Laboratory Evaluation of Representative Disease State Assays on the Abbott ARCHITECT® i2000® Analyzer

We report here the results of our evaluation of representative disease state assays on the Abbott ARCHITECT® i2000® analyzer. The ARCHITECT i-series is a family of immunoassay analyzers under development by Abbott Laboratories (North Chicago, IL, USA). The first instrument available from this family is the i2000 analyzer. The i2000 analyzer is a random access, modular instrument with a maximum throughput of 200 tests per hour and a time to first result of 29 minutes. It is capable of running two step, one step, and automated pretreatment protocols. Modular instrument design allows multiple i2000 analyzers to be combined to form i4000®, i6000®, and i8000® analyzers with maximum throughputs of 400, 600, and 800 tests per hour, respectively. As either an individual module or multiple integrated modules, the instrument is run by one operator using a single system control center (SCC). The SCC has Windows NT based data management software that is touch screen driven. All assays utilize paramagnetic microparticles for analyte capture, and chemiluminescent detection based on a new class of acridinium compound (sulfonyl acridinium carboxamides) with improved aqueous solubility and stability. The i2000 analyzer has onboard, refrigerated storage for up to 25 reagent kits per module, and reagents are available for a wide range of assays. The purpose of our study was to perform a laboratory evaluation of representative thyroid (TSH, Free T4), fertility (Total β-hCG, FSH, LH, Prolactin, Testosterone) and metabolic (Ferritin) assays on the i2000 analyzer. Our evaluation of these assays included sensitivity, precision, and method comparison testing. In addition, we performed a preliminary evaluation of three i2000 tumor marker assays (Total PSA, Free PSA, and CEA) by comparing specimen correlation to other commercially available methods.     

Quantitative small molecule bioanalysis using chip-based nanoESI-MS/MS

A fully automated chip-based nanoelectrospray (nanoESI) system, NanoMate^® 100 (Advion BioSciences, Inc., Ithaca, NY), was evaluated for its application on quantitative bioanalysis of small molecules in support of exploratory pharmacokinetic (PK) studies. The NanoMate^® 100 was compared with the conventional autosampler coupled with liquid chromatography-electrospray (LC-ESI) interface. An API^® 3000 triple quadrupole mass spectrometer (Applied Biosystems, Inc., Foster City, CA) was used for the evaluation. The results show that the NanoMate^® 100 performs comparably to LC-ESI in terms of standard curve fitting, low limit of quantitation (LLOQ), dynamic range, accuracy, and precision. Parallel analyses of exploratory PK study samples show high correlation (R² = 0.971) between the NanoMate^® 100 and the LC-ESI. The NanoMate^® 100 exhibits advantages in carryover, sample consumption, sample cycle time, and the ability to be full automated. Despite these advantages, the necessarily rigorous sample preparation process limits the application of the NanoMate^® 100 for quantitative analysis in areas such as exploratory PK studies, which often involve multiple compounds in one study and require rapid turnaround. However, the NanoMate^® 100 has great potential in qualitative work (e.g., metabolite identification) as well as in high-throughput quantitative analysis of compound in the development stage (i.e., a single analyte with a well-established sample extraction method).     

Resolution-based lower bounds in MaxSAT

The lower bound (LB) implemented in branch and bound MaxSAT solvers is decisive for obtaining a competitive solver. In modern solvers like MaxSatz and MiniMaxSat, the LB relies on the cooperation of the underestimation and inference components. Actually, the underestimation component of some solvers guides the application of the inference component when a conflict is reached and certain structures are found. In this paper we analyze algorithmic and logical aspects of the underestimation components that have been implemented in MaxSatz during its evolution. From an algorithmic point of view, we define novel strategies for selecting unit clauses in UP (the underestimation of LB in UP is the number of independent inconsistent subformulas detected using unit propagation), the extension of UP with failed literal detection, and a clever heuristic for guiding the application of MaxSAT resolution when UP augmented with failed literal detection is applied in the presence of cycles structures. From a logical point of view, we prove that the inconsistent subformulas detected by UP are minimally unsatisfiable, but this property does not hold if failed literal detection is added. In the presence of cycle structures in conflicts detected by UP augmented with failed literal detection, we prove that the application of MaxSAT resolution produces smaller inconsistent subformulas and, besides, generates additional clauses that may be used to improve the LB. The conducted empirical investigation indicates that the LB techniques described in this paper lead to better quality LBs.     

Purifying genomic DNA from whole blood on automated, high-throughput, and moderate-throughput platforms

We describe three new automated methods for purifying genomic DNA from whole blood. The MagneSil^® Blood Genomic, Max Yield System uses MagneSil^® paramagnetic particles (PMPs) in a 96-well format to purify the maximal amount of DNA from a 200-μL blood sample. In contrast, the MagneSil^® ONE, Fixed Yield Blood Genomic System uses MagneSil^® Fixed Yield PMPs to purify a normalized amount of DNA from 60 μL of blood in a 96-well format. These methods are implemented on the Beckman Coulter Biomek^® FX automated workstation. The MagneSil^® KF Genomic System uses MagneSil^® PMPs to purify DNA from 1 to 15 samples of 200-μL blood using the moderate-throughput Thermo Electron KingFisher^® mL instrument.The MagneSil^® Blood Genomic System typically yields > 4 μg per 200 μL of whole blood, depending on the white blood cell content. The MagneSil^® ONE System is best suited where there is a requirement for purification of a narrow concentration range of DNA. This system purifies 1 μg (±50%) of DNA from 60 μL of blood. The MagneSil^® KF System purifies 2 to 6 μg of DNA from 200 μL of blood. DNA purified using all of these methods is suitable for PCR, STR, READIT^® SNP genotype analysis, and multiplexed PCR analysis.     

IQ technology: an automated, high-throughput screening assay for kinase, phosphatase, and protease targets

IQ^® Technology, a homogeneous, universal-detection platform, originally designed for high-throughput screening (HTS) of kinases and phosphatases, has now been applied to protease screening. Representative enzymes from the major classes of proteases have been assayed in the IQ^® format. Enzyme activity and compound inhibition data are presented for such enzymes as Trypsin, Matrix Metalloproteinase 3 (MMP-3) and Calpain 1. The technology has been tested in 96- to 384- to 1536-well microplate formats and is universally suited for automated screening. IQ^® Technology is a direct, noncompetitive assay that does not require antibodies or radioisotopes. Fluorophore-labeled peptides are used as enzyme substrates. Kinase or phosphatase activity is quantified by direct measurement of the phosphorylation state of the substrate. For protease activity, cleavage is quantified with a peptide substrate containing a phospho-residue distal to the fluorphore. Cleavage of the substrate liberates the fluorphore-labeled terminus from the terminus containing the phospho-residue. Protease activity is measured by the change in fluorescence intensity that occurs when a proprietary compound binds specifically to phosphoryl groups on peptides and quenches the fluorescence. IQ^® Technology can be used with any peptide sequence and is insensitive to high concentrations of ATP and substrate. The IQ^® Technology has been validated against a large number of detergents, organics, and other reagents found in reaction mixtures and has been optimized for HTS applications exhibiting representative Z' values of 0.7.     

Thin film characterization and vapor sensing properties of a novel perylenediimide material

A novel N,N′-(glycine tert-butylester)-3,4,9,10-perylenediimide was chosen for the study of Langmuir–Blodgett (LB) thin film characterization and the sensing properties against selected volatile organic vapors. Different number of LB film layer was deposited onto a glass and quartz crystal substrate. The thin film fabrication process was monitored with UV–vis and quartz crystal microbalance (QCM) measurement techniques. The results indicated that absorbance increased linearly with the number of the layers on film. A similar linear relationship between frequency shift and number of the layers was observed by the QCM measurement. It can be concluded that high quality and uniform LB films were produced by using this novel perylenediimide material. Chloroform, toluene, benzene, ethyl alcohol and isopropyl alcohol vapors were selected to test this material's applicability in room temperature as a vapor sensor. This novel material showed a fast, large and reproducible response to chloroform and isopropyl alcohol vapor.     

Total flow time minimization in a flowshop sequence-dependent group scheduling problem

We have developed a mathematical programming model for minimizing total flow time of the flow shop sequence dependent group scheduling (FSDGS) problem, typically classified as F_m|fmls, S_plk, prmu|∑C_j. As the problem is shown to be strongly NP-hard, a tabu search (TS) algorithm as well as a hybrid ant colony optimization (HACO) algorithm have been developed to heuristically solve the problem. A lower bounding (LB) method based on the Branch-and-Price algorithm is also developed to evaluate the quality of the metaheuristic algorithms. In order to compare the performance of metaheuristic algorithms, random test problems, ranging in size from small, medium, to large are created and solved by both the TS and the HACO algorithms. A comparison shows that the HACO algorithm has a better performance than the TS algorithm. The results of the heuristic algorithms are also compared with the results of the LB method to evaluate the quality of the solutions. The LB method presented in this paper can be generalized to solve the FSDGS problem with other objective functions.     

Heraeus cytomat® 6000 Series Incubators and Storage Systems from Kendro Laboratory Products

There are a number of cases in high throughput screening systems where a controlled environment is desired. These include incubation periods for cellbased assays, incubation for protein detection assays such as ELISA or fluorescence assays, and branching assays for mRNA detection. In addition, as the density of wells in microplates increases and well volumes become smaller, evaporation becomes a concern in all assays.The cytomat® 6000 is a robot accessible, automated CO₂ incubator used for cell-based High Throughput Screening systems. The incubator provides superior environmental conditions, due to the unique access door at the back of the instrument, the PlateShuttle. This small access opening insures that the environment inside the system (temperature, humidity and CO₂) is undisturbed as microplates are accessed.The system provides high speed, random (bar-coded) access to all microplate formats for 24, 96, 384 and 1536 well microplates (up to 261-microplate capacity). Other cytomat configurations offer refrigerated and low humidity environments.     

Label-free electrochemical immunosensor for sensitive detection of kanamycin

A novel label-free electrochemical immunosensor for sensitive detection of kanamycin based on water-soluble graphene sheet (WGS)/prussian blue-chitosan (PB-CTS)/nanoporous gold (NPG) composited film has been reported. PB was selected as an electron transfer mediator, and was modified onto the electrode together with WGS through electrostatic adsorption. Then NPG was immobilized onto the as-prepared film for biomolecules anchoring. The electroactivity of PB was greatly enhanced in the presence of WGS and NPG. It could mainly be ascribed to the fact that the good conductivity of WGS and NPG promoted electron transfer and enhanced the sensitivity. kanamycin antibody, as a model, was immobilized onto the composite film for the detection of kanamycin. Under optimum conditions, the amperometric signal of PB decreased linearly with kanamycin concentration (0.02-14 ng mL⁻¹), a linear calibration plot (y = 1.3817 + 4.7544x, r = 0.9993), resulting in a low limit of detection (6.31 pg mL⁻¹). The novel immunosensor for the detection of kanamycin in real sample with satisfactory results has been proved. In addition, this method would be easily adapted for the detection of other residual antibiotics in animal derived foods.     

Automated purification of dye terminator sequencing reactions: an approach to high-throughput capillary electrophoresis sequencing of large templates

Demands for higher quantity and quality of sequence data during genome sequencing projects have led to a need for completely automated reagent systems designed to isolate, process, and analyze DNA samples. While much attention has been given to methodologies aimed at increasing the throughput of sample preparation and reaction setup, purification of the products of sequencing reactions has received less scrutiny despite the profound influence that purification has on sequence quality. Commonly used and commercially available sequencing reaction cleanup methods are not optimal for purifying sequencing reactions generated from larger templates, including bacterial artificial chromosomes (BACs) and those generated by rolling circle amplification. Theoretically, these methods would not remove the original template since they only exclude small molecules and retain large molecules in the sample. If the large template remains in the purified sample, it could understandably interfere with electrokinetic injection and capillary performance. We demonstrate that the use of MagneSil^® paramagnetic particles (PMPs) to purify ABI PRISM^® BigDye^® sequencing reactions increases the quality and read length of sequences from large templates. The high-quality sequence data obtained by our procedure is independent of the size of template DNA used and can be completely automated on a variety of automated platforms.     

Redundant modeling in permutation weighted constraint satisfaction problems

In classical constraint satisfaction, redundant modeling has been shown effective in increasing constraint propagation and reducing search space for many problem instances. In this paper, we investigate, for the first time, how to benefit the same from redundant modeling in weighted constraint satisfaction problems (WCSPs), a common soft constraint framework for modeling optimization and over-constrained problems. Our work focuses on a popular and special class of problems, namely, permutation problems. First, we show how to automatically generate a redundant permutation WCSP model from an existing permutation WCSP using generalized model induction. We then uncover why naively combining mutually redundant permutation WCSPs by posting channeling constraints as hard constraints and relying on the standard node consistency (NC*) and arc consistency (AC*) algorithms would miss pruning opportunities, which are available even in a single model. Based on these observations, we suggest two approaches to handle the combined WCSP models. In our first approach, we propose m\text -NC_{\text c}^*m\text {-NC}_{\text c}^* and m\text -AC_{\text c}^*m\text {-AC}_{\text c}^* and their associated algorithms for effectively enforcing node and arc consistencies in a combined model with m sub-models. The two notions are strictly stronger than NC* and AC* respectively. While the first approach specifically refines NC* and AC* so as to apply to combined models, in our second approach, we propose a parameterized local consistency LB(m,Φ). The consistency can be instantiated with any local consistency Φ for single models and applied to a combined model with m sub-models. We also provide a simple algorithm to enforce LB(m,Φ). With the two suggested approaches, we demonstrate their applicabilities on several permutation problems in the experiments. Prototype implementations of our proposed algorithms confirm that applying 2\text -NC_{\text c}^*,  2\text -AC_{\text c}^*2\text {-NC}_{\text c}^*,\;2\text {-AC}_{\text c}^*, and LB(2,Φ) on combined models allow far more constraint propagation than applying the state-of-the-art AC*, FDAC*, and EDAC* algorithms on single models of hard benchmark problems.     

Mosquito: an accurate nanoliter dispensing technology

Mosquito^® from TTP LabTech Ltd. is an innovative nanoliter dispenser that combines the liquid transfer capability of a fixed head pipette with the elimination of cross-contamination, using disposable tips. For many applications required in genomics, proteomics and drug discovery, Mosquito can reduce assay cost by minimizing reagent and sample usage.     

Comparisons of titer estimation methods for multiplexed pneumococcal opsonophagocytic killing assay

Titer estimation is one of the major components of immunoassay and vaccine development. A multiplexed in vitro opsonization assay (MOPA) is widely accepted to quantitate Streptococcus pneumococcal antibodies to serotype-specific pneumococcal capsular polysaccharide. Titer estimation of vaccine based on OPA is one important component of standardization of OPA, and the selected statistical method is a factor influencing the accuracy and precision of titer estimation. We evaluated five titer estimation methods for pneumococcal OPA in terms of precision and accuracy using three data sets generated by specifically designed experiments with both an eight-dilution and an eleven-dilution design. The bootstrap resampling technique was also used to determine the performance of the estimation. We concluded that the traditional direct method did not perform as well as the other four methods in terms of precision and accuracy of titer estimation. The Spearman–Kärber estimator might be biased upward for OPA titer estimation. The four-parameter logistic model (4PL) method is an alternative choice for OPA titer estimation. The eleven-dilution design provided more information than the eight-dilution design for titer estimation and enhanced precision of estimators. UAB opsotiter, computer software using the statistical language R and Microsoft Excel^®, was developed to implement OPA titer estimation.     

FactSage thermochemical software and databases — recent developments

FactSage^® was introduced in 2001 as the fusion of the F*A*C*T/FACT-Win and ChemSage thermochemical packages. The FactSage package runs on a PC operating under Microsoft Windows^® and consists of a series of information, database, calculation and manipulation modules that enable one to access and manipulate pure substances and solution databases. With the various modules one can perform a wide variety of thermochemical calculations and generate tables, graphs and figures of interest to chemical and physical metallurgists, chemical engineers, corrosion engineers, inorganic chemists, geochemists, ceramists, electrochemists, environmentalists, etc. This paper presents a summary of the recent developments in the FactSage thermochemical software and databases. In the article, emphasis is placed on the new databases and the calculation and manipulation of phase diagrams and complex phase equilibria.     

Environmentally friendly disposable sensors with microfabricated on-chip planar bismuth electrode for in situ heavy metal ions measurement

This paper presents an environmentally friendly disposable heavy metal ion sensor for in situ and online monitoring in the nature and physiological systems. The miniaturized sensor chip consists of a non-toxic microfabricated bismuth (Bi) working electrode that replaces the conventional mercury electrodes, an integrated Ag/AgCl reference electrode, a gold counter electrode, and microfluidic channels. In this work, the electrochemical behavior of the Bi working electrode was characterized in several non-deaerated buffer solutions using cyclic voltammetry. The detection and quantification of Pb (II) and Cd (II) were statically performed using anodic stripping voltammetry inside the microchannels, in the Pb (II) concentration range of 25–400 ppb (R² = 0.991) with limit of detection of 8 ppb for 60 s deposition, and in the Cd (II) concentration range of 28–280 ppb (R² = 0.986) with limit of detection of 9.3 ppb for 90 s deposition. Particularly, the applications of this sensor chip have been reported with the examples of in situ measurement of Cd (II) concentration in soil pore and ground water and online direct measurement of Cd (II) concentration in cell culture media in its native environment.     

Prediction and optimization of unsteady forced convection around a rounded cornered square cylinder in the range of Re

An unsteady two-dimensional laminar forced convection heat transfer around a square cylinder with the rounded corner edge is numerically investigated for Re = 80–180 and non-dimensional corner radius, r = 0.50–0.71 at Pr = 0.71 (Air). A structured non-uniform mesh is used for the computational domain discretization, and the finite-volume-method-based commercial code FLUENT is used for numerical simulation. The heat transfer characteristics over the rounded corner square cylinder are analyzed with average Nusselt number (Nu _avg) at various Re and various corner radii. The heat transfer characteristic is predicted by gene expression programming (GEP), and the GEP-generated explicit equation of Nu _avg is utilized in particle swarm optimization to optimize the corner radii for maximum heat transfer rate. The data required for the training the GEP model have been collected from the authors’ recent published article (Neural Comput Appl, 2015. doi:10.1007/s00521-015-2023-8). It is found that the heat transfer rate of a circular cylinder can be enhanced 12 % by introducing a new cylinder geometry of corner radius r = 0.51.

     

Automatic detection of Salmonella enterica in sprout irrigation water using a nucleic acid sensor

A nucleic acid sensor capable of automated sample and reagent loading, real-time PCR, automated detection, and sample line cleaning was tested. Real-time PCR reactions were performed with Salmonella enterica in autoclaved and spent alfalfa sprout irrigation water. S. enterica boiled cells were detected over a range of approximately 10⁴ to 10⁸ CFU/reaction (rxn). It was possible to generate enough PCR product to visualize a band on a gel at the expected size over approximately five orders of magnitude from 3.2 × 10³ to 10⁸ CFU/rxn. Automated detection experiments yielded correct identification of 9/9 positive control reactions over a range of 10⁴ to 10⁸ CFU/rxn, correctly identified a negative control reaction, and a sample of 3.2 × 10³ CFU/rxn was incorrectly identified as negative. Primer dimers were not seen in positive or negative control reactions with sprout irrigation water, suggesting that it may be possible to improve the detection limit simply by increasing the number of thermal cycles or by lowering the annealing temperature. The system required no interpretation of real-time PCR data by the operator. The entire process of loading, running the PCR, automated data interpretation, and sample line cleaning was completed in under 2 h and 20 min, significantly faster than it would take to ship a sample and have it tested by an independent laboratory.     

A label-free amperometric immunosensor based on horseradish peroxidase functionalized carbon nanotubes and bilayer gold nanoparticles

In this work, a novel label-free amperometric immunosensor has been constructed for detecting α-1-fetoprotein (AFP) based on nanocomposite of horseradish peroxidase (HRP) labeled carbon nanotubes (CNTs). First, the gold nanoparticles (AuNPs) were electrodeposited on the surface of the glass carbon electrode by electrochemical reduction of gold chloride tetrahydrate (HAuCl₄) to immobilize horseradish peroxidase labeled carbon nanotubes (HRP-CNTs). Then HRP-CNTs bioconjugate was immobilized on the surface of the electrodeposited AuNPs layer by the combination of forces (coordination and electrostatic force). Subsequently, it was immersed into gold colloidal nanoparticles (GNPs) solution, which was used to immobilize antibody biomolecules (anti-AFP). Enhanced sensitivity was obtained by using bioconjugates featuring HRP labeled (HRP-CNTs), which had lager specific surface area and good electronic catalysis (current response signal) compared to carbon nanotubes. Under optimized conditions, the linear ranges were from 0.2 to 200 ng mL⁻¹ with a detection limit of 0.067 ng mL⁻¹ (at an S/N of 3). The proposed immunosenor showed good precision, acceptable stability and reproducibility and could be used for the detection AFP in normal human serum, which provided a potential alternative tool for the detection of protein in clinical diagnosis.     

An All-solid-state Cd-selective electrode with a low detection limit

A new all-solid-state Cd²⁺-selective electrode with a low detection limit was prepared by using conjugated thiophene oligomer α-sexithiophene (α-6T) as solid contact deposited between an ionophore-doped poly(vinyl chloride) membrane and a gold disc substrate. The electrode exhibited a Nernstian response for Cd²⁺ ions over a wide concentration range of 10⁻³-10⁻⁷ M with a detection limit as low as 1.3 × 10⁻⁸ M. Results showed that the fabricated potentiometric sensor was suitable for use within the pH range of 2.0-9.0 and exhibited good reproducibility for long-term measurements.