Zero Background Research Articles

Correlative in vivo Fluorescence Imaging and 19F-MRI of Zebrafish Embryos

Monitoring the pathway of a drug in the body and confirming delivery to the desired target is important in drug development. To this aim, we are developing a correlative fluorescence imaging approach in combination with 19F-MRI. Our first attempts to develop a suitable 19F-labeling strategy will be monitored using well-established fluorescence imaging in transparent zebrafish embryos. Later on, 19F-MRI could then be applied to non-transparent organisms. Zero background and high NMR sensitivity render fluorine a promising label to be traced in vivo. Although 19F possesses almost the same magnetic resonance sensitivity as 1H, a mono-atomic fluorine label is far too insensitive for MRI detection in vivo. To enhance the sensitivity, we therefore explored labels possessing many 19F nuclei with, if possible, identical 19F chemical shift. As a first step, we constructed nanoparticles with encapsulated fluorescence dye and perfluorocarbons, using an emulsion diffusion method. Characterization by solid-state NMR and dynamic light scattering demonstrated a successful simultaneous encapsulation of the fluorocarbon and fluorescence dye. In our first confocal microscopy and MRI experiments in zebrafish embryos, we were able to characterize the distribution of the nanoparticles in the organism. Based on these first results, we are currently expanding our correlative 19F-MRI and fluorescence imaging approach to peptide-based drugs. To this aim, we are synthesized artificial amino acids bearing up to 27 equivalent 19F-nuclei, which can then be combined with fluorescence labels to allow tracing peptides in vivo.

Construction of a self-directed replication system for label-free and real-time sensing of repair glycosylases with zero background.

Genomic DNA damage and repair are involved in multiple fundamental biological processes, including metabolism, disease, and aging. Inspired by the natural repair mechanism in vivo, we demonstrate for the first time the construction of a self-directed replication system for label-free and real-time sensing of repair glycosylases with zero background. The presence of DNA glycosylase can catalyze the excision repair of the damaged base, successively autostarting the self-directed replication through recycling polymerization extension and strand-displacement DNA synthesis for the generation of exponentially amplified dsDNAs. The resultant dsDNA products can be label-free and real-time monitored with SYBR Green I as the fluorescent indicator. Owing to the high efficiency of self-directed exponential replication and the absolute zero background resulting from the efficient inhibition of nonspecific amplification induced by multiple primer-dependent amplification, this strategy exhibits high sensitivity with a detection limit of 1 × 10-8 U μL-1 in vitro and 1 cell in vivo, and it can be further used to screen inhibitors, quantify DNA glycosylase from diverse cancer cells, and even monitor various repair enzymes by simply changing the specific damaged base in the DNA template. Importantly, this assay can be performed in a label-free, real-time and isothermal manner with the involvement of only a single type of polymerase, providing a simple, robust and universal platform for repair enzyme-related biomedical research and clinical therapeutics.

Zero background and triple-signal amplified fluorescence aptasensor for antibiotics detection in foods

It's important to eliminate matrix interference for accurate detecting antibiotic residues in complex food samples. In this study, we designed a zero-backgrounded fluorescence aptasensor to achieve on-site detection of antibiotic residues, with chloramphenicol (CAP) as representative analyte. Moreover, a three stir-bars assisted target recycling system (TSBTR) was designed to achieve triple signal amplification and increase the sensitivity. The bars included one magnetic stir-bar modified with two kinds of long DNA chains, and two gold stir-bars modified with Y shape-duplex DNA probes respectively. In the presence of CAP, the target could recurrently react with the probes on the bars and replace a large amount of long DNA chains into supernatant. After then, the bars were taken out and SYBR green dye was added to the solution. The dye can specifically intercalate into the duplex structures of DNA chains to emit fluorescence while not emitting a signal in its free state. Under the optimized experimental conditions, a wide linear response range of 5 orders of magnitude from 0.001 ng mL−1 to 10 ng mL−1 was achieved with a detection limit of 0.033 pg mL−1 CAP. The assay was successfully employed to detect CAP in food samples (milk & fish) with consistent results with ELISA's. High selectivity and sensitivity were attributed to the zero background signal and triple signal-amplification strategy. Moreover, the detection time can be shortened to 40 min due to that three signal amplified process can occur simultaneously. The fluorescent aptasensor was also label- and enzyme-free. All these ensure the platform to be rapid, cost-effective, easily-used, and is especially appropriate for detection antibiotics in food safety.

High-Fidelity Trajectory Optimization with Application to Saddle-Point Transfers

A method to optimize space trajectories subject to impulsive controls is presented. The method employs a high-fidelity model and a multiple-shooting technique. The model accounts for an arbitrary number of gravitational attractions, their corrections due to celestial bodies oblateness, and solar radiation pressure. The peculiarity of this paradigm is that the equations of motion are written in a rotopulsating frame, where two primaries are at rest despite the fact that their motion and the one of the perturbers are given by a real ephemeris model. Direct transcription of the dynamics coupled with a multiple-shooting technique and an efficient computation of the Jacobian of the defects are used to optimize trajectories subject to a finite number of impulsive controls. The method has been applied to find a multitude of solutions to the problem of transferring a spacecraft from the sun–Earth collinear Lagrange points to the sun–Earth gravitational saddle point, where a theoretical zero background acceleration allows testing possible deviations from general relativity. The problem of targeting the sun–Earth saddle point with high accuracy represents a major flight dynamics challenge. The applicative scenario encompasses the possible mission extension option for LISA Pathfinder as special case.

A Study of 48deplCa100MoO4 Scintillation Crystals for the AMoRE-I Experiment

Advanced Molybdenum-based Rare process Experiment (AMoRE) searching neutrinoless double-beta ( $0\nu \beta \beta $ ) decay of 100Mo from 48deplCa100MoO4 (CMO) scintillation crystals has been preparing CMO crystals for the phase-I stage, AMoRE-I. Nine CMO crystals with a total weight of 3.39 kg were produced by the FOMOS-Materials in Russia and delivered to the Yangyang Underground Laboratory (Y2L) in South Korea for about 2 years until the end of 2016. Acceptance tests of all the nine crystals on weight, dimension, transmittance, light output, energy resolution, and internal radioactivities were completed in the middle of 2017. We have set the upper limits of internal radioactivity levels for 228Th, 226Ra, and 227Ac (daughters of 232Th, 238U, and 235U), and the total alpha activity of U/Th in the crystals via a GEANT4 Monte Carlo simulation to achieve zero background in region of interest. We used a delayed-coincidence analysis to separate the alphas from gamma background events. Results of the acceptance tests showed that all the crystals are qualified for the AMoRE-I. We will present the test results of the nine crystals together with a reference crystal.

Aptasensor based on fluorophore-quencher nano-pair and smartphone spectrum reader for on-site quantification of multi-pesticides

Current techniques for the detection of multi-pesticides are limited by technical complexity, sensitivity and cost. There is an urgent demand of developing new specific recognition elements and sensitive signal readouts for on-site monitoring. In this work, we developed a fluorescent aptamer-based lateral flow biosensor (apta-LFB) integrated with fluorophore-quencher nano-pairs and a smartphone spectrum reader to accomplish triple-target detection of chlorpyrifos, diazinon, and malathion. Aptamers serve as alternative recognition elements instead of antibodies in LFB, offering better specificity and stability. A novel fluorophore-quencher nano-pair (quantum dots nanobeads and gold nanostars) was implemented to perform “signal-on” instead of “signal-off”. After optimization, detection limit of chlorpyrifos, diazinon, and malathion were determined to be 0.73 ng/mL, 6.7 ng/mL, and 0.74 ng/mL, respectively. Greatly increased sensitivity may come from the combination of improved signal and zero background. This innovative cascade strategy allowed to lower the detection limit of pesticides residue level in food, which is largely considered satisfactory. The accuracy and practicality of this design for effective on-site quantification of multi-pesticides were further confirmed using 12 vegetable and fruit samples. The estimated recoveries were between 82.4% and 112.8% in spiked vegetable samples, which indicated that the developed method is capable for detecting multi-pesticides in food samples. This sensitive handheld-system is promising to become a powerful tool for practical on-site application of multi-pesticide quantification procedures.

Magnetic particle imaging of islet transplantation in the liver and under the kidney capsule in mouse models.

Islet transplantation (Tx) represents the most promising therapy to restore normoglycemia in type 1 diabetes (T1D) patients to date. As significant islet loss has been observed after the procedure, there is an urgent need for developing strategies for monitoring transplanted islet grafts. In this report we describe for the first time the application of magnetic particle imaging (MPI) for monitoring transplanted islets in the liver and under the kidney capsule in experimental animals. Pancreatic islets isolated from Papio hamadryas were labeled with superparamagnetic iron oxides (SPIOs) and used for either islet phantoms or Tx in the liver or under the kidney capsule of NOD scid mice. MPI was used to image and quantify islet phantoms and islet transplanted experimental animals post-mortem at 1 and 14 days after Tx. Magnetic resonance imaging (MRI) was used to confirm the presence of labeled islets in the liver and under the kidney capsule 1 day after Tx. MPI of labeled islet phantoms confirmed linear correlation between the number of islets and the MPI signal (R2=0.988). Post-mortem MPI performed on day 1 after Tx showed high signal contrast in the liver and under the kidney capsule. Quantitation of the signal supports islet loss over time, which is normally observed 2 weeks after Tx. No MPI signal was observed in control animals. In vivo MRI confirmed the presence of labeled islets/islet clusters in liver parenchyma and under the kidney capsule one day after Tx. Here we demonstrate that MPI can be used for quantitative detection of labeled pancreatic islets in the liver and under the kidney capsule of experimental animals. We believe that MPI, a modality with no depth attenuation and zero background tissue signal could be a suitable method for imaging transplanted islet grafts.

Quantitative Electrochemical DNA Microarray on a Monolith Electrode with Ten Attomolar Sensitivity, 100% Specificity, and Zero Background.

Circulating microRNA are promising diagnostic and prognostic biomarkers of disease in quantitative blood tests. A label-free, PCR-free, electrochemical microarray technology on a monolith electrode is described, with 10 attomolar (aM) sensitivity and responsiveness to binding of <1 zeptomole of target to immobilized ssDNA probes with zero background. Specificity is 100% in a mixture with five nonspecific miRNA each with a 103-fold higher concentration. Direct measurement on plasma-derived miRNA without cDNA conversion and PCR demonstrated multiplexing and near-ideal quantitative correlation with an equivalent pure sample. The dynamic range is a target concentration ranging from 10-2 to 103 femtomolar (fM). This PCR-free novel technology can be applied as a test for cancer diagnosis/prognosis to detect 103 copies of a miRNA sequence in RNA extracted from 100 μL of plasma.

Label-Free Sensitive Detection of DNA Methyltransferase by Target-Induced Hyperbranched Amplification with Zero Background Signal.

DNA methyltransferases (MTases) may specifically recognize the short palindromic sequences and transfer a methyl group from S-adenosyl-l-methionine to target cytosine/adenine. The aberrant DNA methylation is linked to the abnormal DNA MTase activity, and some DNA MTases have become promising targets of anticancer/antimicrobial drugs. However, the reported DNA MTase assays often involve laborious operation, expensive instruments, and radio-labeled substrates. Here, we develop a simple and label-free fluorescent method to sensitively detect DNA adenine methyltransferase (Dam) on the basis of terminal deoxynucleotidyl transferase (TdT)-activated Endonuclease IV (Endo IV)-assisted hyperbranched amplification. We design a hairpin probe with a palindromic sequence in the stem as the substrate and a NH2-modified 3' end for the prevention of nonspecific amplification. The substrate may be methylated by Dam and subsequently cleaved by DpnI, producing three single-stranded DNAs, two of which with 3'-OH termini may be amplified by hyperbranched amplification to generate a distinct fluorescence signal. Because high exactitude of TdT enables the amplification only in the presence of free 3'-OH termini and Endo IV only hydrolyzes the intact apurinic/apyrimidinic sites in double-stranded DNAs, zero background signal can be achieved. This method exhibits excellent selectivity and high sensitivity with a limit of detection of 0.003 U/mL for pure Dam and 9.61 × 10-6 mg/mL for Dam in E. coli cells. Moreover, it can be used to screen the Dam inhibitors, holding great potentials in disease diagnosis and drug development.

The CGL1 (HeLa × Normal Skin Fibroblast) Human Hybrid Cell Line: A History of Ionizing Radiation Induced Effects on Neoplastic Transformation and Novel Future Directions in SNOLAB.

Cellular transformation assays have been utilized for many years as powerful in vitro methods for examining neoplastic transformation potential/frequency and mechanisms of carcinogenesis for both chemical and radiological carcinogens. These mouse and human cell based assays are labor intensive but do provide quantitative information on the numbers of neoplastically transformed foci produced after carcinogenic exposure and potential molecular mechanisms involved. Several mouse and human cell systems have been generated to undertake these studies, and they vary in experimental length and endpoint assessment. The CGL1 human cell hybrid neoplastic model is a non-tumorigenic pre-neoplastic cell that was derived from the fusion of HeLa cervical cancer cells and a normal human skin fibroblast. It has been utilized for the several decades to study the carcinogenic/neoplastic transformation potential of a variety of ionizing radiation doses, dose rates and radiation types, including UV, X ray, gamma ray, neutrons, protons and alpha particles. It is unique in that the CGL1 assay has a relatively short assay time of 18-21 days, and rather than relying on morphological endpoints to detect neoplastic transformation utilizes a simple staining method that detects the tumorigenic marker alkaline phosphatase on the neoplastically transformed cells cell surface. In addition to being of human origin, the CGL1 assay is able to detect and quantify the carcinogenic potential of very low doses of ionizing radiation (in the mGy range), and utilizes a neoplastic endpoint (re-expression of alkaline phosphatase) that can be detected on both viable and paraformaldehyde fixed cells. In this article, we review the history of the CGL1 neoplastic transformation model system from its initial development through the wide variety of studies examining the effects of all types of ionizing radiation on neoplastic transformation. In addition, we discuss the potential of the CGL1 model system to investigate the effects of near zero background radiation levels available within the radiation biology lab we have established in SNOLAB.

The LUCIFER/CUPID-0 demonstrator: searching for the neutrinoless double-beta decay with Zn82Se scintillating bolometers

Future experiments on neutrinoless double beta-decay with the aim of exploring the inverted hierarchy region have to employ detectors with excellent energy resolution and zero background in the energy region of interest. Cryogenic scintillating bolometers turn out to be a suitable candidate since they offer particle discrimination: the dual channel detection of the heat and the scintillation light signal allows for particle identification. In particular such detectors permit for a suppression of α-induced backgrounds, a key-issue for next-generation tonne-scale bolometric experiments.We report on the progress and current status of the LUCIFER/CUPID-0 demonstrator, the first array of scintillating bolometers based on enriched Zn82Se crystals which is expected to start data taking in 2016 and the potential of this detection technique for a future tonne-scale bolometric experiment after CUORE.

In vivo cloning of up to 16 kb plasmids in E. coli is as simple as PCR.

The precise assembly of defined DNA sequences into plasmids is an essential task in bioscience research. While a number of molecular cloning techniques have been developed, many methods require specialized expensive reagents or laborious experimental procedure. Not surprisingly, conventional cloning techniques based on restriction digestion and ligation are still commonly used in routine DNA cloning. Here, we describe a simple, fast, and economical cloning method based on RecA- and RecET-independent in vivo recombination of DNA fragments with overlapping ends using E. coli. All DNA fragments were prepared by a 2-consecutive PCR procedure with Q5 DNA polymerase and used directly for transformation resulting in 95% cloning accuracy and zero background from parental template plasmids. Quantitative relationships were established between cloning efficiency and three factors–the length of overlapping nucleotides, the number of DNA fragments, and the size of target plasmids–which can provide general guidance for selecting in vivo cloning parameters. The method may be used to accurately assemble up to 5 DNA fragments with 25 nt overlapping ends into relatively small plasmids, and 3 DNA fragments into plasmids up to 16 kb in size. The whole cloning procedure may be completed within 2 days by a researcher with little training in cloning. The combination of high accuracy and zero background eliminates the need for screening a large number of colonies. The method requires no enzymes other than Q5 DNA polymerase, has no sequence restriction, is highly reliable, and represents one of the simplest, fastest, and cheapest cloning techniques available. Our method is particularly suitable for common cloning tasks in the lab where the primary goal is to quickly generate a plasmid with a pre-defined sequence at low costs.

Dissipative soliton fiber lasers with higher-order nonlinearity, multiphoton absorption and emission, and random dispersion

We study the generation of dissipative solitons (DSs) in the model of the fiber-laser cavities under the combined action of cubic-quintic nonlinearity, multiphoton absorption and/or multiphoton emission (nonlinear gain), and gain dispersion. A random component of the group-velocity dispersion (GVD) is included too. The DS creation and propagation are studied by means of a variational approximation and direct simulations, which are found to be in reasonable agreement. With a proper choice of the gain, robust DS operation regimes are predicted for different combinations of multiphoton absorption and emission, in spite of the presence of the perturbation in the form of the random GVD. Importantly, the zero background around the solitons remains stable in the presence of the (necessary) linear gain. The solitons are stable too against a certain (realistic) level of noise. Another essential finding is that the quintic gain in the form of three-photon emission (3PE) offers an alternative mechanism for supporting stable solitons, provided that it is not too strong. The DSs coexist in low- and high-amplitude forms, for a given value of their width. The low-amplitude DS is stable, while its high-amplitude counterpart is subject to the blowup instability, in the presence of the 3PE. Interactions between DSs show various scenarios of the creation of breather states through merger of the two solitons.

Effect of Stolt f-k migration filters on ground penetrating radar imaging

Background: Some filters such as (Stolt F-K Migration) and complimentary filters (Time –Zero Adjustment,Background Removal, and Band –Pass) are used to study the effectiveness, digital filters, and to enhance the qualityof radar grams. This filter considered one of the advanced processing filters in general. Materials and Methods:Ground Penetrating Radar (GPR) is a device that transmits short pulses of electromagnetic energy with pulse durationabout 1 ns to 20 ns with a high-frequency range from 10 MHz to 1000 MHz in the ground by a transmitting antenna.The input analogue signals digitized and quantified using an analogue-to-digital the converter in order to be ready forprocessing in the computer to create images called the radar gram. Results: The important feature focused on makingvisible the weak signals and improves certain components required to interpret the data. Conclusions: Applying StoltF-K Migration filter on profile GPR cretaceous limestone rock detected at a depth of 3.75m. Whereas, applying thefilter to the same profile of water pipe at a depth (0.632m)near the road for the faculty of pharmacy at the directionfrom north to south. When applying the filter) to the same profile of difference in the thickness of pavement layerswhich consist of a foundation layer and a layer of asphalt.

Gain characteristics of a metamaterial coated slotted sphere

An analysis about the effects of various metamaterial coatings and background media upon the gain characteristics of a slotted sphere is presented using numerical simulations. It is found that the gain characteristics of a coated slotted sphere can be controlled by varying the electromagnetic parameters of coatings and background media. It is studied that the thickness of different coating layers has a significant effect upon the gain in the forward direction. It is further investigated that a double negative metamaterial coating can be used to greatly enhance the forward direction gain for certain values of the permittivity and the permeability. It is also shown that single near zero and double near zero background media enhance the forward direction gain of a dielectric-coated slotted sphere and reduce the gains for angles closer to polar directions as compared to the free space background.

The LUCIFER Project: Achievements and Near Future Prospects

In the view of exploring the inverted hierarchy region future experiments investigating the neutrinoless double beta decay have to demand for detectors with excellent energy resolution and zero background in the energy region of interest. Cryogenic scintillating bolometers are very suitable detectors for this task since they provide particle discrimination: the simultaneous detection of the phonon and light signal allows us to identify the interacting type of particle and thus guarantees a suppression of $$\alpha $$ -induced backgrounds, the key-issue for next-generation tonne-scale bolometric experiments. The LUCIFER project aims at running the first array of enriched scintillating Zn $$^{\text {82}}$$ Se bolometers (total mass of about 8kg of $$^{\text {82}}$$ Se) with a background level as low as 10 $$^{\text {--3}}$$ counts/(keV kg y) in the energy region of interest. The main effort is currently focused on the finalization of the crystal growth procedure in order to achieve high quality Zn $$^{\text {82}}$$ Se crystals both in terms of radiopurity and bolometric properties. We present results from tests of such crystals operated at mK temperatures which demonstrate the excellent background rejection capabilities of this detection approach towards a background-free demonstrator experiment. Besides, the high purity of the enriched $$^{\text {82}}$$ Se material allows us to establish the most stringent limits on the half-life of the double beta decay of $$^{\text {82}}$$ Se on excited levels.

Complete suppression of the fluorophore fluorescence by combined effect of multiple fluorescence quenching groups: A fluorescent sensor for Cu2+ with zero background signals

The reaction-based fluorescent sensors have attracted increasing attention in the past decades. However, the application of these sensors for accurate sensing was significantly retarded by the background fluorescence from the sensors themselves. In this work, we demonstrated a novel strategy that the background fluorescence of the sensor could be completely eliminated by the combined effect of multiple fluorescence quenching groups. Based on this new strategy, as proof-of-principle study, a fluorescent sensor (CuFS) for Cu2+ was judiciously developed. In CuFS, three types of fluorescence quenching groups were directly tethered to a commonly used coumarin fluorophore. The fluorescence of coumarin fluorophore in CuFS was completely suppressed by the combined effect of these fluorescence quenching groups. Upon treatment with 22 μM Cu2+, sensor CuFS achieved a dramatic fluorescence enhancement (fluorescence intensity enhanced up to 811-fold) centered at 469 nm. The detection limits was determined to be 12.3 nM. The fluorescence intensity enhancement also showed a good linearity with the Cu2+ concentration in the range of 12.3 nM to 2 μM. By fabricating test strips, sensor CuFS can be utilized as a simple tool to detect Cu2+ in water samples. Furthermore, the fluorescent sensor was successfully applied in detecting different concentration of Cu2+ in living cells.

A novel and simple method for rapid generation of recombinant porcine adenoviral vectors for transgene expression.

Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620±49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes.

Reflection and transmission properties of a wire grid embedded in a SNG or SZ medium

The reflection and transmission properties of a wire grid embedded in lossless Single Negative (SNG) or Single Zero (SZ) medium have been analytically studied. The wires of the grid are taken to be lossless as well as lossy. It is shown that a lossy grid has approximately identical reflection characteristics for the Transverse Magnetic incident wave with Epsilon Negative and Mu Negative background medium. The lossy grid with Epsilon Zero background medium acts as a perfect reflector for the normally incident wave. Likewise, it is further studied that a lossy wire grid embedded in Mu Zero medium can be cloaked for the incoming normally incident wave.

Stability and dynamics of nonautonomous systems with pulsed nonlinearity

We study the dynamics of a class of nonautonomous systems with pulsed nonlinearity that consist of a periodic sequence of linear and nonlinear autonomous systems, each one acting alone in a different time or space interval. We focus on the investigation of control capabilities of such systems in terms of altering their fundamental dynamical properties by appropriate parameter selections. For the case of single oscillators, the stability of the zero solution as well as the phase space topology is shown to drastically depend on parameters such as the frequency of the linear oscillations and the durations of the linear and nonlinear intervals. In cases of chain of coupled oscillators with pulsed onsite nonlinearity, it is shown that appropriate parameter selections can stabilize an otherwise unstable zero background allowing for the existence of dynamically robust localized excitations, whose evolution properties can now be explicitly determined and controlled.