Complementary Detection Research Articles

Clostridium spp. discrimination with a simple bead-based fluorescence assay

C. chauvoei is the causative agent of blackleg, an endogenous bacterial infection which usually affects cattle and other ruminants. Due to the fact that the symptoms of this severe disease are very similar to the phenotype caused by an infection with C. septicum, a reliable differentiation of C. chauvoei from other Clostridium spp. is mandatory. Traditional microbiological detection methods are time consuming and the proper specification is hampered by the overgrowing tendency of swarming C. septicum colonies when both species are in the clinical sample. Thus, there is an urgent need to improve and simplify the specific detection of C. chauvoei and C. septicum. We report an easy and fast Clostridium spp. discrimination method via a magnetic bead-based fluorescence assay. To that end, the target DNA was amplified using 16S-23S rDNA spacer region specific primers. These PCR products were employed to generate single-stranded capture probe DNA, which was immobilized on magnetic beads. Functionalized magnetic particles exhibit numerous advantages, like their simple manipulation in combination with a huge binding capacity of biomolecules and make therefore excellent biosensors. In this context, the discrimination between C. chauvoei and C. septicum was realized by means of hybridization with complementary detection probe DNA. Finally, fluorescence spectroscopy allowed the signal readout. With this approach a precise discrimination between C. chauvoei, C. septicum and C. carnis was accomplished.

Dose spectra from energetic particles and neutrons

AbstractDose spectra from energetic particles and neutrons (DoSEN) are an early‐stage space technology research project that combines two advanced complementary radiation detection concepts with fundamental advantages over traditional dosimetry. DoSEN measures not only the energy but also the charge distribution (including neutrons) of energetic particles that affect human (and robotic) health in a way not presently possible with current dosimeters. For heavy ions and protons, DoSEN provides a direct measurement of the lineal energy transfer (LET) spectra behind shielding material. For LET measurements, DoSEN contains stacks of thin‐thick Si detectors similar in design to those used for the Cosmic Ray Telescope for the Effects of Radiation. With LET spectra, we can now directly break down the observed spectrum of radiation into its constituent heavy‐ion components and through biologically based quality factors that provide not only doses and dose rates but also dose equivalents, associated rates, and even organ doses. DoSEN also measures neutrons from 10 to 100 MeV, which requires enough sensitive mass to fully absorb recoil particles that the neutrons produce. DoSEN develops the new concept of combining these independent measurements and using the coincidence of LET measurements and neutron detection to significantly reduce backgrounds in each measurement. The background suppression through the use of coincidence allows for significant reductions in size, mass, and power needed to provide measurements of dose, neutron dose, dose equivalents, LET spectra, and organ doses. Thus, we introduce the DoSEN concept: a promising low‐mass instrument that detects the full spectrum of energetic particles, heavy ions, and neutrons to determine biological impact of radiation in space.

Comprehensive sample analysis using high performance liquid chromatography with multi-detection

Herein we assess the separation space offered by a liquid chromatography system with an optimised uni-dimensional separation for the determination of the key chemical entities in the highly complex matrix of a tobacco leaf extract. Multiple modes of detection, including UV–visible absorbance, chemiluminescence (acidic potassium permanganate, manganese(IV), and tris(2,2′-bipyridine)ruthenium(III)), mass spectrometry and DPPH radical scavenging were used in an attempt to systematically reduce the data complexity of the sample whilst obtaining a greater degree of molecule-specific information. A large amount of chemical data was obtained, but several limitations in the ability to assign detector responses to particular compounds, even with the aid of complementary detection systems, were observed. Thirty-three compounds were detected via MS on the tobacco extract and 12 out of 32 compounds gave a peak height ratio (PHR) greater than 0.33 on one or more detectors. This paper serves as a case study of these limitations, illustrating why multidimensional chromatography is an important consideration when developing a comprehensive chemical detection system.

Advances in turbulent mixing techniques to study microsecond protein folding reactions

Recent experimental and computational advances in the protein folding arena have shown that the readout of the one-dimensional sequence information into three-dimensional structure begins within the first few microseconds of folding. The initiation of refolding reactions has been achieved by several means, including temperature jumps, flash photolysis, pressure jumps, and rapid mixing methods. One of the most commonly used means of initiating refolding of chemically denatured proteins is by turbulent flow mixing with refolding dilution buffer, where greater than 99% mixing efficiency has been achieved within 10's of microseconds. Successful interfacing of turbulent flow mixers with complementary detection methods, including time-resolved Fluorescence Spectroscopy (trFL), Förster Resonance Energy Transfer, Circular Dichroism, Small-Angle X-ray Scattering, Hydrogen Exchange followed by Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy, Infrared Spectroscopy (IR), and Fourier Transform IR Spectroscopy, has made this technique very attractive for monitoring various aspects of structure formation during folding. Although continuous-flow (CF) mixing devices interfaced with trFL detection have a dead time of only 30 µs, burst phases have been detected in this time scale during folding of peptides and of large proteins (e.g., CheY and TIM barrels). Furthermore, a major limitation of the CF mixing technique has been the requirement of large quantities of sample. In this brief communication, we will discuss the recent flurry of activity in micromachining and microfluidics, guided by computational simulations, which are likely to lead to dramatic improvements in time resolution and sample consumption for CF mixers over the next few years.

Detecting subarachnoid hemorrhage: Comparison of combined FLAIR/SWI versus CT

ObjectivesAim of this study was to compare the utility of susceptibility weighted imaging (SWI) with the established diagnostic techniques CT and fluid attenuated inversion recovery (FLAIR) in their detecting capacity of subarachnoid hemorrhage (SAH), and further to compare the combined SWI/FLAIR MRI data with CT to evaluate whether MRI is more accurate than CT. MethodsTwenty-five patients with acute SAH underwent CT and MRI within 6 days after symptom onset. Underlying pathology for SAH was head trauma (n=9), ruptured aneurysm (n=6), ruptured arteriovenous malformation (n=2), and spontaneous bleeding (n=8). SWI, FLAIR, and CT data were analyzed. The anatomical distribution of SAH was subdivided into 8 subarachnoid regions with three peripheral cisterns (frontal-parietal, temporal-occipital, sylvian), two central cisterns and spaces (interhemispheric, intraventricular), and the perimesencephalic, posterior fossa, superior cerebellar cisterns. ResultsSAH was detected in a total of 146 subarachnoid regions. CT identified 110 (75.3%), FLAIR 127 (87%), and SWI 129 (88.4%) involved regions. Combined FLAIR and SWI identified all 146 detectable regions (100%). FLAIR was sensitive for frontal-parietal, temporal-occipital and Sylvian cistern SAH, while SWI was particularly sensitive for interhemispheric and intraventricular hemorrhage. ConclusionsBy combining SWI and FLAIR, MRI yields a distinctly higher detection rate for SAH than CT alone, particularly due to their complementary detection characteristics in different anatomical regions. Detection strength of SWI is high in central areas, whereas FLAIR shows a better detection rate in peripheral areas.

Bacteria Screening, Viability, And Confirmation Assays Using Bacteriophage-Impedimetric/Loop-Mediated Isothermal Amplification Dual-Response Biosensors

Here, we integrate two complementary detection strategies for the identification and quantification of Escherichia coli based on bacteriophage T4 as a natural bioreceptor for living bacteria cells. The first approach involves screening and viability assays, employing bacteriophage as the recognition element in label-free electrochemical impedance spectroscopy. The complementary approach is a confirmation by loop-mediated isothermal amplification (LAMP) to amplify specifically the E. coli Tuf gene after lysis of the bound E. coli cells, followed by detection using linear sweep voltammetry. Bacteriphage T4 was cross-linked, in the presence of 1,4-phenylene diisothiocyanate, on a cysteamine-modified gold electrode. The impedimetric biosensor exhibits specific and reproducible detection with sensitivity over the concentration range of 10(3)-10(9) cfu/mL, while the linear response of the LAMP approach was determined to be 10(2)-10(7) cfu/mL. The limit of detection (LOD) of 8 × 10(2) cfu/mL in less than 15 min and 10(2) cfu/mL within a response time of 40 min were achieved for the impedimetric and LAMP method, respectively. This work provides evidence that integration of the T4-bacteriophage-modified biosensor and LAMP can achieve screening, viability, and confirmation in less than 1 h.

EFFECT OF FIBER DISTANCE ON VARIOUS SAC-OCDMA DETECTION TECHNIQUES

Optical code division multiple access system (OCDMA) has been gaining importance with increasing demands of high speed and large capacity for communication in optical networks. OCDMA system is totally asynchronous, that does not require any clock signals for synchronization in the network. Therefore, OCDMA provide a network that is simpler and offers the potential for scalability to higher levels of connectivity. OCDMA encoding /decoding process also provides a level of security directly implemented in the physical layer. Using OCDMA technique high spectral efficiency is achieved, hence fiber bandwidth is used very efficiently with throughput in excess of Tbit/s. Among several kinds of OCDMA systems, spectra amplitude coding (SAC) scheme attracts increasing interest because multiple access interference (MAI) can be eliminated and preserve the orthogonality between users in the system. This paper presents comparison of three important SAC-OCDMA detection techniques, namely Direct detection, Complementary subtraction and AND subtraction. The design of encoder and decoder modules for SAC-OCDMA system used in this paper is based on Fiber Bragg Gratings (FBGs). Here conventional single mode fiber (SMF) is used as the transmission link and the performance metric studied is Quality factor (Q) in multiple access environments of various user systems for these three detection techniques. Finally Effect of increasing number of fiber distance on the direct detection technique using NRZ and RZ data formats have been studied. Simulated results show that AND subtraction technique gives better Quality-factor (Q) than the complementary subtraction and direct detection techniques for the signal with same power.

Field-Scale Transport and Transformation of Carboxymethylcellulose-Stabilized Nano Zero-Valent Iron

The fate of nano zerovalent iron (nZVI) during subsurface injection was examined using carboxymethylcellulose (CMC) stabilized nZVI in a very large three-dimensional physical model aquifer with detailed monitoring using multiple, complementary detection methods. A fluorescein tracer test in the aquifer plus laboratory column data suggested that the very-aggressive flow conditions necessary to achieve 2.5 m of nZVI transport could be obtained using a hydraulically constrained flow path between injection and extraction wells. However, total unoxidized nZVI was transported only about 1 m and <2% of the injected nZVI concentration reached that distance. The experimental data also indicated that groundwater flow changed during injection, likely due to hydrogen bubble formation, which diverted the nZVI away from the targeted flow path. The leading edge of the iron plume became fully oxidized during transport. However, within the plume, oxidation of nZVI decreased in a fashion consistent with progressive depletion of aquifer "reductant demand". To directly quantify the extent of nZVI transport, a spectrophotometric method was developed, and the results indicated that deployment of unoxidized nZVI for groundwater remediation will likely be difficult.

Ultra-high resolution and long scan depth optical coherence tomography with full-phase detection for imaging the ocular surface

We used a unique combination of four state-of-the-art technologies to achieve a high performance spectral domain optical coherence tomography system suitable for imaging the entire ocular surface. An ultra-high resolution, extended depth range, full-phase interferometry, and high-speed complementary metal-oxide semiconductor transistor camera detection provided unprecedented performance for the precise quantification of a wide range of the ocular surface. We demonstrated the feasibility of this approach by obtaining high-speed and high-resolution images of a model eye beyond the corneal–scleral junction. Surfaces determined from the images with a segmentation algorithm demonstrated excellent accuracy and precision.

VMM detection using privilege rings and benchmark execution times

This paper proposes two complementary virtual machine monitor VMM detection methods. These methods can be used to detect any VMM that is designed for ×86 architecture. The first method works by finding probable discrepancies in hardware privilege levels of the guest operating system's kernel on which user applications run. The second method works by measuring the execution times of a set of benchmark programs and comparing them with the stored execution times of the same programmes previously ran on a trusted physical machine. Unlike other methods, our proportional execution time technique could not be easily thwarted by VMMs. In addition, using proportional execution times, there is no need for a trusted external source of time during detection. It is shown experimentally that the deployment of both methods together can detect the existence of four renowned VMMs, namely, Xen, VirtualBox, VMware, and Parallels, on both types of processors that support virtualisation technology VT-enabled or do not support it VT-disabled.

Specific nucleic acid detection based on fluorescent light-up probe from fluorogens with aggregation-induced emission characteristics

We report a new fluorescent light-up probe design strategy by conjugating fluorogens with aggregation-induced emission (AIE) characteristics, e.g. tetraphenylethene with a single-stranded oligonucleotide for homogeneous specific complementary DNA detection in solution.

Tampered Data Recovery in WSNs through Dynamic PCA and Variable Routing Strategies

Wireless sensor networks (WSNs) are highly sensible to data integrity attacks, which have an important impact on a number of relevant deployments and services. This paper introduces a tolerance approach to fight against data modification attacks in WSNs, which is based on a missing data imputation scheme. The proposal relies on two principal contributions: (1) a multivariate statistical technique where the dynamics of the sensor measurements for the monitored area are captured through the use of dynamic PCA (DPCA), and (2) a variable routing strategy that improves the recovering performance by spreading the effects of the data tampering attack. On the other hand, a complementary multivariate statistical anomaly detection module is implemented to determine the occurrence of data tampering attacks and trigger the subsequent reaction procedure to recover the affected data. As shown by the results obtained, the proposed tolerance approach improves the robustness of a WSN against data tampering attacks, and so its survivability and normal operation over time.

Two-Step Detector for RADARSAT-2's Experimental GMTI Mode

Ground moving target indication (GMTI) from space has currently raised interest from wide area traffic monitoring as well as for military surveillance activities. This paper presents theoretical and real data GMTI results of RADARSAT-2's Moving Object Detection Experiment based on data gathered during the commissioning phase in February 2008. The proposed constant false alarm rate target detection is based on a novel and thorough analysis of the multilook test statistics. The practical and relevant case in which a target, for instance a passenger car, occurs in only l-out-of-n ground resolution multilook cells is analyzed. This general case is analyzed for the determination of adequate detection thresholds as well as the anticipated probability of detection Pd particularly with regard to a varying degree of terrain heterogeneity and target characteristics. The overall false alarm rate Pfa is significantly reduced by a complementary detection step using the along-track interferometric phase. The validity of these theoretical findings are corroborated by real two-channel space-based synthetic aperture radar-GMTI data of civilian vehicles of opportunity, whose main parameters have been estimated and compared with the derived Cramer-Rao Bounds.

An electrochemical assay of polynucleotide kinase activity based on streptavidin–gold nanoparticles and enzymatic amplification

An electrochemical method for the determination of polynucleotide kinase (PNK) activity has been proposed with a dual signal amplification strategy. The method relies on the phosphorylation-induced DNA digestion and two-step signal amplification using streptavidin–gold nanoparticle biocomplexes and alkaline phosphatase. In the presence of T4 PNK, the 5′-terminus of the immobilized PNK substrate probe was phosphorylated, and the substrate probe would be cleaved by λ exonuclease after being hybridized with the complementary detection probe biotinylated in the 3′-hydroxyl terminus. As a result, the detection probe would be released, with an electrochemical signal decrease, due to less biotinylated alkaline phosphatase loading on the electrode surface. The electrochemical signal exhibited a linear correlation to the logarithm of PNK concentration ranging from 0.01 U mL−1 to 5 U mL−1 with the detection limit of 0.01 U mL−1. The inhibiting effect of (NH4)2SO4 on the activity of PNK was also evaluated.

Complementary Detection of Embryotoxic Properties of Substances in the Neural and Cardiac Embryonic Stem Cell Tests

In developmental toxicity testing, in vitro screening assays are highly needed to increase efficiency and to reduce animal use. A promising in vitro assay is the cardiac embryonic stem cell test (ESTc), in which the effect of developmental toxicants on cardiomyocyte differentiation is assessed. Recently, we developed a neural differentiation variant of the stem cell test (neural embryonic stem cell test [ESTn]). In both of these models, we have previously performed a series of transcriptomic studies to characterize gene expression changes (1) across time during normal differentiation and (2) in response to a series of developmental toxicants in the ESTn and ESTc. Here, using the cumulative of these studies, we compared gene expression profiles of ESTn and ESTc over time as well as model-specific changes induced by seven compounds, comprising six known in vivo developmental toxicants and one negative control. Time-related gene expression profiles showed similarities between the two EST systems. However, specific genes could be identified changing over time differently in each model related to the two different lineages of differentiation. Interestingly, compound-induced gene expression changes were generally model specific, especially for methylmercury and flusilazole, which were predicted better in ESTn and ESTc, respectively. Valproic acid-induced gene expression changes were most comparable out of the six developmental toxicants between the ESTn and ESTc. Direct transcriptomic comparisons between the ESTn and ESTc indicate that combined transcriptomic analyses support and complement each other. Therefore, a combined approach incorporating ESTc and ESTn may improve developmental toxicant detection over individual assays.

A study of the performance of the transit detection tool DST in space-based surveys

Context. Transit detection algorithms are mathematical tools used for detecting planets in the photometric data of transit surveys. In this work we study their application to space-based surveys. Aims: Space missions are exploring the parameter space of the transit surveys where classical algorithms do not perform optimally, either because of the challenging signal-to-noise ratio of the signal or its non-periodic characteristics. We have developed an algorithm addressing these challenges for the mission CoRoT. Here we extend the application to the data from the space mission Kepler. We aim at understanding the performances of algorithms in different data sets. Methods: We built a simple analytical model of the transit signal and developed a strategy for the search that improves the detection performance for transiting planets. We analyzed Kepler data with a set of stellar activity filtering and transit detection tools from the CoRoT community that are designed for the search of transiting planets. Results: We present a new algorithm and its performances compared to one of the most widely used techniques in the literature using CoRoT data. Additionally, we analyzed Kepler data corresponding to quarter Q1 and compare our results with the most recent list of planetary candidates from the Kepler survey. We found candidates that went unnoticed by the Kepler team when analyzing longer data sets. We study the impact of instrumental features on the production of false alarms and false positives. These results show that the analysis of space mission data advocates the use of complementary detrending and transit detection tools also for future space-based transit surveys such as PLATO.

Exciton correlations and input–output relations in non-equilibrium exciton superfluids

The photoluminescence (PL) measurements on photons and the transport measurements on excitons are the two types of independent and complementary detection tools to search for possible exciton superfluids in electron–hole semi-conductor bilayer systems. In fact, it was believed that the transport measurements can provide more direct evidences on superfluids than the spectroscopic measurements. It is important to establish the relations between the two kinds of measurements. In this paper, using quantum Heisenberg–Langevin equations, we establish such a connection by calculating various exciton correlation functions in the putative exciton superfluids. These correlation functions include both normal and anomalous greater, lesser, advanced, retarded, and time-ordered exciton Green functions and also various two exciton correlation functions. We also evaluate the corresponding normal and anomalous spectral weights and the Keldysh distribution functions. We stress the violations of the fluctuation and dissipation theorem among these various exciton correlation functions in the non-equilibrium exciton superfluids. We also explore the input–output relations between various exciton correlation functions and those of emitted photons such as the angle resolved photon power spectrum, phase sensitive two mode squeezing spectrum and two photon correlations. Applications to possible superfluids in the exciton–polariton systems are also mentioned. For a comparison, using conventional imaginary time formalism, we also calculate all the exciton correlation functions in an equilibrium dissipative exciton superfluid in the electron–electron coupled semi-conductor bilayers at the quantum Hall regime at the total filling factor νT=1. We stress the analogies and also important differences between the correlations functions in the two exciton superfluid systems.

Synthesis and Chromatography-Free Purification of PNA-PEO Conjugates for the Functionalisation of Gold Sensors

Peptide Nucleic Acids (PNAs) linked to high molecular weight (MW) poly(ethylene oxide) (PEO) derivatives could be useful conjugates for the direct functionalisation of gold surfaces dedicated to Surface Plasmon Resonance (SPR)-based DNA sensing. However their use is hampered by the difficulty to obtain them through a convenient and economical route. In this work we compared three synthetic strategies to obtain PNA-high MW PEO conjugates composed of (a) a 15-mer PNA sequence as the probe complementary to genomic DNA of Mycobacterium tuberculosis, (b) a PEO moiety (2 or 5 KDa MW) and (c) a terminal trityl-protected thiol necessary (after acidic deprotection) for grafting to gold surfaces. The 15-mer PNA was obtained by solid-phase synthesis. Its amino terminal group was later condensed to bi-functional PEO derivatives (2 and 5 KDa MW) carrying a Trt-cysteine at one end and a carboxyl group at the other end. The reaction was carried out either in solution, using HATU or PyOxim as coupling agents, or through the solid-phase approach, with 49.6%, 100% and 5.2% yield, respectively. A differential solvent extraction strategy for product purification without the need for chromatography is described. The ability of the 5 KDa PEO conjugate to function as a probe for complementary DNA detection was demonstrated using a Grating-Coupling Surface Plasmon Resonance (GC-SPR) system. The optimized PEO conjugation and purification protocols are economical and simple enough to be reproduced also within laboratories that are not highly equipped for chemical synthesis.

SEARCHING FOR YOUNG JUPITER ANALOGS AROUND AP COL:L-BAND HIGH-CONTRAST IMAGING OF THE CLOSEST PRE-MAIN-SEQUENCE STAR

The nearby M-dwarf AP Col was recently identified by Riedel et al. 2011 as a pre-main sequence star (age 12 - 50 Myr) situated only 8.4 pc from the Sun. The combination of its youth, distance, and intrinsically low luminosity make it an ideal target to search for extrasolar planets using direct imaging. We report deep adaptive optics observations of AP Col taken with VLT/NACO and Keck/NIRC2 in the L-band. Using aggressive speckle suppression and background subtraction techniques, we are able to rule out companions with mass m >= 0.5 - 1M_Jup for projected separations a>4.5 AU, and m >= 2 M_Jup for projected separations as small as 3 AU, assuming an age of 40 Myr using the COND theoretical evolutionary models. Using a different set of models the mass limits increase by a factor of ~2. The observations presented here are the deepest mass-sensitivity limits yet achieved within 20 AU on a star with direct imaging. While Doppler radial velocity surveys have shown that Jovian bodies with close-in orbits are rare around M-dwarfs, gravitational microlensing studies predict that ~17% of these stars host massive planets with orbital separations of 1-10 AU. Sensitive high-contrast imaging observations, like those presented here, will help to validate results from complementary detection techniques by determining the frequency of gas giant planets on wide orbits around M-dwarfs.

Mass spectrometry based analysis of nucleotides, nucleosides, and nucleobases—application to feed supplements

In this work, accurate MS-based methods for quantitative profiling of nucleotides, nucleosides, and nucleobases in yeast extracts used as additives in animal feedstuff are presented. Reversed-phase chromatography utilizing a stationary phase compatible with 100% aqueous mobile phases resulted in superior analytical figures of merit than HILIC or ion-pair reversed-phase separation. The novel separation method was combined with both molecular and elemental mass spectrometry. By use of RP-LC-MS-MS, excellent limits of detection <1 μmol L(-1) could be obtained for all the compounds investigated. The elemental speciation analysis approach enabled determination of nucleotides by phosphorus detection. Sensitivity of LC-ICP-MS was 1-2 orders of magnitude lower than that of LC-MS-MS. Quantitative analysis of yeast products using complementary MS detection furnished values in good agreement.