Multifunctional polyether architectures : versatile materials for surface attachment and functionalization

Chapter 1 of this thesis comprises a review of polyether polyamines, i.e., combinations of polyether scaffolds with polymers bearing multiple amino moieties. Focus is laid on controlled or living polymerization methods. Furthermore, fields in which the combination of cationic, complexing, and pH-sensitive properties of the polyamines and biocompatibility and water-solubility of polyethers promise enormous potential are presented. Applications include stimuli-responsive polymers with a lower critical solution temperature (LCST) and/or the ability to gel, preparation of shell cross-linked (SCL) micelles, gene transfection, and surface functionalization.rnIn Chapter 2, multiaminofunctional polyethers relying on the class of glycidyl amine comonomers for anionic ring-opening polymerization (AROP) are presented. In Chapter 2.1, N,N-diethyl glycidyl amine (DEGA) is introduced for copolymerization with ethylene oxide (EO). Copolymer microstructure is assessed using online 1H NMR kinetics, 13C NMR triad sequence analysis, and differential scanning calorimetry (DSC). The concurrent copolymerization of EO and DEGA is found to result in macromolecules with a gradient structure. The LCSTs of the resulting copolymers can be tailored by adjusting DEGA fraction or pH value of the environment. Quaternization of the amino moieties by methylation results in polyelectrolytes. Block copolymers are used for PEGylated gold nanoparticle formation. Chapter 2.2 deals with a glycidyl amine monomer with a removable protecting group at the amino moiety, for liberation of primary amines at the polyether backbone, which is N,N-diallyl glycidyl amine (DAGA). Its allyl groups are able to withstand the harsh basic conditions of AROP, but can be cleaved homogeneously after polymerization. Gradient as well as block copolymers poly(ethylene glycol)-PDAGA (PEG-PDAGA) are obtained. They are analyzed regarding their microstructure, LCST behavior, and cleavage of the protecting groups. rnChapter 3 describes applications of multi(amino)functional polyethers for functionalization of inorganic surfaces. In Chapter 3.1, they are combined with an acetal-protected catechol initiator, leading to well-defined PEG and heteromultifunctional PEG analogues. After deprotection, multifunctional PEG ligands capable of attaching to a variety of metal oxide surfaces are obtained. In a cooperative project with the Department of Inorganic and Analytical Chemistry, JGU Mainz, their potential is demonstrated on MnO nanoparticles, which are promising candidates as T1 contrast agents in magnetic resonance imaging. The MnO nanoparticles are solubilized in aqueous solution upon ligand exchange. In Chapter 3.2, a concept for passivation and functionalization of glass surfaces towards gold nanorods is developed. Quaternized mPEG-b-PqDEGA diblock copolymers are attached to negatively charged glass surfaces via the cationic PqDEGA blocks. The PEG blocks are able to suppress gold nanorod adsorption on the glass in the flow cell, analyzed by dark field microscopy.rnChapter 4 highlights a straightforward approach to poly(ethylene glycol) macrocycles. Starting from commercially available bishydroxy-PEG, cyclic polymers are available by perallylation and ring-closing metathesis in presence of Grubbs’ catalyst. Purification of cyclic PEG is carried out using α-cyclodextrin. This cyclic sugar derivative forms inclusion complexes with remaining unreacted linear PEG in aqueous solution. Simple filtration leads to pure macrocycles, as evidenced by SEC and MALDI-ToF mass spectrometry. Cyclic polymers from biocompatible precursors are interesting materials regarding their increased blood circulation time compared to their linear counterparts.rnIn the Appendix, A.1, a study of the temperature-dependent water-solubility of polyether copolymers is presented. Macroscopic cloud points, determined by turbidimetry, are compared with microscopic aggregation phenomena, monitored by continuous wave electron paramagnetic resonance (CW EPR) spectroscopy in presence of the amphiphilic spin probe and model drug (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO). These thermoresponsive polymers are promising candidates for molecular transport applications. The same techniques are applied in Chapter A.2 to explore the pH-dependence of the cloud points of PEG-PDEGA copolymers in further detail. It is shown that the introduction of amino moieties at the PEG backbone allows for precise manipulation of complex phase transition modes. In Chapter A.3, multi-hydroxyfunctional polysilanes are presented. They are obtained via copolymerization of the acetal-protected dichloro(isopropylidene glyceryl propyl ether)methylsilane monomer. The hydroxyl groups are liberated through acidic work-up, yielding versatile access to new multifunctional polysilanes.

Steady-state function of the ubiquitous mammalian Na/H exchanger (NHE1) in relation to dimer coupling models with 2Na/2H stoichiometry

We describe the steady-state function of the ubiquitous mammalian Na/H exchanger (NHE)1 isoform in voltage-clamped Chinese hamster ovary cells, as well as other cells, using oscillating pH-sensitive microelectrodes to quantify proton fluxes via extracellular pH gradients. Giant excised patches could not be used as gigaseal formation disrupts NHE activity within the patch. We first analyzed forward transport at an extracellular pH of 8.2 with no cytoplasmic Na (i.e., nearly zero-trans). The extracellular Na concentration dependence is sigmoidal at a cytoplasmic pH of 6.8 with a Hill coefficient of 1.8. In contrast, at a cytoplasmic pH of 6.0, the Hill coefficient is <1, and Na dependence often appears biphasic. Results are similar for mouse skin fibroblasts and for an opossum kidney cell line that expresses the NHE3 isoform, whereas NHE1(-/-) skin fibroblasts generate no proton fluxes in equivalent experiments. As proton flux is decreased by increasing cytoplasmic pH, the half-maximal concentration (K(1/2)) of extracellular Na decreases less than expected for simple consecutive ion exchange models. The K(1/2) for cytoplasmic protons decreases with increasing extracellular Na, opposite to predictions of consecutive exchange models. For reverse transport, which is robust at a cytoplasmic pH of 7.6, the K(1/2) for extracellular protons decreases only a factor of 0.4 when maximal activity is decreased fivefold by reducing cytoplasmic Na. With 140 mM of extracellular Na and no cytoplasmic Na, the K(1/2) for cytoplasmic protons is 50 nM (pH 7.3; Hill coefficient, 1.5), and activity decreases only 25% with extracellular acidification from 8.5 to 7.2. Most data can be reconstructed with two very different coupled dimer models. In one model, monomers operate independently at low cytoplasmic pH but couple to translocate two ions in "parallel" at alkaline pH. In the second "serial" model, each monomer transports two ions, and translocation by one monomer allosterically promotes translocation by the paired monomer in opposite direction. We conclude that a large fraction of mammalian Na/H activity may occur with a 2Na/2H stoichiometry.

Development and Characterization of an in vitro Four-species Anaerobic Dental Biofilm Model

Dental caries is the most common chronic disease worldwide. It is characterized by the demineralization of tooth enamel caused by acid produced by cariogenic dental bacteria growing on tooth surfaces, termed bacterial biofilms. Cariogenesis is a complex biological process that is influence by multiple factors and is not attributed to a sole causative agent. Instead, caries is associated with multispecies microbial biofilm communities composed of some bacterial species that directly influence the development of a caries lesion and other species that are seemingly benign but must contribute to the community in an uncharacterized way. Clinical analysis of dental caries and its microbial populations is challenging due to many factors including low sensitivity of clinical measurement tools, variability in saliva chemistry, and variation in the microbiota. Our laboratory has developed an in vitro anaerobic biofilm model for dental carries to facilitate both clinical and basic research-based analyses of the multispecies dynamics and individual factors that contribute to cariogenicity. The rational for development of this system was to improve upon the current models that lack key elements. This model places an emphasis on physiological relevance and ease of maintenance and reproducibility. The uniqueness of the model is based on integrating four critical elements: 1) a biofilm community composed of four distinct and representative species typically associated with dental caries, 2) a semi-defined synthetic growth medium designed to mimic saliva, 3) physiologically relevant biofilm growth substrates, and 4) a novel biofilm reactor device designed to facilitate the maintenance and analysis. Specifically, human tooth sections or hydroxyapatite discs embedded into poly(methyl methacrylate) (PMMA) discs are incubated for an initial 24 hr in a static inverted removable substrate (SIRS) biofilm reactor at 37°C under anaerobic conditions in artificial saliva (CAMM) without sucrose in the presence of 1 X 106 cells/ml of each Actinomyces odontolyticus, Fusobacterium nucleatum, Streptococcus mutans, and Veillonella dispar. During days 2 and 3 the samples are maintained continually in CAMM with various exposures to 0.2% sucrose; all of the discs are transferred into fresh medium every 24 hr. To validate that this model is an appropriate in vitro representation of a caries-associated multispecies biofilm, research aims were designed to test the following overarching hypothesis: an in vitro anaerobic biofilm composed of four species (S. mutans, V. dispar, A. odontolyticus, and F. nucleatum) will form a stable biofilm with a community profile that changes in response to environmental conditions and exhibits a cariogenic potential. For these experiments the biofilms as described above were exposed on days 2 and 3 to either CAMM lacking sucrose (no sucrose), CAMM with 0.2% sucrose (constant sucrose), or were transferred twice a day for 1 hr each time into 0.2% sucrose (intermittent sucrose). Four types of analysis were performed: 1) fluorescence microscopy of biofilms stained with Syto 9 and hexidium idodine to determine the biofilm architecture, 2) quantitative PCR (qPCR) to determine the cell number of each species per cm2, 3) vertical scanning interferometry (VSI) to determine the cariogenic potential of the biofilms, and 4) tomographic pH imaging using radiometric fluorescence microscopy after exposure to pH sensitive nanoparticles to measure the micro-environmental pH. The qualitative and quantitative results reveal the expected dynamics of the community profile when exposed to different sucrose conditions and the cariogenic potential of this in vitro four-species anaerobic biofilm model, thus confirming its usefulness for future analysis of primary and secondary dental caries.

Seawater carbonate chemistry and boron incorporation of planktic foraminifer Orbulina universa during experiments, 2011

Culture experiments with living planktic foraminifers reveal that the ratio of boron to calcium (B/Ca) in Orbulina universa increases from 56 to 92 µmol mol-1 when pH is raised from 7.61 +/- 0.02 to 8.67 +/- 0.03 (total scale). Across this pH range, the abundances of carbonate, bicarbonate, and borate ions also change (+ 530, - 500, and + 170 µmol kg-1, respectively). Thus specific carbonate system control(s) on B/Ca remain unclear, complicating interpretation of paleorecords. B/Ca in cultured O. universa also increases with salinity (55-72 µmol mol-1 from 29.9-35.4 per mil) and seawater boron concentration (62-899 µmol mol-1 from 4-40 ppm B), suggesting that these parameters may need to be taken into account for paleorecords spanning large salinity changes (~ 2 per mil) and for samples grown in seawater whose boron concentration ([B]SW) differs from modern by more than 0.25 ppm. While our results are consistent with the predominant incorporation of the charged borate species B(OH)4 into foraminiferal calcite, the behavior of the partition coefficient KD (defined as [B/Ca]calcite/B(OH)4/HCO3seawater) cannot be explained by borate incorporation alone, and suggests the involvement of other pH-sensitive ions such as CO3 For a given increase in seawater B(OH)4, the corresponding increase in B/Ca is stronger when B(OH)4 is raised by increasing [B]SW than when it is raised by increasing pH. These results suggest that B incorporation controls should be reconsidered. Additional insight is gained from laser-ablation ICP-MS profiles, which reveal variable B/Ca distributions within individual shells.

Multifunctional core–shell Co–SiO2 nanowires via electrodeposition and sol–gel techniques

In this work, we propose a new strategy for the synthesis of multifunctional nanowires using a combination of sol–gel and electrodeposition techniques, based on a two-step procedure. First of all, nanotubes of SiO2 are synthesized via a sol–gel technique using polycarbonate membranes as templates. Homogenous nanotubes are obtained after centrifugation and thermal annealing. Afterwards, a ferromagnetic cobalt core is grown using potentiostatic electrodeposition. Finally, the core–shell Co–SiO2 nanowires are released by dissolving the template using wet-etching. These nanodevices can be used for many detection and sensing purposes. As a proof of concept, we have developed a pH nanosensor by including a pH-sensitive organic dye in the SiO2 shell. The sensing principle is based on the optical response of the organic dye towards pH when added to a solution. The magnetic core allows the recovery of the nanosensors after use. These nanowires can therefore be used as recoverable pH nanosensors. By changing the dye molecule to another molecule or receptor, the procedure described in the paper can be used to synthesize nanodevices for many different applications.

Two hemoglobin genes in Arabidopsis thaliana: The evolutionary origins of leghemoglobins

We cloned two hemoglobin genes from Arabidopsis thaliana. One gene, AHB1, is related in sequence to the family of nonsymbiotic hemoglobin genes previously identified in a number of plant species (class 1). The second hemoglobin gene, AHB2, represents a class of nonsymbiotic hemoglobin (class 2) related in sequence to the symbiotic hemoglobin genes of legumes and Casuarina. The properties of these two hemoglobins suggest that the two families of nonsymbiotic hemoglobins may differ in function from each other and from the symbiotic hemoglobins. AHB1 is induced, in both roots and rosette leaves, by low oxygen levels. Recombinant AHB1 has an oxygen affinity so high as to make it unlikely to function as an oxygen transporter. AHB2 is expressed at a low level in rosette leaves and is low temperature-inducible. AHB2 protein has a lower affinity for oxygen than AHB1 but is similar to AHB1 in having an unusually low, pH-sensitive oxygen off-rate.

Identification of the proton pathway in bacterial reaction centers: Both protons associated with reduction of QB to QBH2 share a common entry point

The reaction center from Rhodobacter sphaeroides uses light energy for the reduction and protonation of a quinone molecule, QB. This process involves the transfer of two protons from the aqueous solution to the protein-bound QB molecule. The second proton, H+(2), is supplied to QB by Glu-L212, an internal residue protonated in response to formation of QA− and QB−. In this work, the pathway for H+(2) to Glu-L212 was studied by measuring the effects of divalent metal ion binding on the protonation of Glu-L212, which was assayed by two types of processes. One was proton uptake from solution after the one-electron reduction of QA (DQA→D+QA−) and QB (DQB→D+QB−), studied by using pH-sensitive dyes. The other was the electron transfer kAB(1) (QA−QB→QAQB−). At pH 8.5, binding of Zn2+, Cd2+, or Ni2+ reduced the rates of proton uptake upon QA− and QB− formation as well as kAB(1) by ≈an order of magnitude, resulting in similar final values, indicating that there is a common rate-limiting step. Because D+QA− is formed 105-fold faster than the induced proton uptake, the observed rate decrease must be caused by an inhibition of the proton transfer. The Glu-L212→Gln mutant reaction centers displayed greatly reduced amplitudes of proton uptake and exhibited no changes in rates of proton uptake or electron transfer upon Zn2+ binding. Therefore, metal binding specifically decreased the rate of proton transfer to Glu-L212, because the observed rates were decreased only when proton uptake by Glu-L212 was required. The entry point for the second proton H+(2) was thus identified to be the same as for the first proton H+(1), close to the metal binding region Asp-H124, His-H126, and His-H128.

Structural activity of a cloned potassium channel (ROMK1) monitored with the atomic force microscope: The “molecular-sandwich” technique

The atomic force microscope (AFM) was used to continuously follow height changes of individual protein molecules exposed to physiological stimuli. A AFM tip was coated with ROMK1 (a cloned renal epithelial potassium channel known to be highly pH sensitive) and lowered onto atomically flat mica surface until the protein was sandwiched between AFM tip and mica. Because the AFM tip was an integral part of a highly flexible cantilever, any structural alterations of the sandwiched molecule were transmitted to the cantilever. This resulted in a distortion of the cantilever that was monitored by means of a laser beam. With this system it was possible to resolve vertical height changes in the ROMK1 protein of ≥0.2 nm (approximately 5% of the molecule’s height) with a time resolution of ≥1 msec. When bathed in electrolyte solution that contained the catalytic subunit of protein kinase A and 0.1 mM ATP (conditions that activate the native ion channel), we found stochastically occurring height fluctuations in the ROMK1 molecule. These changes in height were pH-dependent, being greatest at pH 7.6, and lowering the pH (either by titration or by the application of CO2) reduced their magnitude. The data show that overall changes in shape of proteins occur stochastically and increase in size and frequency when the proteins are active. This AFM “molecular-sandwich” technique, called MOST, measures structural activity of proteins in real time and could prove useful for studies on the relationship between structure and function of proteins at the molecular level.

Biodegradable polyanhydrides as drug delivery systems

Polyanhydrides are useful biodegradable vehicles for controlled drug delivery. In aqueous media the breaking of the anhydride bonds resulting in gradually polymer fragments collapse and release drugs in a controlled manner. In this study, two new biodegradable polyanhydrides copolymers were synthesised using a melt-polycondensation method. The first is poly (bis (p-carboxyphenoxy)-2-butene-co-sebacic acid) (CP2B: SA), which has double bonds along the polymer backbone. The second is crosslinked poly (glutamic acid-sebacic acid-co-sebacic acid) (GluSA: SA), where the conjugated unit of glutamic acid with sebacic acid (glutamic acid-SA) acted as a crosslinking fragment in producing the crosslinking polymer. The two polymers were applied to preparation of microspheres with bovine serum albumin (BSA) as a model protein, using both double emulsion solvent evaporation and spray drying methods. The characterisation of the microspheres, morphology, particle size, and drug loading, was studied. The in vitro hydrolytic degradation of polymers and blank microspheres was monitored using IR, GPC, and DSC. In vitro drug release behaviour was also studied. Though the studies showed cleavages of anhydride bonds occurred rapidly (<5 days), bulks of the polymer microspheres could be observed after a few weeks to a month; and only around 10-35% of the protein was detectable in a four-week period in vitro. We found the pH of the medium exerts a large impact on the release of the protein from the microspheres. The higher the pH, the faster the release. Therefore the release of the protein from the polyanhydride microspheres was pH-sensitive due mainly to the dissolution of monomers from the microspheres.

Bacteria growth, enzyme activities and TEP during KOSMOS 2011 mesocosm experiment in the Raunefjord

Marine bacteria are the main consumers of freshly produced organic matter. Many enzymatic processes involved in the bacterial digestion of organic compounds were shown to be pH sensitive in previous studies. Due to the continuous rise in atmospheric CO2 concentration, seawater pH is presently decreasing at a rate unprecedented during the last 300 million years but the consequences for microbial physiology, organic matter cycling and marine biogeochemistry are still unresolved. We studied the effects of elevated seawater pCO2 on a natural plankton community during a large-scale mesocosm study in a Norwegian fjord. Nine Kiel Off-Shore Mesocosms for Future Ocean Simulations (KOSMOS) were adjusted to different pCO2 levels ranging initially from ca. 280 to 3000 µatm and sampled every second day for 34 days. The first phytoplankton bloom developed around day 5. On day 14, inorganic nutrients were added to the enclosed, nutrient-poor waters to stimulate a second phytoplankton bloom, which occurred around day 20. Our results indicate that marine bacteria benefit directly and indirectly from decreasing seawater pH. During the first phytoplankton bloom, 5-10% more transparent exopolymer particles were formed in the high pCO2 mesocosms. Simultaneously, the efficiency of the protein-degrading enzyme leucine aminopeptidase increased with decreasing pH resulting in up to three times higher values in the highest pCO2/lowest pH mesocosm compared to the controls. In general, total and cell-specific aminopeptidase activities were elevated under low pH conditions. The combination of enhanced enzymatic hydrolysis of organic matter and increased availability of gel particles as substrate supported up to 28% higher bacterial abundance in the high pCO2 treatments. We conclude that ocean acidification has the potential to stimulate the bacterial community and facilitate the microbial recycling of freshly produced organic matter, thus strengthening the role of the microbial loop in the surface ocean.

Extruded colour-based plastic film for the measurement of dissolved CO2

Dissolved CO2 measurements are usually made using a Severinghaus electrode, which is bulky and can suffer from electrical interference. In contrast, optical sensors for gaseous CO2, whilst not suffering these problems, are mainly used for making gaseous (not dissolved) CO2 measurements, due to dye leaching and protonation, especially at high ionic strengths (>0.01 M) and acidity (<pH 4). This is usually prevented by coating the sensor with a gas-permeable, but ion-impermeable, membrane (GPM). Herein, we introduce a highly sensitive, colourimetric-based, plastic film sensor for the measurement of both gaseous and dissolved CO2, in which a pH-sensitive dye, thymol blue (TB) is coated onto particles of hydrophilic silica to create a CO2-sensitive, TB-based pigment, which is then extruded into low density polyethylene (LDPE) to create a GPM-free, i.e. naked, TB plastic sensor film for gaseous and dissolved CO2 measurements. When used for making dissolved CO2 measurements, the hydrophobic nature of the LDPE renders the film: (i) indifferent to ionic strength, (ii) highly resistant to acid attack and (iii) stable when stored under ambient (dark) conditions for >8 months, with no loss of colour or function. Here, the performance of the TB plastic film is primarily assessed as a dissolved CO2 sensor in highly saline (3.5 wt%) water. The TB film is blue in the absence of CO2 and yellow in its presence, exhibiting 50% transition in its colour at ca. 0.18% CO2. This new type of CO2 sensor has great potential in the monitoring of CO2 levels in the hydrosphere, as well as elsewhere, e.g. food packaging and possibly patient monitoring.

Investigation of properties affecting controlled release of macromolecules from PLGA microspheres

Poly(lactide-co-glycolide), or PLGA, microspheres offer a widely-studied biodegradable option for controlled release of therapeutics. An array of fabrication methodologies have been developed to produce these microspheres with the capacity to encapsulate therapeutics of various types; and produce microspheres of a wide range of sizes for different methods of delivery. The encapsulation, stability, and release profiles of therapeutic release based on physical and thermodynamic properties has also been studied and modeled to an extent. Much research has been devoted to tailoring formulations for improved therapeutic encapsulation and stability as well as selective release profiles. Despite the breadth of available research on PLGA microspheres, further analysis of fundamental principles regarding the microsphere degradation, formation, and therapeutic encapsulation is necessary. This work aims to examine additional fundamental principles related to PLGA microsphere formation and degradation from solvent-evaporation of preformed polymer. In particular, mapping the development of the acidic microenvironment inside the microsphere during degradation and erosion is discussed. Also, the effect of macromolecule size and conformation is examined with respect to microsphere diameter and PLGA molecular weight. Lastly, the effects of mechanical shearing and protein exposure to aqueous media during microsphere formation are examined. In an effort to better understand the acidic microenvironment development across the microsphere diameter, pH sensitive dye conjugated to protein that undergoes conformational change at different acidic pH values was encapsulated in PLGA microspheres of diameters ranging from 40 µm to 80 µm, and used in conjunction with fluorescence resonance energy transfer to measure the radial pH change in the microspheres. Qualitative analysis of confocal micrographs was used to correlate fluorescence intensity with pH value, and obtain the radial pH across the center of the microsphere. Therapeutic encapsulation and release from polymeric microspheres is governed by an interconnected variety of factors, including the therapeutic itself. The globular protein bovine serum albumin, and the elongated and significantly smaller enzyme, lysozyme, were encapsulated in PLGA microspheres ranging from 40 µm to 80 µm in diameter. The initial surface morphology upon microsphere formation, release profiles, and microsphere erosion characteristics were explored in an effort to better understand the effect of protein size, conformation, and known PLGA interaction on the formation and degradation of PLGA microspheres and macromolecule release, with respect to PLGA molecular weight and microsphere diameter. In addition to PLGA behavior and macromolecule behavior, the effect of mechanical stresses during fabrication was examined. Two similar solvent extraction techniques were compared for the fabrication of albumin loaded microspheres. In particular, the homogeneity of the microspheres as well as capacity to retain encapsulated albumin were compared. This preliminary study paves the way for a more rigorous treatment of the effect of mechanical forces present in popular microsphere fabrication. Several factors affecting protein release from PLGA microspheres are examined herein. The technique explored for spatial resolution of the pH inside the microsphere proved mildly effective in producing a reliable method of mapping microsphere pH changes. However, notable trends with respect to microsphere size, PLGA molecular weight, and microsphere porosity were observed. Proposed methods of improving spatial resolution of the acidic microenvironment are also provided. With respect to microsphere formation, studies showed that albumin and lysozyme had little effect on the internal homogeneity of the microsphere. Rather, ionic interactions with PLGA played a more significant role in the encapsulation and release of each macromolecule. Studies also showed that higher instances of mechanical stress led to less homogeneous microspheres with lower protein encapsulation. This suggests that perhaps instead of or in addition to modifying the microsphere formation formulation, the fabrication technique itself should be more closely considered in achieving homogeneous microspheres with desired loading.

A simple and sensitive colorimetric pH meter based on DNA conformational switch and gold nanoparticle aggregation

A simple and rapid colorimetric pH meter has been developed based on the conformational switch of i-motif DNA and non-crosslinking AuNP aggregation, the average accuracy of the nano-meter was found to be +/- 0.04 pH unit across the physiological operating range.

Highly sensitive electrochemical sensor for nitric oxide using the self-assembled monolayer of 1,8,15,22-tetraaminophthalo-cyanatocobalt(II) on glassy carbon electrode

Glassy carbon (GC) electrode modified with a self-assembled monolayer (SAM) of 1,8,15,22-tetraaminophthalocyanatocobalt(II) (4α-CoIITAPc) was used for the selective and highly sensitive determination of nitric oxide (NO). The SAM of 4α-CoIITAPc was formed on GC electrode by spontaneous adsorption from DMF containing 1 mM 4α-CoIITAPc. The SAM showed two pairs of well-defined redox peaks corresponding to CoIII/CoII and CoIIIPc−1/CoIIIPc−2 in 0.2 M phosphate buffer (PB) solution (pH 2.5). The SAM modified electrode showed excellent electrocatalytic activity towards the oxidation of nitric oxide (NO) by enhancing its oxidation current with 310 mV less positive potential shift when compared to bare GC electrode. In amperometric measurements, the current response for NO oxidation was linearly increased in the concentration range of 3×10−9 to 30×10−9 M with a detection limit of 1.4×10−10 M (S/N=3). The proposed method showed a better recovery for NO in human blood serum samples.

Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Olanzapine in Human Urine

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method was developed to determine olanzapine (OLZ) in human urine. After solid-phase extraction with SPE cartridge, the urine sample was analysed on a C-18 column (Symmetry 3.5 mu m, 50 x 4.6 mm i.d) interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of ammonium acetate (pH 7.8)-acetonitrile (10:90, v/v). The method was linear over a concentration range of 1-100 ngml(-1). The lower limit of quantitation was 1 ngml(-1). The intra-day and inter-day relative standard deviation across three validation runs over the entire concentration range was < 11.5 %. The accuracy determined at three concentrations (8.0, 50.0 and 85.0 ngml(-1) OLZ) was within +/- 1.21 % in terms of relative errors.