observed. Furthermore, we observed the fluorescence-emitting bacteria-aptamer complex immobilized on silver film after adding RNA aptamers hybridized with fluorophore, FAM-conjugated 16 dT-linker molecules. As a result, the RNA aptamers-immobilized slide system developed in this study was a useful new tool to rapidly monitor individual food pathogens.     

Integration of on-chip isotachophoresis and functionalized hydrogels for enhanced-sensitivity nucleic Acid detection

We introduce an on-chip electrokinetic assay to perform high-sensitivity nucleic acid (NA) detection. This assay integrates electrokinetic sample focusing using isotachophoresis (ITP) with a background signal-removal strategy that employs photopatterened, DNA-functionalized hydrogels. In this multistage assay, ITP first enhances hybridization kinetics between target NAs and end-labeled complementary reporters. After enhanced hybridization, migration through a DNA-functionalized hydrogel region removes excess reporters through affinity interactions. We demonstrate our assay on microRNAs, an important class of low-abundance biomarkers. The assay exhibits 4 orders of magnitude dynamic range, near 1 pM detection limits starting from less than 100 fg of microRNA, and high selectivity for mature microRNA sequences, all within a 10 min run time. This new microfluidic framework provides a unique quantitative assay for NA detection.     

基于LBL声信标的AUV快速精确定位

长基线声学定位系统是水下机器人广泛应用的外部导航设备。以对“CR-02”6000m自治水下机器人（简称AUV）技术的深入开发为背景,提出在原有长基线（LBL）定位系统的基础上增加导航功能的方案。由于海水介质非均匀性与复杂的时空变化特性,给基于测距的位置计算带来很大困难。采用平均声速法计算耗时小而误差大,波阵面定位法误差小而耗时大。为解决这个矛盾,根据AUV深度传感器给出的深度信息,采用本征声线快速计算方法解算AUV的水平面位置。与波阵面法、平均声速法进行了综合比较。仿真实验表明该方法具有优良的性能,满足AUV导航需要。     

Rapid electronic detection of probe-specific microRNAs using thin nanopore sensors

Small RNA molecules have an important role in gene regulation and RNA silencing therapy, but it is challenging to detect these molecules without the use of time-consuming radioactive labelling assays or error-prone amplification methods. Here, we present a platform for the rapid electronic detection of probe-hybridized microRNAs from cellular RNA. In this platform, a target microRNA is first hybridized to a probe. This probe:microRNA duplex is then enriched through binding to the viral protein p19. Finally, the abundance of the duplex is quantified using a nanopore. Reducing the thickness of the membrane containing the nanopore to 6 nm leads to increased signal amplitudes from biomolecules, and reducing the diameter of the nanopore to 3 nm allows the detection and discrimination of small nucleic acids based on differences in their physical dimensions. We demonstrate the potential of this approach by detecting picogram levels of a liver-specific miRNA from rat liver RNA.     

Rapid detection of Escherichia coli by using antibody-conjugated silver nanoshells

Silver shells of 20 nm thickness have been deposited on silica particles of 200 nm diameter with narrow size distribution. Silver nanoshells dispersed in water exhibit a strong surface plasmon resonance band at 443 nm. This band was found to be very sensitive to rabbit immunoglobulin G antibodies, which were anchored on the nanoshells. These in turn could be utilized to detect the presence of small (approximately 5) to large numbers (approximately 10(9)) of Escherichia coli in water. The protocol presented here proves to be a specific, rapid, reliable, and inexpensive method to detect E. coli.     

微流控LAMP芯片构建及在MRSA快速检测中的应用研究

构建一种基于环介导等温扩增(loop-mediated isothermal amplification,LAMP),集细菌在线裂解、核酸提取、目标基因扩增和产物检测一体化的用于病原菌快速检测的集成式微流控芯片。以耐甲氧西林金黄色葡萄球菌(methicillin-resistant staphylococcus,MRSA)为模式菌,以mec A为靶基因,在优化条件下用芯片实现对病原菌的在线检测,完成对101~106cfu MRSA的在线裂解、LAMP扩增和产物测定,采用荧光原位检测可得101~105cfu的检测范围和101cfu的检出限。该微流控LAMP芯片结构简单,操作便捷,可在1 h内实现对MRSA mec A基因的快速检测,具有较高的灵敏度和特异性,为下一步临床生物样本病原菌快速检测微流控芯片系统的构建奠定前期研究基础。