Enhanced sensitivity electrochemical assay of low-molecular-weight heparins using rotating polyion-sensitive membrane electrodes

Use of a novel rotating polycation-sensitive polymer membrane electrode yields sensors that can serve as simple potentiometric titration endpoint detectors for the determination of three FDA approved low-molecular-weight heparin (LMWH) anticoagulant drugs (Fragmin, Normiflo, and Lovenox). The rotating electrode configuration dramatically improves the reproducibility and increases the sensitivity for LMWH determinations by protamine titration. At a rotation speed of 3000 rpm, electrodes with optimized thin (50 µm) polymer membranes doped with dinonylnaphthalene sulfonate (DNNS) respond to low levels of protamine (<2 µg mL^–1) with good precision (±1 mV, N=10), when protamine is infused continuously into a Tris-buffer solution, pH 7.4. When infusing protamine (at 5 µg min^–1) continuously into solutions containing Fragmin, a clear endpoint is obtained, with the amount of protamine required to reach this endpoint proportional to the level of Fragmin present. A detection limit of less than 0.02 U mL^–1 Fragmin can be obtained via this new method, approximately one order of magnitude lower than that previously reported based on a non-rotating polycation electrode. Similar low detection limits can be achieved for potentiometric titrations of Normiflo and Lovenox. Such titrations can also be carried out in undiluted plasma samples containing the various LMWH species. In this case, detection of the LMWHs at clinically relevant concentrations (>0.2 U mL^–1) can be readily achieved.     

Potentiometric flow injection sensing system for determination of heparin based on current-controlled release of protamine

A flow injection system incorporated with a polycation-sensitive polymeric membrane electrode in the flow cell is proposed for potentiometric determination of heparin. An external current in nano-ampere scale is continuously applied across the polymeric membrane for controlled release of protamine from the inner filling solution to the sample solution, which makes the electrode membrane regenerate quickly after each measurement. The protamine released at membrane–sample interface is consumed by heparin injected into the flow cell via their strong electrostatic interaction, thus decreasing the measured potential, by which heparin can be detected. Under optimized conditions, a linear relationship between the potential peak height and the concentration of heparin in the sample solution can be obtained in the range of 0.1–2.0 U mL⁻¹, and the detection limit is 0.06 U mL⁻¹. The proposed potentiometric sensing system has been successfully applied to the determination of heparin in undiluted sheep whole blood.     

Physical adsorption of protamine for heparin assay using a quartz crystal microbalance and electrochemical impedance spectroscopy

This study attempted to determine absolute heparin concentration in phosphate buffer solution (PBS, pH 7.4) by using quartz crystal microbalance (QCM) as an affinity biosensor. Electrochemical impedance spectroscopy (EIS) was also used to investigate immobilization of protamine and heparin assay. In addition, the effectiveness of physical adsorption in immobilizing protamine was confirmed by examining the preparation conditions, including the incubation time and protamine concentration. It induced maximum decrease (ca. −100 Hz) in oscillating frequency of QCM by applying 20 mg/ml protamine and 20 min for incubation in PBS. Heparin adsorption onto protamine-modified electrode in PBS revealed an exponential-like binding curve and long duration for reaching the steady state in frequency response of QCM. Moreover, two linear calibration curves were obtained judging from the initial slope (df/dt) and the frequency change (Δf) of QCM obtained after a binding interval (600 s) for heparin concentrations from 0 to 3.0 and 7.0 U/ml, respectively. In EIS analysis, calibration curves with linear concentration range of 0-3.0 U/ml were obtained for heparin in PBS when ferrocyanide was used as an electroactive marker.     

Fluoride determination in some mouth wash preparations by a novel La(III) graphite coated membrane sensor based on amitraz

Solution studies on the binding properties of N-2,4-dimethylphenyl-N′-ethylformamidine (amitraz) toward nine lanthanide ions including lanthanum, cerium, neodium, samarium, europium, gadolinium, terbium, dysprosium, ytterbium and some other transition and heavy metal ions such as copper, lead, cobalt, nickel ions, showed a selective 1:1 complexation between amitraz and lanthanum ions. Consequently, amitraz was applied as an ion carrier in construction of a novel poly(vinyl chloride) membrane sensor for La(III). The sensor has a linear dynamic range of 1.0 × 10⁻¹ to 1.0 × 10⁻⁷ M with a Nernstian slope of 19.8 ± 0.2 mV per decade and a detection limit of 8.0 × 10⁻⁸ M. The proposed sensor displays a fast response time (<8 s), and can be used for at least 2 months without any considerable divergences in the potentials. The La(III) membrane sensor revealed comparatively good selectivity with respect to most of cations including alkaline, alkaline earth, and some transition and heavy metal ions. It could be used in a pH range of 3.0-9.0. The proposed membrane electrode was used as an indicator electrode in the potentiometric titration of La(III) ions with an EDTA solution, and also in the determination of fluoride concentration in some mouth wash preparations.     

Aluminum(III) selective potentiometric sensor based on morin in poly(vinyl chloride) matrix

Al³⁺ selective sensor has been fabricated from poly(vinyl chloride) (PVC) matrix membranes containing neutral carrier morin as ionophore. Best performance was exhibited by the membrane having composition as morin:PVC:sodium tetraphenyl borate:tri-n-butylphosphate in the ratio 5:150:5:150 (w/w, mg). This membrane worked well over a wide activity range of 5.0 × 10⁻⁷ to 1.0 × 10⁻¹ M of Al³⁺ with a Nernstian slope of 19.7 ± 0.1 mV/decade of Al³⁺ activity and a limit of detection 3.2 × 10⁻⁷ M. The response time of the sensor is ∼5 s and membrane could be used over a period of 2 months with good reproducibility. The proposed sensor works well over a pH range (3.5-5.0) and demonstrates good discriminating power over a number of mono-, di- and trivalent cations. The sensor can also be used in partially non-aqueous media having up to 20% (v/v) methanol, ethanol or acetone content with no significant change in the value of slope or working activity range. The sensor has also been used in the potentiometric titration of Al³⁺ with EDTA and for its determination in zinc plating mud and red mud.     

An amperometric biosensor based on peroxidases from Brassica napus for the determination of the total polyphenolic content in wine and tea samples

An amperometric biosensor based on peroxidases from Brassica napus hairy roots (PBHR) used to determine the total polyphenolic content in wine and tea samples is proposed by the first time. The method employs carbon paste (CP) electrodes filled up with PBHR, ferrocene (Fc), and multi-walled carbon nanotubes embedded in a mineral oil (MWCNT + MO) at a given composition (PBHR-Fc-MWCNT + MO). The biosensor was covered externally with a dialysis membrane, which was fixed at the electrode body side part with a Teflon laboratory film and an O-ring. Calibration curves obtained from steady-state currents as a function of the concentration of a polyphenolic standard reference compound such as t-resveratrol (t-Res) or caffeic acid (CA) were then used to estimate the total polyphenolic content in real samples. The reproducibility and the repeatability were of 7.0% and 4.1% for t-Res (8.4% and 5.2% for CA), respectively, showing a good biosensor performance. The calibration curves were linear in a concentration range from 0.05 to 52 mg L⁻¹ and 0.06 to 69 mg L⁻¹ for t-Res and CA, respectively. The lowest polyphenolic compound concentration values measured experimentally for a signal to noise ratio of 3:1 were 0.023 mg L⁻¹ and 0.020 mg L⁻¹ for t-Res and CA, respectively.     

Copper(I)-bathocuproine complex as carrier in iodide-selective electrode

A PVC membrane electrode for iodide ions based on Cu(I)-bathocuproine as ionophore in membrane composition is prepared. The electrode exhibits a linear response over a wide concentration range 5.0×10⁻⁶ to 2.0×10⁻¹ mol l⁻¹ with a detection limit 1.0×10⁻⁶ mol l⁻¹. The proposed membrane electrode shows Nernstian behavior with a slope of −56.8 mV/decade, a fast response time 10 s and a lifetime at least 3 months. Iodide-selective electrode reveals good selectivities for iodide ion over a wide variety of the other anions and can be used in pH range of 3-9. It can also be used as an indicator electrode in potentiometric titration of iodide ion.     

Local factor analysis of rank-deficient reaction systems

A variety of biochemical and physical properties of proteins are regulated by calcium ion (Ca²⁺) binding with varying specificity and affinity. Calcium ion binding can adjust the phospholipid-protein interactions through changing the properties of phospholipid membrane. As an attractive detection technique, whole column imaging detection (WCID) coupled to capillary isoelectric focusing (cIEF) displays several advantages in the study of protein-ligand and protein-protein interactions, including fast and high-efficient separation, high resolution, and simple operation. In this study, a cIEF-WCID method was evaluated for studying the effect of Ca²⁺ binding on protein structural changes and phospholipid-protein interactions. Four proteins with different isoelectirc point (pI), trypsin inhibitor (pI = 4.5), β-lactoglobulin B (pI = 5.2), phosphorylase b (pI = 6.3), and trypsinogen (pI = 9.3), were used for this purpose. The targeted proteins exhibited altered cIEF profiles due to protein conformation changes resulting from the Ca²⁺ binding. The study showed that Ca²⁺ can be buried in the phospholipid membrane, modify the membrane property, and change the phospholipid-protein interactions. The utility of the cIEF-WCID technique demonstrates that the calcium binding plays a crucial role in the protein structural changes and the phospholipid-protein interactions, and elucidates the possible mechanisms involved in calcium-protein binding and calcium bound phospholipid-protein interactions.     

Al(III)-selective electrode based on newly synthesized xanthone derivative as neutral ionophore

The suitability of a xanthone derivative, 1-hydroxy-3-methyl-9H-xanthen-9-one (HMX) as a neutral ionophore for the preparation of a polyvinylchloride (PVC) membrane electrode for aluminum(III) ions was investigated. The prepared electrode exhibits a Nernstian response for Al³⁺ ions over a wide concentration range (1.0 × 10⁻⁶ to 1.6 × 10⁻¹ M) with a limit of detection 6.0 × 10⁻⁷ M. It has a relatively fast response time and can be used for at least three months without any considerable divergence in potentials. The proposed membrane electrode revealed very good selectivity for Al³⁺ ions over a wide variety of other cations and could be used at a working pH range of 3.0-8.5. It was used as an indicator electrode in potentiometric titration of aluminum ions with EDTA and in the determination of Al³⁺ in different real samples.     

Highly selective thiocyanate membrane electrode based on butane-2,3-dione bis(salicylhydrazonato)zinc(II) complex

A highly selective poly(vinyl chloride) (PVC) membrane electrode based on butane -2,3-dione bis(salicylhydrazonato) zinc(II) [Zn (BDSH)] complex as carrier for thiocyanate-selective electrode is reported. The influence of membrane composition, pH and possible interfering anions were investigated on the response properties of the electrode. The sensor responds to thiocyanate in linear range from 1.0 × 10⁻⁶ to 1.0 × 10⁻¹ M with a slope −56.5 ± 1.1 mV decade⁻¹, over a wide pH range of 3.5-8.5. The limit of detection of the electrode was 7.0 × 10⁻⁷ M SCN⁻. Selectivity coefficients determined with fixed interference method (FIM) indicate a good discriminating ability towards SCN⁻ ion in comparison to other anions. The proposed sensor has a fast response time of about 5-15 s and can be used for at least 3 months without any considerable divergence in potential. It was applied as indicator electrode in titration of thiocyanate with Ag⁺ and in potentiometric determination of thiocyanate in saliva and urine samples.     

Contribution to vapor generation-inductively coupled plasma spectrometric techniques for determination of sulfide in water samples

Vapor generation-inductively coupled plasma-optical emission spectrometry was used for the determination of sulfide in water samples preserved by the addition of a zinc acetate and sodium hydroxide solution. Hydrogen sulfide and acid-volatile sulfides were transformed, by acidification, to a gaseous phase in a vapor generator and subsequently detected by inductively coupled plasma optical emission spectrometry. Compounds interfering with iodometric titration and spectrophotometric determination were examined as potential chemical interferents. The proposed method provides results comparable to iodometric titration in the tested concentration range 0.06-22.0 mg L⁻¹. Limit of detection for the determination of hydrogen sulfide by this method is 0.03 mg L⁻¹.     

A newly synthesized macrocyclic dithioxamide receptor for phosphate sensing

Polyvinyl chloride (PVC) based membranes using macrocyclic dithioxamide receptor (I) derived from isophthaloyl dichloride and dithioxamide have been prepared and explored as HPO₄²⁻-selective sensors. Effect of various plasticizers viz., bis(2-ethylhexyl) sebacate (DOS), dibutylphosphate (DBP), tri-n-butylphosphate (TBP), O-nitrophenyl octyl ether (NPOE), tris(2-ethylhexyl)phosphate (TEHP) and a cation excluder, tridodecylmethylammonium chloride (TDDMACl) was studied in detail and improved performance was observed at several instances. Optimum performance was observed with the membrane having (I)-PVC-TDDMACl-NPOE in the ratio 2:33:1.5:63.5 (w/w). The sensor works satisfactorily in the concentration range 1.7 × 10⁻⁶ to 1.0 × 10⁻² M (detection limit 0.2 ppm) with Nernstian compliance (29.6 mV/decade of activity) at pH 8.0 with a fast response time of about 8 s. The potentiometric selectivity coefficient values as determined by the matched potential method (MPM) and the fixed interference method (FIM) indicate selective response for HPO₄²⁻ in presence of interfering ions. The sensor exhibits adequate shelf life (∼2 months) with good reproducibility (S.D. ± 0.4 mV). The sensor was also used successfully in the potentiometric titration of HPO₄²⁻ with Ba²⁺.     

Highly selective and sensitive copper membrane electrode based on a new synthesized Schiff base

Bis(2-hydroxyacetophenone)butane-2,3-dihydrazone (BHAB) was used as new N-N Schiffs base which plays the role of an excellent ion carrier in the construction of a Cu(II) membrane sensor. The best performance was obtained with a membrane composition of 30% poly(vinyl chloride), 55% o-nitrophenyloctyl ether (NPOE), 7% BHAB and 8% oleic acid (OA). This sensor shows very good selectivity and sensitivity towards copper ion over a wide variety of cations, including alkali, alkaline earth, transition and heavy metal ions. The effect of membrane composition and pH and influence of additive anionic on the response properties of electrode were investigated. The electrode exhibits a Nernstian behavior (with slope of 29.6 mV per decade) over a very wide concentration range (5.0 × 10⁻⁸ to 1.0 × 10⁻² mol L⁻¹) with a detection limit of 3.0 × 10⁻⁸ mol L⁻¹ (2.56 ng mL⁻¹). It shows relatively fast response time, in whole concentration range (<15 s), and can be used for at least 12 weeks in the pH range of 2.8-5.8. The proposed sensor was successfully used to determination of copper in different water samples and as indicator electrode in potentiometric titration of copper ion with EDTA.     

A cerium(III) selective polyvinyl chloride membrane sensor based on a Schiff base complex of N,N'-bis[2-(salicylideneamino)ethyl]ethane-1,2-diamine

A polyvinyl chloride (PVC) based membrane sensor for cerium ions was prepared by employing N,N′-bis[2-(salicylideneamino)ethyl]ethane-1,2-diamine as an ionophore, oleic acid (OA) as anion excluder and o-nitrophenyloctyl ether (o-NPOE) as plasticizer. The plasticized membrane sensor exhibits a Nernstian response for Ce(III) ions over a wide concentration range (1.41 × 10⁻⁷ to 1.0 × 10⁻² M) with a limit of detection as low as 8.91 × 10⁻⁸ M. It has a fast response time (<10 s) and can be used for 4 months. The sensor revealed a very good selectivity with respect to common alkali, alkaline earth and heavy metal ions. The response of the proposed sensor is independent of pH between 3.0 and 8.0. It was used as an indicator electrode in potentiometric titration of fluoride, carbonate and oxalate anions and determination of cerium in simulated mixtures.     

Lead(II) ion-selective electrode based on polyaminoanthraquinone particles with intrinsic conductivity

A new polyvinylchloride membrane electrode was facilely prepared by using polyaminoanthraquinone (PAAQ) microparticles with an intrinsically electrical conductivity as a lead(II) ionophore. It is found that the electrode performance will significantly be improved with adding 1 wt% PAAQ microparticles and decreasing the membrane thickness. A 90 μm-thick membrane electrode consisting of PAAQ(salt):polyvinyl chloride:dioctylphthalate:sodium tetraphenylborate of 1:33:66:1 (wt) but without any traditional lead(II) ionophore achieved the optimal performance and exhibited a good Nernstian response for Pb(II) ions over a wide concentration range from 2.5 × 10⁻⁶ to 0.1 M with a slope of 28.9 mV/decade and a detection limit down to 776 nM. A reasonably short response time of 12 s was revealed together with a long lifetime over a period of around 4 months in a wide pH range between 2.8 and 5.2. A fixed interference method indicated that the electrode has an excellent selectivity for lead(II) ion over alkali, alkaline earth and other heavy metal ions. The proposed electrode has been also found to be a powerful indicator electrode for potentiometric titration of Pb(II) ions with EDTA. The electrode can be used to accurately monitor the Pb(II) pollution in environmental waters.     

Determination of molybdenum in environmental samples

Molecular imprinted membrane of indole-3-ethanol (IE) was prepared by hybridization of IE imprinted polymer powder and polysulfone (PSf) membrane. The IE imprinted polymer by covalent imprinting method was synthesized with copolymerization of indole-3-ethyl methacrylate (IEMA) and divinylbenzene (DVB). The cross-linked P(IEMA-co-DVB) was ground to be powders having less than 63 μm size and then hybridized within PSf membrane by using phase inversion process. The resultant imprinted powder showed binding capacities of 1.8, 7.2, 0 and 0 μmol g⁻¹ for IE, indole, 8-hydroxyquinoline and pyrrole in aqueous solution, respectively, and after hybridization with the PSf membrane, the value was 46, 26, 0 and 0 μmol g⁻¹. As a result, it was found that the IE imprinted powder alone showed non-selectively binding to the IE, but, the hybridized powder within the PSf membrane bound selectively the IE. Evidence was presented that hydrophobic interaction of the PSf matrix caused the selective and efficient binding. We also showed separation behavior of the hybrid membranes and discussed on the binding selectivity of the IE molecule. In view point of hybrid effect of the PSf membrane and the cross-linked imprinted powder, the results of the separation of these substrates were considered.     

Flow injection conductometric system with gas diffusion separation for the determination of Kjeldahl nitrogen in milk and chicken meat

A simple flow injection (FI) conductometric system with gas diffusion separation was developed for the determination of Kjeldahl nitrogen (or proteins) in milk and chicken meat. The sample was digested according to the Kjeldahl standard method and the digest was diluted and directly injected into the donor stream consisting of 4 M NaOH. In alkaline medium, ammonium was converted to ammonia, which diffused through the PTFE membrane to dissolve in an acceptor stream (water). Dissociation of ammonia caused a change in conductance of the acceptor solution, which was linearly proportional to the concentration of ammonium originally present in the injected solution. A conductometric flow through cell and an amplifier circuit was fabricated, which helped improve sensitivity of the conductometric detection system. With using a plumbing Teflon tape as a gas diffusion membrane and without thermostating control of the system, a linear calibration graph in range of 10-100 mg L⁻¹ N-NH₄ was obtained, with detection limit of 1 mg L⁻¹ and good precision (relative standard deviation of 0.3% for 11 replicate injections of 50 mg L⁻¹ N-NH₄). The developed method was validated by the standard Kjeldahl distillation/titration method for the analysis of milk and chicken meat samples. The proposed system had sample throughput of 35 h⁻¹ and consumed much smaller amounts of chemical than the standard method (275 mg vs 17.5 g of NaOH per analysis, respectively).     

Selective thiocyanate poly(vinyl chloride) membrane based on a 1,8-dibenzyl-1,3,6,8,10,13-hexaazacyclotetradecane-Ni(II) perchlorate

A highly selective poly(vinyl chloride) (PVC) membrane electrode based on 1,8-dibenzyl-1,3,6,8,10,13-hexaazacyclotetradecane-Ni(II) as a membrane carrier with unique selectivity toward thiocyanate is reported. The influence of membrane composition, pH and foreign anions were investigated. The sensor exhibits a Nernstian response for thiocyanate over a wide concentration range of 3.3×10⁻⁶ to 0.10 M, with a slope 58.4±0.3 mV per decade. The limit of detection is 3.0×10⁻⁶ M SCN⁻. The sensor has a response time of <20 s and can be used for at least 2 months without any considerable divergence in potential. The proposed electrode shows fairly a good discriminating ability towards SCN⁻ ion in comparison to other anions. It was successfully applied to direct determination of thiocyanate in urine and saliva and it was also used as an indicator electrode in titration of thiocyanate with Ag⁺ ions.     

Antioxidant and Antisteatotic Activities of a New Fucoidan Extracted from Ferula hermonis Roots Harvested on Lebanese Mountains

Fucoidan is a fucose-rich sulfated polysaccharide with attractive therapeutic potential due to a variety of biological activities, including antioxidant action. Fucoidan is typically found in the cell wall of marine brown algae, but extra-algal sources have also been discovered. In the present work, for the first time we extracted a water soluble fucoidan fraction from the roots of the terrestrial shrub Ferula hermonis. This fucoidan fraction was termed FUFe, and contained fucose, glucose, sulfate, smaller amounts of monosaccharides such as galactose and mannose, and a minor quantity of proteins. FUFe structural features were investigated by FTIR, ¹H NMR and ¹³C NMR spectroscopy. The antioxidant property of FUFe was measured by DPPH, ABTS and FRAP assays, which revealed a high radical scavenging capacity that was confirmed in in vitro cellular models. In hepatic and endothelial cells, 50 μg/mL FUFe could reduce ROS production induced by intracellular lipid accumulation. Moreover, in hepatic cells FUFe exhibited a significant antisteatotic action, being able to reduce intracellular triglyceride content and to regulate the expression of key genes of hepatic lipid metabolism. Altogether, our results candidate FUFe as a possible bioactive compound against fatty liver disease and related vascular damage.     

Ultratrace determination of phosphorus in ultrapurified water by a slope comparison method

The analytical method for the determination of phosphorus in ultrapurified water was developed. Ultrapurified water was evaporated to concentrate phosphorus and the final sample volume for analysis was 10 ml. In 0.55 mol l⁻¹ HCl, orthophosphate forms molybdophosphate, and then the molybdophosphate forms ion associate with Malachite Green (MG), which can be collected on a tiny membrane filter (diameter: 5 mm, and effective filtering diameter: 2 mm). After the ion associate on the membrane filter is dissolved together with the membrane filter in 1 ml of methyl cellosolve (MC), the absorbance of MC solution is measured at 627 nm by a flow injection-spectrophotometric detection technique. When 10 ml of the sample solution was used for the procedures and absorbance measurement, the calibration graph is linear up to about 500 ng l⁻¹ of phosphorus and the detection limit was 8 ng l⁻¹ (S/N=3). For the determination of phosphorus in an ultrapurified water, 10-40 ml of sample solutions were transferred into poly(tetrafluoroethylene) (PTFE) beaker and evaporated to 5 ml or to dryness. To them, 0.003 mol l⁻¹ HCl was added to get 10 ml of final solution, which was used as sample. Phosphate is determined by comparing the slope of the varied sample volume after evaporation/concentration with a slope of the standard calibration graph (a slope comparison method: SCM). The SCM enables to evaluate the concentration of phosphate in ultrapurified waters more sensitively and accurately.