Subfemtomole detection limit for amino acid determination with laser-induced crossed-beam thermal lens detection.
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Amino Acids↗
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Results for “Limit of Detection”
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An electrochemical enzyme immunoassay for chicken luteinizing hormone: extension of the detection limit by adequate control of the nonspecific adsorption.
A noncompetitive heterogeneous enzyme immunoassay for the determination of chicken luteinizing hormone (LH) was equipped with an electrochemical endpoint in order to further enhance its sensitivity. The immunological principle of the original ELISA remained essentially unchanged, except for the fact that the peroxidase label was replaced by alkaline phosphatase, since in the upgraded version of the assay, p-aminophenyl phosphate was to be used as the substrate of alkaline phosphatase. Enzyme-generated p-aminophenol was injected into a flow-injection system and detected amperometrically in a thin-layer flow cell with a glassy carbon electrode at 0.325 V vs Ag/AgCl. A classical problem associated with this type of solid-phase immunoassay is the adsorption of proteins other than the capture antibody to the solid phase. The detection sensitivity is therefore often limited by a large background signal observed in the absence of antigen. In the present study, an experiment was designed to examine in each step of the assay the contribution of each of the potential sources of background current. It was shown that the major contribution to the background current was caused by the nonspecific adsorption of biotinylated secondary antibody. Adsorption of the secondary antibody (biotinylated goat anti-rabbit IgG) to the capture antibody (mouse anti-chicken LHbeta) was clearly a case of specific aspecificity, whereas adsorption to the solid phase itself had to be treated as a nonspecific aspecificity. Addition of 0.25% mouse serum to the secondary antibody as a source of mouse immunoglobulin could overcome the cross-reaction and markedly reduced adsorption to capture antibody. The second part of nonspecific adsorption was eliminated by using combinations of Tween 20 and bovine serum albumin as blocking agents. Controlling the adsorption of the biotinylated secondary antibody in this way decreased the detection limit from 39 pg/ml in the original assay to 2.5 pg/ml in the electrochemical version. This way, the plasma volume of samples containing on the order of 1 ng/ml LH was reduced to less than 10 microl. The linear range was 2.5-625 pg/ml. The method allowed us to measure LH in buffer and in adult and juvenile chicken plasma.
Adsorption↗
An extension of the transparent-motion detection limit using speed-tuned global-motion systems.
When transparent motion is defined purely by direction differences, no more than two signal directions can be detected simultaneously. This limit appears to occur because higher signal intensities are required to detect transparent motion compared with uni-directional motion (Edwards, M., & Greenwood, J. A. (2005). The perception of motion transparency: A signal-to-noise limit. Vision Research, 45, 1877-1884). Increasing the effective signal intensities should therefore increase the number of signals that can be detected. We achieved this by adding speed differences, dividing transparent-motion signals between two speed-tuned global-motion systems. When some signals moved at appropriate low speeds and others at high speeds, up to three signals were detected. This is consistent, at least in part, with the signal-to-noise processing basis of the transparency limit. Differences in contrast polarity were also used to assess whether the limit could be extended using stimulus features without independent global-motion systems. A modest improvement in performance was obtained, suggesting that there may be multiple routes to extending the transparent-motion limit.
Computer Graphics↗
A new single-locus cytogenetic mapping system for maize (Zea mays L.): overcoming FISH detection limits with marker-selected sorghum (S. propinquum L.) BAC clones.
The development of a cytogenetic map for maize (Zea mays L.) is shown to be feasible by means of a combination of resources from sorghum and oat that overcome limitations of single-copy gene detection. A maize chromosome-addition line of oat, OMAd9.2, provided clear images of optically isolated pachytene chromosomes through a chromosome spread and painting technique. A direct labeled oligonucleotide fluorescence in situ hybridization (FISH) probe MCCY specifically stained the centromere. The arm ratio (long/short) for maize chromosome 9 in the addition line was 1.7, comparable to the range of 1.6-2.1 previously reported for maize chromosome 9. A sorghum (Sorghum propinquum L.) BAC library was screened by hybridization with each of three maize core-bin-marker (CBM) probes: umc109 (CBM9.01), umc192/bz1 (CBM9.02), and csu54b (CBM9.08). A single BAC clone for each marker was chosen; designated sCBM9.1, sCBM9.2, or sCBM9.8; and used as a FISH probe on pachytene spreads from OMAd9.2. In each case, discrete FISH signals were observed, and their cytogenetic positions were determined to be 9S.79 (at position 79% of the length of chromosome 9 short arm) for sCBM9.1, 9S.65 for sCBM9.2, and approximately 9L.95 for sCBM9.8. These map positions were co-linear with linkage-map positions for these and other loci common to the linkage and cytogenetic maps. This work represents a major breakthrough for cytogenetic mapping of the maize genome, and also provides a general strategy that can be applied to cytogenetic mapping of other plant species with relatively large and complex genomes.
Avena↗
Potentiometry at trace levels in confined samples: ion-selective electrodes with subfemtomole detection limits.
We explore here for the first time the direct potentiometric detectability of calcium, lead, and silver ions in amounts on the order of 300 attomoles at 100 picomolar concentrations without any preconcentration, analyte recycling, or electrocatalytic signal enhancement. The results presented here place zero-current potentiometry among the most sensitive electrochemical methods available.
Calcium↗
A note on comparing exposure data to a regulatory limit in the presence of unexposed and a limit of detection.
In some occupational health studies, observations occur in both exposed and unexposed individuals. If the levels of all exposed individuals have been detected, a two-part zero-inflated log-normal model is usually recommended, which assumes that the data has a probability mass at zero for unexposed individuals and a continuous response for values greater than zero for exposed individuals. However, many quantitative exposure measurements are subject to left censoring due to values falling below assay detection limits. A zero-inflated log-normal mixture model is suggested in this situation since unexposed zeros are not distinguishable from those exposed with values below detection limits. In the context of this mixture distribution, the information contributed by values falling below a fixed detection limit is used only to estimate the probability of unexposed. We consider sample size and statistical power calculation when comparing the median of exposed measurements to a regulatory limit. We calculate the required sample size for the data presented in a recent paper comparing the benzene TWA exposure data to a regulatory occupational exposure limit. A simulation study is conducted to investigate the performance of the proposed sample size calculation methods.
Algorithms↗
Penalized Cumulative Probability Model for a Continuous Outcome Subject to Detection Limits.
Mixed-type outcome data occur when the outcome variable's distribution is a mixture of both continuous and discrete ordinal variables. Such mixed-type outcomes are common in biomedical, psychological, and the health sciences, particularly for variables having either a detection or quantitation limit. When interest lies in identifying a combination of genomic features associated with a mixed-type outcome, any method used would require a variable selection strategy for high-dimensional data. Unfortunately, few variable selection methods exist for modeling a mixed-type outcome when the covariate space is high dimensional. This study develops a high-dimensional penalized cumulative probability model (CPM), to allow for the identification of genomic features associated with mixed-type outcome of interest. We demonstrated how such model may be estimated using the iterative penalization procedure-the generalized monotone incremental forward stagewise (GMIFS) algorithm. The Model-X knockoffs procedure was combined with the estimation algorithm to control the false discovery rates (FDR) when performing variable selection. Through extensive simulation studies, our penalized CPM was shown to outperform alternative methods in terms of controlled variable selection performance by achieving high statistical power with the FDR being controlled at the target level. We demonstrate the utility of our method by applying it to predict estimated glomeruli filtration rate (eGFR) in kidney transplant recipients at 24 months post-transplant using baseline gene expression data as predictors. Our CPM model identified five genes associated with this mixed-type outcome which have important links to renal disease, which may provide prognostic guidance for kidney transplantation recipients.
Models, Statistical↗
What limits detection and resolution of short-wavelength sinusoidal gratings across the retina?
Peripheral resolution acuity for achromatic gratings is known to be limited by the density of the underlying ganglion cell sampling array. After confirming isolation of the short-wavelength sensitive (SWS) system using chromatic adaptation methods, we wished to determine if resolution is limited by blue/yellow ganglion cell sampling (evidenced by a superiority of detection over resolution acuity and the perception of aliasing) and thus directly related to SWS-driven ganglion cell density. We measured detection and resolution acuity between 0 degrees and 35 degrees, using blue sinusoidal gratings superimposed on a yellow adapting background which ranged in intensity from 2.5 to 4.7logTrolands. At all locations, a break could be observed in the acuity vs. illumination curves followed by a plateau. Detection and resolution acuity were the same for low background intensities, but resolution acuity was lower than detection at higher intensities, accompanied by observations of chromatic aliasing. SWS resolution is sampling-limited across the retina and agrees well with predicted performance based on anatomical estimates of small bistratified ganglion cell density.
Adolescent↗
A generic sandwich-type biosensor with nanomolar detection limits.
A quantitative and highly sensitive, yet simple and rapid, biosensor system was developed for the detection of nucleic acid sequences that can also be adapted to the detection of antigens. A dipstick-type biosensor with liposome amplification, based on a sandwich assay format with optical detection, was combined with a simple coupling reaction that allows the transformation of the generic biosensor components to target specific ones by a mere incubation step. This biosensor platform system was developed and optimized, and its principle was proven using DNA oligonucleotides that provided a nucleic acid biosensor for the specific detection of RNA and DNA sequences. However, the coupling reaction principle chosen can also be used for the immobilization of antibodies or receptor molecules, and therefore for the development of immunosensors and receptor-based biosensors. The generic biosensor consists of liposomes entrapping sulforhodamine B that are coated with streptavidin on the outside, and polyethersulfone membranes with anti-fluorescein antibodies immobilized in the detection zone. In order to transform the generic biosensor into a specific DNA/RNA biosensor, two oligonucleotides that are able to hybridize to the target sequence were labeled with a biotin and a fluorescein molecule, respectively. By simultaneously incubating the liposomes, both oligonucleotides, and the target sequence in a hybridization buffer for 20-30 min at 42 degrees C, a sandwich complex was formed. The mixture was applied to the polyethersulfone membrane. The complex was captured in the detection zone and quantified using a hand-held reflectometer. The system was tested using RNA sequences from B. anthracis, C. parvum and E. coli. Quantitation of concentrations between 10 fmol and 1000 fmol (10-1000 nM) was possible without altering any biosensor assay conditions. In addition, no changes to hybridization conditions were required when using authentic nucleic acid sequence-based amplified RNA sequences, and the generic biosensor compared favorably with those previously developed specifically for the RNA sequences. Therefore, the universal biosensor described is an excellent tool, for use in laboratories or at test sites, for rapidly investigating and quantifying any nucleic acid sequence of interest, as well as potentially any antigen of interest that can be bound by two antibodies simultaneously.
Animals↗
Amplified enzyme-linked immunoassay of human proinsulin in serum (detection limit: 0.1 pmol/L).
We describe an amplified enzyme-linked immunoassay of human proinsulin in serum that detects intact proinsulin and both the 32/33 and 65/66 split forms. The method uses the IgG fraction of a polyclonal antibody raised in a guinea pig against intact proinsulin, which we used to coat plastic microtiter plates. A sandwich was formed with proinsulin by using a monoclonal antibody against C-peptide labeled with alkaline phosphatase. We quantified the reaction by using the enzyme amplification procedure, which detected as little intact proinsulin as 0.1 pmol/L. We found no cross-reactivity with C-peptide in the assay, and decreased recovery attributable to the presence of insulin could be demonstrated only with a 30-fold excess of this hormone over proinsulin.
Alkaline Phosphatase↗
Ion detection limitations to mass resolution in matrix-assisted laser desorption time-of-flight mass spectrometry.
The ion detection process in a discrete-dynode electron multiplier can result in significant mass resolution losses in time-of-flight mass spectrometry (TOF-MS) for higher mass-to-charge (m/z) ion species. This resolution loss is attributed to propagation time delays and signal broadening in the ion detector. This is presumed to be due to the generation of a distribution of secondary ion species produced initially upon impact of a primary ion with the first dynode surface of the ion detector. Comparisons are made between the signals produced by a standard discrete dynode ion detector (which amplifies the negatively charged species produced by impact of a primary ion) and a detector modified to respond to only the positively charged secondary ion species produced by a primary ion impact. Ion signals for higher m/z ions with the standard detector geometry are seen to be due to a narrow signal component, most likely due to the generation of secondary electrons and/or very low mass secondary ions (H-), and a broad signal component, apparently due to secondary ions which take significant amounts of time to traverse the low potential fields between the first and second detector dynode. This results in ion signal tailing for higher m/z ion species. Numerical subtraction of the ion signal obtained with the standard and modified detector geometries (singly protonated molecular ion species of equine myoglobin) results in an improvement in mass resolution, such that a new adduct ion species (from trifluoroacetic acid) can be resolved.
Animals↗
Gel-based nonradioactive single-strand conformational polymorphism and mutation detection: limitations and solutions.
Single-strand conformation polymorphism (SSCP) for screening mutations/single-nucleotide polymorphisms (SNPs) is a simple, cost-effective technique, saving an expensive exercise of sequencing each and every PCR reaction product and assisting in choosing only the amplicons of interest with expected mutation. The principle of detection of small changes in DNA sequences is based on the changes in single-strand DNA conformations. The changes in electrophoretic mobility that SSCP detects are sequence-dependent. The limitations faced in SSCP range from the routine polyacrylamide gel electrophoresis (PAGE) problems to the problems of resolving mutant DNA bands. Both these problems could be solved by controlling PAGE conditions and by varying physical and environmental conditions such as pH, temperature, voltage, gel type and percentage, addition of additives or denaturants, and others. Despite much upgrading of the technology for mutation detection, SSCP continues to remain the method of choice to analyze mutations and SNPs in order to understand genomic variations, spontaneous and induced, and the genetic basis of diseases.
Animals↗
Assay detection limits: concept, definition, and estimation.
This paper emphasizes the need for standardizing the definition and estimation of the minimum detectable analyte concentration in an assay. It reviews the key papers that are most frequently quoted in the literature to illustrate the variety of procedures currently in use.
Biometry↗
[Expiratory flow limitation detected by negative expiratory pressure in patients with chronic obstructive pulmonary disease].
OBJECTIVE: To evaluate the relationship between expiratory flow limitation (EFL) and chronic dyspnea and the effect of bronchodilator on EFL in patients with chronic obstructive pulmonary disease (COPD). METHODS: Thirty-three ambulatory patients with COPD (46 - 78 yrs; male 31, female 2) were included in this study. The severity of chronic dyspnea was rated according to the dyspnea scale proposed by the Medical Research Council (MRC). EFL was measured by applying negative pressure at the mouth during tidal expiration before and after bronchodilation test (inhalation of 400 microg salbutamol). RESULTS: EFL was detected in 12 (36%) of the 33 COPD patients in both seated and supine positions and in 11 (33%) only in supine position. There was a significant difference in the percent predicted forced expired volume in one second (FEV(1%)pred) between subgroups of the patients with or without EFL (t = 7.601, P < 0.01). The mean values of FEV(1%)pred in non-EFL group and EFL group was (66 +/- 16)% and (31 +/- 10)%, respectively, and the value was lowest in patients who showed EFL both in seated and supine positions [(24 +/- 7)%]. Both three-point EFL and five-point EFL were significantly correlated with FEV(1) (r = -0.836 and -0.818, respectively, all P < 0.01). There was a significant correlation between MRC dyspnea scale and three-point EFL and five-point EFL (r = 0.903 and 0.912, respectively, all P < 0.01). In the multiple regression analysis, five-point EFL was a better predictor of dyspnea than FEV(1) (regression coefficient was 0.679 and -0.265, respectively, P < 0.01 and 0.029, respectively). EFL persisted after salbutamol in all of the 23 patients with EFL under baseline conditions. CONCLUSIONS: EFL as measured by negative expiratory pressure (NEP) technique may be more useful in the evaluation of dyspnea in COPD patients than routine lung function measurements. The EFL in COPD patients is irreversible after bronchodilator administration.
Aged↗
Determination of the detection limit of the polymerase chain reaction for chicken infectious anemia virus.
Chicken infectious anemia virus (CIAV) DNA in infected cell cultures and chicken tissues was detected using a polymerase chain reaction (PCR) assay. The complete CIAV genome of several strains was amplified in two segments with two sets of primer pairs. The DNA segments of four CIAV strains and full-length Cux-1 strain DNA were cloned. After amplification, 100 original genome equivalents were detected by Southern hybridization. The sensitivity of the assay was enhanced considerably by performing a reamplification with nested primers. This modification permitted the detection of one molecule of CIAV DNA. Some problems of the assay and its possible application are discussed.
Anemia↗
A note on inhibition test and electrophoretic detection limits of antibiotics used in British animal husbandry.
A four plate microbiological inhibition test (the FPT) and a bioelectrophoretic method were evaluated for their ability to detect a range of antibiotic agents, which may be present as residues in animal tissues following their therapeutic use in animal husbandry. Both methods exhibited a wide range of sensitivities and several of the tested antibiotics could not be detected by either method. The pattern of responses across the bacterial plates in the FPT could not be used to identify agents and the bioelectrophoretic inhibition zone diameters were generally too large to allow the use of Rs values for identification. The Bacillus subtilis pH 7.2 plate with trimethoprim added was as effective as the four bacterial plates used in the FPT in antibiotic detection.
Animals↗
Oscillometric instrument for the non-invasive detection of low-level microbial activity. Part 2: Growth characteristics, detection limits and impedimetric assays.
Instrument performance and application was evaluated with a variety of microbial suspensions. Data were compared with those obtainable from current commercial apparatus in terms of resolution and detection times. Trials with growing and non-growing cultures indicated periodic synchronous ion exchange processes. Media composition, particularly salt and nutrient ratios, remain crucial to early and rapid detection of viable microbial activity at low concentrations. With appropriate conditions very significant improvements in detection times with typical detection thresholds of 300 min for start concentrations of 10(2) are shown. It is concluded that with regard to resolution and stability, the system out-performs current systems and is superior in terms of potential test costs, sample handling and sample transport.
Asparaginase↗
Detection limit enhancement of fluorescent heterogeneities in turbid media by dual-interfering excitation.
We report on a quantitative comparison between the single-source and the dual-interfering-source configurations for the detection of fluorescent heterogeneities embedded in a piecewise highly scattering homogeneous fluorescent background. The study is based on simulations with analytical solutions of the frequency-domain fluorescent diffuse photon density waves and practical signal-to-noise ratio considerations. Results show that dual-interfering sources outperform single-source techniques for the detection of heterogeneities in terms of fluorophore concentration and lifetime contrast. To detect the same inhomogeneity, less concentration and lifetime contrast is required with dual-interfering sources.
Coloring Agents↗