Polymerase chain reaction coupling with magnetic nanoparticles-based biotin-avidin system for amplification of chemiluminescent detection signals of nucleic acid

A novel method was established through the detection of chemiluminescent signals of nucleic acid hybridization based on magnetic nanoparticles (MNPs) and PCR. 5' amino- modified specific probes were immobilized on the surface of silanized MNPs by Schiff reaction between amino and aldehyde group. The probes were used to capture the synthetic biotin-dUTP-labeled DNA fragments which were obtained by polymerase chain reaction (PCR). Then these complexes were bonded with streptavidin-modified alkaline phosphatase (SA-AP). Finally the chemiluminescent signals were detected by adding 3-(2'-spiroadamantane)- 4-methoxy -4-(3"-phosphoryloxy) phenyl-1, 2-dioxetane (AMPPD) which was the substrate reagent of AP. The concentration of probes which were immobilized on the surface of MNPs was studied, how to reduce the adsorption of SA-AP on the surface of MNPs was also researched. It was shown that 12.5 pmol of probes were immobilized on 1 mg of MNPs. Aldehyde-MNPs modified with probes could adsorb SA-AP, affecting the sensitivity of chemiluminescene consequently. Reduction of aldehyde group by sodium borohydride and blocking the bare position of MNPs with bovine serum albumin (BSA) could decrease the background of chemiluminescence, and this method has good specificity in detection of chloramphenicol acetyltransferase (CAT) gene.     

Surface plasmon resonance detection of biological warfare agent Staphylococcal enterotoxin B using high affinity monoclonal antibody

A novel sensitive method was developed for the detection as well as quantification of Staphylococcal enterotoxin B (SEB) using surface plasmon resonance (SPR). It is well known that the amount of SEB needed to cause the intoxication to human beings is very less and this concentration (0.02 μg/kg) is highly dangerous, hence, it is used as biological warfare agent. Thus, the need to develop a reliable and potential detection system against SEB is warranted. In the present work, SEB antibody was immobilized on carboxymethyldextran modified gold chip. The immobilization of SEB antibody and interaction of antigen with immobilized antibody were in-situ characterized by SPR and electrochemical impedance spectroscopy. A sample solution containing SEB antigen was injected in a working channel and the results revealed linearity in the concentration from 2.0 to 32.0 pM with a detection limit of 1.0 pM. By using kinetic evaluation software, K_D (equilibrium constant) and Bmax (maximum binding capacity of analyte) values were calculated and found to be 13 pM and 424.23, respectively. Moreover, the thermodynamic parameter, change in Gibb's free energy was deduced and found to be −62.08 kJ/mol and this value shows the spontaneous interaction between SEB antigen and SEB antibody. In order to optimize the detection method, temperature and pH variation studies were also performed. Interference study was conducted to know the selectivity for the antigen-antibody interaction of SEB. The selectivity efficiency of SEB, SEC, SEA and SED were 100, 27.15, 20.01 and 12.05%, respectively towards SEB antibody.     

Autonomous detection of aerosolized biological agents by multiplexed immunoassay with polymerase chain reaction confirmation

The autonomous pathogen detection system (APDS) is an automated, podium-sized instrument that continuously monitors the air for biological threat agents (bacteria, viruses, and toxins). The system has been developed to warn of a biological attack in critical or high-traffic facilities and at special events. The APDS performs continuous aerosol collection, sample preparation, and detection using multiplexed immunoassay followed by confirmatory PCR using real-time TaqMan assays. We have integrated completely reusable flow-through devices that perform DNA extraction and PCR amplification. The fully integrated system was challenged with aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii, and botulinum toxoid. By coupling highly selective antibody- and DNA-based assays, the probability of an APDS reporting a false positive is extremely low.     

Foveal contour interaction: detection and discrimination

Contour interaction, the detrimental effect of flanking features on the discrimination of optotypes, has been studied mainly close to the visual acuity limit. We were interested to know how these results compare with those for the detection of targets. According to the simplest model of contour interaction, comparable detection effects would be expected. The case for low-level masking would be further strengthened if the form and nature of the dependence on flank separation and flank polarity followed that typically found in studies of lateral spatial masking [Vision Res. 33, 993 (1993)]. Landolt Cs subtending a visual angle of 0.25 degrees, 0.5 degrees, and 1.0 degrees were presented and contrast thresholds for detecting the presence of the Landolt C and discriminating its orientation were measured in five normal subjects as a function of flank separation and flank polarity. The results obtained for the relationship between detection and discrimination depend on the size of the target used. For small letters, discrimination but not detection was significantly affected by flanking bars. For large letters, detection and discrimination were affected to the same extent. However, in this case the effectiveness of opposite-polarity flanks and the finding that facilitation occurred at close, not far, flank separations suggests that the simplest explanation in terms of masking may not be applicable.     

Quantitative detection of protein arrays

We introduce a quantitative method that utilizes scanning electron microscopy for the analysis of protein chips (SEMPC). SEMPC is based upon counting target-coated gold particles interacting specifically with ligands or proteins arrayed on a derivative microscope glass slide by utilizing backscattering electron detection. As model systems, we quantified the interactions of biotin and streptavidin and of an antibody with its cognate hapten. Our method gives quantitative molecule-counting capabilities with an excellent signal-to-noise ratio and demonstrates a broad dynamic range while retaining easy sample preparation and realistic automation capability. Increased sensitivity and dynamic range are achieved in comparison to currently used array detection methods such as fluorescence, with no signal bleaching, affording high reproducibility and compatibility with miniaturization. Thus, our approach facilitates the determination of the absolute number of molecules bound to the chip rather than their relative amounts, as well as the use of smaller samples.     

Selective photoelectrochemical detection of DNA with high-affinity metallointercalator and tin oxide nanoparticle electrode

Selective detection of double-stranded DNA (ds-DNA) in solution was achieved by photoelectrochemistry using a high-affinity DNA intercalator, Ru(bpy)2dppz (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) as the signal indicator and tin oxide nanoparticle as electrode material. When Ru(bpy)2dppz alone was irradiated with 470-nm light, anodic photocurrent was detected on the semiconductor electrode due to electron injection from its excited state into the conduction band of the electrode. The current was sustained in the presence of oxalate in solution, which acted as a sacrificial electron donor to regenerate the ground-state metal complex. After addition of double-stranded calf thymus DNA into the solution, photocurrent dropped substantially. The drop was attributed to the intercalation of Ru(bpy)2dppz into DNA and, consequently, the reduced mass diffusion of the indicator to the electrode, as well as electrostatic repulsion between oxalate anion and negative charges on DNA. The degree of signal reduction was a function of the DNA concentration, thus forming the basis for real-time DNA detection. The signal reduction was selective for ds-DNA, as no such effect was observed for single-stranded polynucleotides such as poly-G, poly-C, poly-A, and poly-U. The detection limit of calf thymus ds-DNA reached 1.8 x 10(-10) M in solution.     

Quantitative analysis of molecular interaction in a microfluidic channel: the T-sensor

The T-sensor is a recently developed microfluidic chemical measurement device that exploits the low Reynolds number flow conditions in microfabricated channels. The interdiffusion and resulting chemical interaction of components from two or more input fluid streams can be monitored optically, allowing measurement of analyte concentrations on a continuous basis. In a simple form of T-sensor, the concentration of a target analyte is determined by measuring fluorescence intensity in a region where the analyte and a fluorescent indicator have interdiffused. An analytical model has been developed that predicts device behavior from the diffusion coefficients of the analyte, indicator, and analyte--indicator complex and from the kinetics of the complex formation. Diffusion coefficients depend on the local viscosity which, in turn, depends on local concentrations of all analytes. These relationships, as well as reaction equilibria, are often unknown. A rapid method for determining these unknown parameters by interpreting T-sensor experiments through the model is presented.     

Environmental monitoring for biological threat agents using the autonomous pathogen detection system with multiplexed polymerase chain reaction

We have developed and field-tested a now operational civilian biodefense capability that continuously monitors the air in high-risk locations for biological threat agents. This stand-alone instrument, called the Autonomous Pathogen Detection System (APDS), collects and selectively concentrates particles from the air into liquid samples and analyzes the samples using multiplexed PCR amplification coupled with microsphere array detection. During laboratory testing, we evaluated the APDS instrument's response to Bacillus anthracis and Yersinia pestis by spiking the liquid sample stream with viable spores and cells, bead-beaten lysates, and purified DNA extracts. APDS results were also compared to a manual real-time PCR method. Field data acquired during 74 days of continuous operation at a mass-transit subway station are presented to demonstrate the specificity and reliability of the APDS. The U.S. Department of Homeland Security recently selected the APDS reported herein as the first autonomous detector component of their BioWatch antiterrorism program. This sophisticated field-deployed surveillance capability now generates actionable data in one-tenth the time of manual filter collection and analysis.     

Impedance spectroscopy and affinity measurement of specific antibody–antigen interaction

We use impedance spectroscopy technique to develop a rapid and sensitive antigen biosensor. The biosensor is based on the immobilization of antibodies onto an 11-Mercaptoundecanoic acid self-assembled monolayer. The high insulating properties of the thiol can be measured with cyclic voltammetry and impedance spectroscopy with a redox couple. The, the modified gold electrode is activated with 1-ethyl-3 (3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) ester for antibody immobilisation. The affinity interaction of the antigen–antibody can be measured with quartz crystal microbalance technique. Dissociation and association constant are determined by applying a Fortran program fitting for the experimental data.     

Quantitative analysis of microRNA in blood serum with protein-facilitated affinity capillary electrophoresis

MicroRNAs (miRNAs) are small (～22 nt) regulatory RNAs that are frequently deregulated in cancer and have shown promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Here we present a protein-facilitated affinity capillary electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum using single-stranded DNA binding protein (SSB) and double-stranded RNA binding protein (p19) as separation enhancers. The method utilizes either the selective binding of SSB to a single-stranded DNA/RNA probe or the binding of p19 to miRNA-RNA probe duplex. For the detection of ultralow amounts of miRNA without polymerase chain reaction (PCR) amplification in blood samples we apply off-line preconcentration of synthetic miRNA-122 from serum by p19-coated magnetic beads followed by online sample stacking in the ProFACE assay. The detection limit is 0.5 fM or 30?000 miRNA molecules in 1 mL of serum as a potential source of nai?ve miRNAs.     

面制品中痕量铝的快速高灵敏光电化学检测方法的建立

本实验采用新型的光电化学检测手段实现了对于面制品中痕量铝的快速高灵敏检测。实验中通过优化相应检测条件,探索出一种针对面制品中痕量铝检测的高灵敏、高稳定性的新的定量检测方法,并将该方法运用于实际样品检测,结果令人满意。     

Near-simultaneous and real-time detection of multiple analytes in affinity microcolumns

A miniaturized immunoassay system based on beads in poly(dimethylsiloxane) microchannels for analyzing multiple analytes has been developed. The method involves real-time detection of soluble molecules binding to receptor-bearing microspheres, sequestered in affinity column format inside a microfluidic channel. Identification and quantitation of analytes occurs via direct fluorescence measurements or fluorescence resonance energy transfer. A preliminary account of this work based on single-analyte format has been published in this journal (Buranda, T.; Huang, J.; Perez-Luna, V. H.; Schreyer, B.; Sklar, L. A.; Lopez, G. P. Anal. Chem. 2002, 74, 1149-1156). We have extended the work to a multianalyte model system composed of discrete segments of beads that bear distinct receptors. Near-simultaneous and real-time detection of diverse analytes is demonstrated. The importance of this work is established in the exploration of important factors related to the design, assessment, and utility of affinity microcolumn sensors. First, beads derivatized with surface chemistry suitable for the attachment of fluorescently labeled biomolecules of interest are prepared and characterized in terms of functionality and receptor site densities by flow cytometry. Second, calibrated beads are incorporated in microfluidic channels. The analytical device that emerges replicates the basic elements of affinity chromatography with the advantages of microscale and real-time direct measurement of bound analyte on beads rather than the indirect determination from eluted sample typical of affinity chromatography. In addition, the two-compartment analysis of the assay data as demonstrated in single-analyte columns provides a template upon which the dynamics of multiple-analyte assays can be characterized using existing theoretical models and be tested experimentally. The assay can potentially detect subfemtomole quantities of protein with high signal-to-noise ratio and a large dynamic range spanning nearly 4 orders of magnitude in analyte concentration in microliter to submicroliter volumes of analyte fluid. The approach has the potential to be generalized to a host of bioaffinity assay methods including analysis of protein complexes (e.g., biomolecular indicators of diseases). Proof-of-principle analytes include FLAG peptide and carcinoembryonic antigen detected at physiologically relevant concentration levels.     

Mechanism of interaction of magnetic fields with biological systems

A review is presented of the known mechanisms by which magnetic fields can interact with biological systems. The basic equations for exerting forces by both AC and DC magnetic fields on charged particles and dipoles are reviewed. Examples are given that allow order-of-magnitude estimates of the induced current densities and forces to be obtained. These estimates are compared with values known to induce measurable changes in the behavior of biological systems     

Depth of interaction detection for -ray imaging

A novel design for an inexpensive depth of interaction capable detector for γ-ray imaging has been developed. The design takes advantage of the strong correlation between the width of the scintillation light distribution in monolithic crystals and the interaction depth of γ-rays. We present in this work an inexpensive modification of the commonly used charge dividing circuits which enables the instantaneous and simultaneous computation of the second order moment of light distribution. This measure provides a good estimate for the depth of interaction and does not affect the determination of the position centroids and the energy release of γ-ray impact. The method has been tested with a detector consisting of a monolithic LSO block sized and a position-sensitive photomultiplier tube H8500 from Hamamatsu. The mean spatial resolution of the detector was found to be for the position centroids and for the DOI. The best spatial resolutions were observed at the center of the detector and yielded for the position centroids and for the DOI.     

RAPTOR: a fluoroimmunoassay-based fiber optic sensor for detection of biological threats

The RAPTOR was developed to meet the need for a small, portable, easily operated biosensor for the detection of biological threats in the field. This device has evolved over a number of years to reach its current level of maturity. This paper describes details of the RAPTOR's design, including the recent upgrades to the fluidics and optics subsystems, as well as design improvements that have increased both system reliability and sensitivity. Working with these system upgrades, we also investigated biochemical methods that further improve assay sensitivity. The use of NeutrAvidin/biotin chemistry to improve immobilization of the capture antibody, coupled with use of the fluorophore Alexa Fluor 647 to label the tracer antibody, has resulted in a two- to four-fold signal enhancement. These gains, in combination with redesigned optics and instrumentation, have resulted in approximately a log-order improvement over earlier systems. These improvements are shown for an assortment of analytes of interest, including Staphylococcal enterotoxin type-B, Francisella tularensis, Bacillus anthracis, and Bacillus globigii spores.     

Improved photoelectrochemical detection of mercury (II) with a TiO2-modified composite photoelectrode

The spectrophotometric change of a mercury (II) (Hg²⁺) selective small molecule chemosensor has been successfully converted into a photovoltaic response upon ligating Hg²⁺. The photon excitation was followed by charge separation facilitated by TiO₂ and polyaniline (PANI), resulting in an electron transfer to an electrical back contact. The photoresponse of the Hg²⁺ selective chromophore was converted to an electron current equivalent to the amount of Hg²⁺ in solution. The favourable properties of a Hg²⁺ sensitive chemosensor was combined with the semiconductor capabilities of TiO₂ to construct a sensor that is capable of generating a current in the presence of Hg²⁺ under illumination. A composite of the fluorescent chemosensor rhodamine 6G hydrozone derivative (RS) and PANI was immobilized on indium tin oxide (ITO) plates coated with TiO₂ and subjected to photovoltammetric measurements. The photovoltammetric responses of the coated layers were investigated to determine the sensitivity and selectivity of the immobilized sensor to Hg²⁺ in the presence of background ions. The photo-response increased linearly with increasing Hg²⁺ concentration from 10 to 200 μg L⁻¹ with a limit of quantification (LOQ) of 4 μg L⁻¹. The pH independence for the photoresponse was limited by the TiO₂ layer and was optimal between pH 6 and 7.     

Defect engineering of nanostructures: Insights into photoelectrochemical water splitting

Development of long-term and sustainable energy economy is one of the most significant technical challenges facing humanity. Photoelectrochemical (PEC) water splitting is regarded as the most attractive approach for conversion of solar energy to chemical energy, with H₂ and O₂ as the energy carriers. Defect engineering of photocatalytic materials has been proved effective in improving their performances in PEC water splitting process involving three basic steps, i.e., light absorption, charge transfer/separation, and surface catalytic reaction. In this paper, recent developments in using various techniques to introduce, characterize and regulate defects are summarized, based on which the important roles played by defects are highlighted in the development of high-performance defect engineered photoelectrodes for PEC water splitting application. Moreover, current challenges and future perspectives in the field of defect engineering of nanostructures for photoelectrodes are discussed.