Recombinant Gaussia luciferase. Overexpression,purification, and analytical application of a bioluminescent reporter for DNA hybridization

The cDNA for Gaussia luciferase (GLuc), the enzyme responsible for the bioluminescent reaction of the marine copepod Gaussia princeps, has been cloned recently. GLuc (MW = 19 900) catalyzes the oxidative decarboxylation of coelenterazine to produce coelenteramide and light. We report the first quantitative anaytical study of GLuc and examine its potential as a new reporter for DNA hybridization. A plasmid encoding both a biotin acceptor peptide-GLuc fusion protein as well as the enzyme biotin protein ligase (BPL) is engineered by using GLuc cDNA as a starting template. BPL catalyzes the covalent attachment of a single biotin to the fusion protein in vivo. Purification of GLuc is then accomplished by affinity chromatography using immobilized monomeric avidin. Moreover, the in vivo biotinylation enables subsequent complexation of GLuc with streptavidin (SA), thereby avoiding chemical conjugation reactions that are known to inactivate luciferases. Purified GLuc can be detected down to 1 amol with a signal-to-background ratio of 2 and a linear range extending over 5 orders of magnitude. The background luminescence of coelenterazine is the main limiting factor for even higher detectability of GLuc. Furthermore, the GLuc-SA complex is used as a detection reagent in a microtiter well-based DNA hybridization assay. The analytical range extends from 1.6 to 800 pmol/L of target DNA. Biotinylated GLuc produced from 1 L of bacterial culture is sufficient for 150,000 hybridization assays.     

Development of a bioluminescence resonance energy-transfer assay for estrogen-like compound in vivo monitoring

A new bioluminescence resonance energy transfer (BRET) homogeneous assay to evaluate the presence of estrogen-like compounds has been developed and optimized. The assay is based on the direct evaluation of estrogen alphareceptor (ERalpha) homodimerization as a result of estrogen-like compound binding. ERalpha monomer was genetically fused either to Renilla luciferase (Rluc) or to enhanced yellow fluorescent protein (EYFP). In the presence of estrogens, ERalpha dimerization brings Rluc and EYFP molecules close enough for an energy transfer. An in vitro BRET assay was first developed using purified fusion proteins (ERalpha-Rluc and ERalpha-EYFP) expressed in Escherichia coli to evaluate and optimize the analytical performances of the assay in the presence of 17-beta estradiol. The "in vivo" BRET quantitative assay was then developed by coexpressing the two fusion proteins in live HepG2 cells. The assay can be performed in 96-well microplate format with a 30-min incubation and allows detection with adequate accuracy and precision of as low as 1 nM of 17-beta estradiol. This new "in vivo" BRET assay allows evaluating the estrogen-like activity and synthetic xenoestrogens from biological and environmental samples.     

pHluorin-based in vivo assay for hydrolase screening

pHluorin, a pH-sensitive mutant of green fluorescent protein (GFP), acts as a sensor for intracellular pH shifts, triggered by hydrolytic enzymes. This principle was used to develop a pHluorin-based in vivo assay for hydrolase screening. The presented assay was evaluated for Escherichia coli (E. coli) cells, producing heterologous pHluorin and an esterase from Geobacillus stearothermophilus which is considered as a model hydrolase. Subsequently, the utility of this detection system was also demonstrated with recombinantly expressed hydantoinase and amidase in E. coli. This in vivo assay also shows capability for readout with flow cytometric devices. Population shifts of pHluorin-expressing E. coli cells were easily recognized due to pH changes caused by substrate hydrolysis.     

Biodegradable optode-based nanosensors for in vivo monitoring

Optode-based fluorescent nanosensors are being developed for monitoring important disease states such as hyponatremia and diabetes. However, traditional optode-based sensors are composed of nonbiodegradable polymers such as poly(vinyl chloride) (PVC) raising toxicity concerns for long-term in vivo use. Here, we report the development of the first biodegradable optode-based nanosensors that maintain sensing characteristics similar to those of traditional optode sensors. The polymer matrix of these sensors is composed of polycaprolactone (PCL) and a citric acid ester plasticizer. The PCL-based nanosensors yielded a dynamic and reversible response to sodium, were tuned to respond to extracellular sodium concentrations, and had a lifetime of at least 14 days at physiological temperature. When in the presence of lipase, the nanosensors degraded within 4 h at lipase concentrations found in the liver but were present after 3 days at lipase concentrations found in serum. The development of biodegradable nanosensors is not only a positive step towards their future use in in vivo applications, but they also represent a new sensor platform that can be extended to other sensing mechanisms.     

High-throughput screening assay for biological hydrogen production

This paper describes a screening assay, compatible with high-throughput bioprospecting or molecular biology methods, for assessing biological hydrogen (H2) production. While the assay is adaptable to various physical configurations, we describe its use in a 96-well, microtiter plate format with a lower plate containing H2-producing cyanobacteria strains and controls and an upper, membrane-bottom plate containing a color indicator and a catalyst. H2 produced by cells in the lower plate diffuses through the membrane into the upper plate, causing a color change that can be quantified with a microplate reader. The assay is reproducible, semiquantitative, sensitive down to at least 20 nmol of H2, and largely unaffected by oxygen, carbon dioxide, or volatile fatty acids at levels appropriate to biological systems.     

Cumulative quantity control charts for monitoring production processes

Two commonly used statistical quality control charts, the c-chart and u-chart, are unsatisfactory for monitoring high-yield processes with low defect rates. To overcome this difficulty, a new type of control chart called the cumulative quantity control chart (CQC-chart) is introduced in this paper. The CQC-chart can be used no matter whether the process defect rate is low or not, and when the process defect rate is low or moderate the CQC-chart does not have the shortcoming of the c- and u-charts of showing up false alarm signals too frequently. The CQC-chart does not require rational subgrouping of samples (which is necessary for the c- and u-charts), and is appropriate for monitoring automated manufacturing processes.     

A joint monitoring scheme for automatically controlled processes

It has been proposed to integrate statistical process control techniques with Automatic Process Control (APC) or engineering process control schemes to ensure continuous quality improvement. The data streams collected from an APC-controlled process have been shown to be critically dependent on the process settings. This paper proposes a univariate approach which combines the information provided by both the process output and input control actions based on a uniformly most powerful test. The proposed control chart can be customized to be sensitive to large or small shifts in a linearly-controlled process with an optimal efficiency in either transient or steady-state conditions. The presented numerical study is restricted to consider only APC-controlled stationary disturbances.     

Enhanced EWMA charts for monitoring the process coefficient of variation

The coefficient of variation (CV) is an important quality characteristic when the process variance is a function of the process mean for a production process. In this paper, we develop an auxiliary information–based (AIB) estimator for estimating the squared CV, along with its approximated mean and variance. This estimator is then used to devise new one-sided EWMA charts for monitoring the increases or decreases in the squared CV of a normal process, named the AIB-EWMA CV charts. In addition, the sensitivities of these control charts are also enhanced with the fast initial response feature. The Monte Carlo simulation method is used to compute the run length characteristics of the proposed CV charts. Based on detailed run length comparisons, it is found that the proposed AIB-EWMA CV charts are uniformly and substantially better than the existing EWMA CV charts when detecting different kinds of upward/downward shifts in the squared CV. The proposed charts are also applied to a real dataset to support the proposed theory.     

Validation of paper-based assay for rapid blood typing

We developed and validated a new paper-based assay for the detection of human blood type. Our method involves spotting a 3 μL blood sample on a paper surface where grouping antibodies have already been introduced. A thin film chromatograph tank was used to chromatographically elute the blood spot with 0.9% NaCl buffer for 10 min by capillary absorption. Agglutinated red blood cells (RBCs) were fixed on the paper substrate, resulting in a high optical density of the spot, with no visual trace in the buffer wicking path. Conversely, nonagglutinated RBCs could easily be eluted by the buffer and had low optical density of the spot and clearly visible trace of RBCs in the buffer wicking path. Different paper substrates had comparable ability to fix agglutinated blood, while a more porous substrate like Kleenex paper had enhanced ability to elute nonagglutinated blood. Using optimized conditions, a rapid assay for detection of blood groups was developed by spotting blood to antibodies absorbed to paper and eluted with 200 μL of 0.9% NaCl buffer directly by pipetting. RBCs fixation on paper accurately detected blood groups (ABO and RhD) using ascending buffer for 10 min or using a rapid elution step in 100/100 blood samples including 4 weak AB and 4 weak RhD samples. The assay has excellent reproducibility where the same blood group was obtained in 26 samples assessed in 2 different days. Agglutinated blood fixation on porous paper substrate provides a new, simple, and sensitive assay for rapid detection of blood group for point-of-care applications.     

Electrochemical characterization and in vivo biocompatibility of a thick-film printed sensor for continuous in vivo monitoring

This paper assessed the material biocompatibility and investigated the temporal modulation in electrochemical performance of printed thick-film electrochemical sensing devices (ESDs) that can serve as the basis of various enzymatic sensor in detecting an electrochemically potent species. The sensors were placed in phosphate buffered saline (PBS), human serum, or implanted subcutaneously in rats, free or in stainless steel cages. The exudate collection allowed the evaluation of inflammatory cell populations, up to 21 days. The ferrous/ferric redox electrode reactions were used to assess the electrode elements performance for up to 49 days. Following testing, scanning electron microscopy (SEM) evaluated cell surface adhesion, while fibrous capsules were examined by histology. It was determined that the exudates leukocyte concentration due to the presence of sensors was comparable to the empty cage controls. For the length of the study, the sensors functionality appeared not to be influenced by the in vivo environment, when tested ex vivo, without the surrounding fibrous capsule. Surface imaging (SEM) indicated temporal focal dissolution of the Ag/AgCl electrodes with no apparent local toxicity. We concluded that the ESDs were biocompatible and their ex vivo functionality was not lost when maintained in vivo for up to 49 days.     

Deviance residual-based control charts for monitoring the beta-distributed processes

Beta-distributed process outputs are common in manufacturing industry because they range from 0 to 1 based on inputs like yield. Under the normality assumption, Shewarts control charts and Hotelling's control charts based on the deviance residual have been applied to monitor the process mean of the beta-distributed process outputs. The normality assumption can be violated according to the shape of the beta distribution. Therefore, without the normality assumption, we propose antirank control charts, exponentially weighted moving average (EWMA) control charts and cumulative sum (CUSUM) control charts. The proposed control charts outperform the existing control charts in the experimental results. The previous research has been focused on monitoring the process mean only. For the first time, in order to monitor the process variance of the beta-distributed process outputs, we propose the multivariate exponentially weighted mean squared deviation (MEWMS) chart, the first norm distance of the MEWMS deviation from its expected value (MEWMSL₁) chart, the chart based on MEWMS deviation with the approximated distribution of trace (MEWMS_AT), the multivariate trace sum squared deviation (MTSSD) chart and the multivariate matrix sum squared deviation (MMSSD) chart based on the deviance residual. The proposed control charts are compared and recommended in terms of the experimental results. This research can be a guideline for practitioners who monitor the deviance residual.     

A note on GLR charts for monitoring count processes

Various generalized likelihood ratio (GLR) charts have been proposed to monitor count processes such as binomial, Bernoulli, Poisson, and multinomial processes. The advantages of GLR charts are that designing the chart is relatively easy, estimates of the process change‐point and shift size are available for post‐signal diagnosis, and they are effective in detecting a wide range of shifts in the process parameter. However, for some special cases of the observations, such as observing all defective items or all non‐defective items, the GLR chart statistic for monitoring a count process has been said to be undefined. We show that the GLR chart statistic is always well defined.     

The dynamic T 2 chart for monitoring feedback-controlled processes

As manufacturing quality has become a decisive factor in global market competition, statistical quality techniques such as Statistical Process Control (SPC) are widely used in industry. With advances in information, sensing, and data collection technology, large volumes of data are routinely available in processes employing Automatic Process Control (APC) and Engineering Process Control (EPC). Although there is a growing need for SPC monitoring in these feedback-controlled environments, an effective implementation scheme is still lacking. This research provides a monitoring method, termed the dynamic T1 chart that improves the detection of assignable causes in feedback-controlled processes.     

Generalized linear model based monitoring methods for high-yield processes

Emerge in technology brought well-organized manufacturing systems to produce high-quality items. Therefore, monitoring and control of products have become a challenging task for quality inspectors. From these highly efficient processes, produced items are mostly zero-defect and modeled based on zero-inflated distributions. The zero-inflated Poisson (ZIP) and zero-inflated Negative Binomial (ZINB) distributions are the most common distributions, used to model the high-yield and rare health-related processes. Therefore, data-based control charts under ZIP and ZINB distributions (i.e., Y-ZIP and Y-ZINB) are proposed for the monitoring of high-quality processes. Usually, with the defect counts, few covariates are also measured in the process, and the generalized linear model based on the ZIP and ZINB distributions are used to estimate their parameters. In this study, we have designed monitoring structures (i.e., PR-ZIP and PR-ZINB) based on the ZIP and ZINB regression models which will provide the monitoring of defect counts by accounting the single covariate. Further, proposed model-based charts are compared with the existing data-based charts. The simulation study is designed to access the performance of monitoring methods in terms of run length properties and a case study on the number of flight delays between Atlanta and Orlando during 2012–2014 is also provided to highlight the importance of the stated research.     

A modified CUSUM control chart for monitoring industrial processes

Control charts are the most popular tool of statistical process control for monitoring variety of processes. The detection ability of these control charts can be improved by introducing various transformations. In this study, we have enhanced the performance of CUSUM charts by introducing a link relative variable transformation technique. Link relative variable converts the original process variable in a form which is relative to its mean. So, the link relative represents the relative positioning of the observations. Average run length (ARL ) is used to compare our technique with the previous studies. The comparison shows the overall good detection performance of our scheme for a span of shifts in the mean. A real‐world example from the electrical engineering process is also included to demonstrate the application of proposed control chart.     

Mass spectrometry "sensor" for in vivo acetylcholine monitoring

Developing sensors for in vivo chemical monitoring is a daunting challenge. An alternative approach is to couple sampling methods with online analytical techniques; however, such approaches are generally hampered by lower temporal resolution and slow analysis. In this work, microdialysis sampling was coupled with segmented flow electrospray ionization mass spectrometry (ESI-MS) to perform in vivo chemical monitoring. The use of segmented flow to prevent Taylor dispersion of collected zones and rapid analysis with direct ESI-MS allowed 5 s temporal resolution to be achieved. The MS "sensor" was applied to monitor acetylcholine in the brain of live rats. The detection limit of 5 nM was sufficient to monitor basal acetylcholine as well as dynamic changes elicited by microinjection of neostigmine, an inhibitor of acetycholinesterase, that evoked rapid increases in acetycholine and tetrodotoxin, a blocker of Na(+) channels, that lowered the acetylcholine concentration. The versatility of the sensor was demonstrated by simultaneously monitoring metabolites and infused drugs.     

Calibration and evaluation of a transportable in vivo monitoring system for accident monitoring of internal contamination

A transportable in vivo monitoring system has been constructed and calibrated. The system uses two hyper pure germanium detectors--one for measuring whole body activities, by measuring activity in the torso, and the second for determining activities of radioiodine in the thyroid. The optimum counting geometries have been determined and the system has been calibrated for subjects of different ages and builds. The complete system is transported in two trailers which are pulled by ordinary motor vehicles. The minimum detectable activity (MDA) for 137Cs in whole body for a 10 min counting interval at the 95% confidence level is 200 Bq. The MDA for a count of 131I in thyroid is 20 Bq. The system is capable of detecting activities that are equivalent to a dose of 1 mSv for a wide range of radionuclides.