A novel immunoassay system and bioseparation process based on thermal phase separating polymers

Poly-N-isopropylacrylamide (polyNIPAAm), a water-soluble, thermally precipitating synthetic polymer, has been conjugated together with a monoclonal antibody (MAb) and utilized in a novel separation method for an immunoassay. The PolyMPAAm precipitates out of water above a critical temperature of 31°C, enabling a polymerbound immune complex to be separated from the solution. The principal advantages of this method are that it utilizes a homogeneous incubation for the antigen-antibody reaction, plus, it has the ability to assay large-molecular-weight antigens with sensitivities equivalent to other nonisotopic heterogeneous immunoassays. In addition, since the polymer-immune complex may be reversibly redissolved by cooling, the method may be used both to concentrate the signal and isolate the analyte. This general technique may also be used for a wide variety of separation processes in addition to immunoassays, in which a specific component in a biological fluid, industrial process stream, or body of water is to be isolated for analysis, recovery, or disposal. Thus, product recovery and/or toxin or pollutant removal processes are possible with this methodology.     

Large-scale 0–1 linear programming on distributed workstations

We present a methodology which uses a collection of workstations connected by an Ethernet network as a parallel processor for solving large-scale linear programming problems. On the largest problems we tested, linear and super-linear speedups have been achieved. Using the branch-and-cut approach of Hoffman, Padberg and Rinaldi, eight workstations connected in parallel solve problems from the test set documented in the Crowder, Johnson and Padberg 1983Operations Research article. Very inexpensive, networked workstations are now solving in minutes problems which were once considered not solvable in economically feasible times. In this peer-to-peer (as opposed to master-worker) implementation, interprocess communication was accomplished by using shared files and resource locks. Effective communication between processes was accomplished with a minimum of overhead (never more than 8% of total processing time). The implementation procedures and computational results will be presented.Supported in part by a grant from the Digital Equipment Corporation.Supported in part by grants from the Office of Naval Research and the National Science Foundation (ECS-8615438).     

Determination of the entropy-factor of the gold donor level in silicon by resistivity and DLTS measurements

The parameters of the gold donor level inp-type silicon are determined from the temperature dependence of the free carrier concentration and from DLTS measurements. The entropy-factorX _T is determined to be 20±2. In addition, the capture cross-section for holes _p =5.5×10^–15 cm² and the reaction enthalpyH _T –H _v =0.35eV for the exchange of holes between the gold donor level and the valence band are obtained in the present investigation.     

A superoxo-ferrous state in a reduced oxy-ferrous hemoprotein and model compounds

Cryoreduction of the [FeO2]6 (n = 6 is the number of electrons in 3d orbitals on Fe and pi* orbitals on O2) dioxygen-bound ferroheme through irradiation at 77 K generates an [FeO2]7 reduced oxy-heme. Numerous investigations have examined [FeO2]7 centers that have been characterized as peroxo-ferric centers, denoted [FeO2]per7, in which a ferriheme binds a dianionic peroxo-ligand. The generation of such an intermediate can be understood heuristically if the [FeO2]6 parent is viewed as a superoxo-ferric center and the injected electron localizes on the O-O moiety. We here report EPR/ENDOR experiments which show quite different properties for the [FeO2]7 centers produced by cryoreduction of monomeric oxy-hemoglobin (oxy-GMH3) from Glycera dibranchiata, which is unlike mammalian "globins" in having a leucine in place of the distal histidine; of frozen aprotic solutions of oxy-ferrous octaethyl porphyrin; and of the oxy-ferrous complex of the heme model, cyclidene. These [FeO2]7 centers are characterized as "superoxo-ferrous" centers ([FeO2]sup7), with nearly unit spin density localized on a superoxo moiety which is end-on coordinated to a low-spin ferrous ion. This assignment is based on their g tensors and 17O hyperfine couplings, which are characteristic of the superoxide ion coordinated to a diamagnetic metal ion, and on the absence of detectable ENDOR signals either from the in-plane 14N ligands or from an exchangeable H-bond proton. Such a center would arise if the electron that adds to the [FeO2]6 superoxo-ferric parent localizes on the Fe ion, to make a superoxo-ferrous moiety. Upon annealing to T > 150 K, the [FeO2]sup7 species converts to peroxo/hydroperoxo-ferric ([FeO2H]7) intermediates. These experiments suggest that the primary reduction product is [FeO2]sup7 and that the internal redox transition to [FeO2]per7/[FeO2H]7 states is driven at least in part by H-bonding/proton donation by the environment.     

Controlled release of 2‐heptanone using starch gel and polycaprolactone matrices and polymeric films

Varroa jacobsoni is a parasitic mite that is threatening the honeybee industry in many parts of the world. 2‐Heptanone, a natural product made by honeybees at low concentrations, is effective at elevated concentrations in controlling mite populations in honeybee colonies, especially when released over a 42 day period. An extrusion process was used to encapsulate 14, 18, and 25% 2‐heptanone in a polycaprolactone (PCL) matrix. Less than 18% of the 2‐heptanone was encapsulated in the PCL matrix. The high vapor permeability of PCL to 2‐heptanone resulted in a high flux rate and limited the usefulness of PCL as an encapsulation matrix for controlled‐release devices. A starch gel containing three times its weight in 2‐heptanone was prepared from starch‐based microcellular foam (MCF). The gel had compressive, tensile, and flexural strength values in the range of 0.56 to 1.9 MPa. 2‐Heptanone quickly evaporated from non‐laminated gels. However, when the gel was laminated with different polymeric films, a wide range of flux rates was obtained. The T₅₀ for gels laminated or coated with poly(vinyl alcohol) (PVAL, 99% hydrolyzed) and ethylene‐vinyl alcohol copolymer (EVAL) was 72 and 1030 days, respectively. The most promising film was a starch/glycerol film that released 50% of the 2‐heptanone (T₅₀) in approximately 13 days. Copyright © 2007 John Wiley & Sons, Ltd.     

Nano-scale templating using honeycomb-like poly(amidoamine-organosilicon) (PAMAMOS) dendrimer networks

Two different methods for preparation of Cu²⁺-poly(amidoamine-organosilicon) (PAMAMOS) dendrimer-based network nanocomplexes were developed and small angle neutron scattering (SANS) and X-ray photoelectron spectroscopy (XPS) were used for elucidation of fine structure of the obtained products. It was found that the in situ preparation method, by which less than the limiting amount of Cu²⁺ for the given dendrimer generation was complexed with dissolved dendrimer before cross-linking, enabled precise templating of copper into the nanoscopic polyamidoamine (PAMAM) network domains only. The limiting amount of Cu²⁺ for networks with generation 4 PAMAM domains was found to be between 5 (by SANS) and 8 (by XPS) mass %, in good agreement with the 9 mass % value calculated for idealized perfect dendrimer structure. It was also found that exceeding this concentration limit resulted in partitioning of Cu²⁺ ions into the organosilicon (OS) network domains as well, probably because of possible complexation of cations with siloxane oxygens. Consistent with this, the diffusion method always resulted in random distribution of Cu²⁺ cations throughout the bulk of the penetrated network layer(s), but the depth of this penetration was time-dependent and it followed Type II diffusion kinetics. This enables convenient control of layer thickness and preparation of thin layers of metal-network nanocomplexes. XPS data strongly suggest preferential Cu²⁺-tert. N complexation with in the PAMAM network domains, and indicate an unexpected oxido-reduction process above a certain copper concentration (approximately 2 tert. N per Cu ion) that leads to the formation of Cu¹⁺ and N⁺ species.     

H-bonding interactions and control of thiolate nucleophilicity and specificity in model complexes of zinc metalloproteins

It is shown in model complexes designed to mimic the binding site of zinc-thiolate proteins that a single hydrogen bond between an amide N-H and a Zn-coordinated thiolate reduces its reactivity toward electrophiles by up to 2 orders of magnitude. In addition, we show that a single N-H...S hydrogen bond is sufficient to achieve near 100% regiospecificity of reaction between a strong, and hence inherently indiscriminate, alkylating agent like trimethyloxonium tetraflouroborate and a single sulfur in a dithiolate construct. The importance of these results in understanding how systems such as the zinc fingers of the GATA family and the E. coli DNA repair protein Ada, which share the same pseudotetrahedral structure and tetracysteinyl ligation around the zinc, can fulfill such widely divergent (structural vs reactive) roles and how specificity of reaction in such multi-thiolate containing systems can be achieved is discussed.     

Determination of228Ra in drinking water

A procedure for the analysis of²²⁸Ra in drinking water has been developed. The procedure involves separation of radium by an initial coprecipitation with lead sulfate. The isolated Pb(Ra)SO₄ is then dissolved in sodium diethylenetriamine pentaacetate (DTPA). Radium-228 is co-precipitated from this solution with barium sulfate while the DTPA supernate which contains pre-existing²²⁸Ac is discarded. The purified Ba(Ra)SO₄ precipitate is then allowed to ingrow, generating²²⁸Ac, which is then dissolved in DTPA, isolating both²²⁶Ra and²²⁸Ra in the precipitate while²²⁸ Ac remains in the aqueous supernate. The supernate is partitioned against di-(2-ethylhexyl phosphoric acid), HDEHP, dissolved in n-heptane, which retains the²²⁸Ac. Actinium-228 is then stripped from the organic phase by partitioning against 1M HNO₃. Finally, the²²⁸Ac is coprecipitated onto cerium oxalate. The precipitate is collected on a filter and counted in a low-background beta counter. Radium-228 standards with concentrations ranging from 0.044 to 1.6 Bq were used to establish the detector counting efficiency for²²⁸Ac in cerium oxalate samples, as well as monitoring the chemical yield and absorption factors. The resultant average value of 30.3±2.1 cpm/Bq (uncertainty given at 95% level of confidence) was obtained. Various²²⁸Ra cross checks from U. S. Environmental Protection Agency (EPA) with concentrations of 0.063–0.52 Bq/l were analyzed in order to assess the performance of the procedure. The minimum detectable concentration (MDC) of²²⁸Ra in water with this procedure is 0.015 Bq/l. This is based on a one liter aliquot of sample, a 100 min couting period, and a 3 hour decay interval between the end of²²⁸Ac ingrowth and midpoint of counting. Decontamination factor studies were performed to determine the extent of the carry-over of²³⁸U,²²⁶Ra,²¹⁰Po, and⁹⁰Sr into the final fraction.