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Analytica Chimica Acta (v.564, #2)
A spatially resolved nucleic acid biochip based on a gradient of density of immobilized probe oligonucleotide by Sung Han Park; Ulrich Krull (pp. 133-140).
The potential for a new biochip design based on a continuous gradient of density of immobilized single-stranded DNA oligonucleotide probes (ssDNA) is explored. This gradient resolved information platform (GRIP) can provide sequence identification based on the spatial location and extent of hybridization by a target sequence. Surfaces based on indium–tin oxide (ITO) on glass were first functionalized by 3-aminopropyltriethoxysilane (APTES) followed by attachment of glutaraldehyde, prior to immobilization of oligonucleotide probe that was terminated with amine. The use of Cy3 and Cy5 dye-labelled ssDNA probes and targets allowed estimation of density and correlation of the location of binding of labelled targets. Probe molecules of 20 mer lengths were loaded to produce density gradients in the range of 1.0–200ng/mm2. The biochips could resolve a mixture of fully complementary five base-pair mismatched targets by the location of binding on the surface. Thermal control provided additional selectivity. Thermal cycling and washing provided for regeneration of the surface, and the fluorescence intensities showed no deterioration in at least five cycles of hybridization reactions.
Keywords: DNA; Hybridization; Biosensor; Fluorescence; Gradient; Selectivity
Purification and partial characterization of CMP-Neu5Ac synthetase from rat brain by J.C. Feo-Manga; L.B. Rodríguez-Aparicio; M.A. Ferrero; A. Reglero (pp. 141-150).
N-Acetyl-neuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-Neu5Ac synthetase), which catalyzes the formation of cytidine-5′-monophospho- N-acetyl-neuraminic acid (CMP-Neu5Ac) from cytidine-5′-triphosphate (CTP) and N-acetyl-neuraminic acid (Neu5Ac), was purified from rat brains aged 8–9 days, which presented the highest specific activity, and partially characterized. Partial protein fractionation in the crude extract was achieved by using 40–60% ammonium sulphate. Subsequently, CMP-Neu5Ac synthetase was purified by column chromatography on Sephacryl S-200 (gel filtration), Yellow-86-Agarose (affinity) and Phenyl-Sepharose (hydrophobic affinity). The pure enzyme had a specific activity of 3.6555U/mg of protein and was purified 1662-fold, with an 18% yield. The purified CMP-Neu5Ac synthetase had a molecular weight of about 46±1kDa. Its purity was confirmed by sodium dodecyl sulphate and polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). The active enzyme chromatographed on a gel filtration column at 190kDa, suggesting it exists in its native form as a tetramer. The greatest activity of enzyme was observed a temperature of 40°C for a period of 45min of incubation, revealing a certain thermal stability. The enzyme was found to remain stable in the pH range 8.5–9.5 at 40°C, specifically at pH 9.0 for a 45min incubation period. The enzyme was blocked by thiol-modifying reagents and such heavy metal cations as Mn2+, Cu2+, Sn2+, Co2+, Zn2+ and Hg2+, but was not inhibited by thiol-containing reagents like reduced glutathione (GSH), mercaptoethanol and cysteine. Finally, in the presence of 0.01M of dithiothreitol (DTT) or 0.06M of NaF, the enzyme showed activity losses of approximately 20 and 17%, respectively.
Keywords: CMP-sialic acid synthetase; Rat brain; Enzyme purification; pH stability; Thermal stability
Novel dye-embedded core-shell nanoparticles as surface-enhanced Raman scattering tags for immunoassay by Ji-Lai Gong; Jian-Hui Jiang; Hai-Feng Yang; Guo-Li Shen; Ru-Qin Yu; Yukihiro Ozaki (pp. 151-157).
An approach to prepare novel glass-coated gold core-shell nanoparticles embedded with Raman active molecules has been developed. This strategy eliminates the need of a coupling agent and enables efficient embedding of most of commercially available Raman active dyes, even those with weak affinity to a gold surface. Our experiments have demonstrated the hypothesis that the glass encapsulation chemistry is based on the reaction between silanol groups hydrolyzed by TEOS and citrate anion groups adsorbed onto the gold nanoparticles leading to the formation of a silica layer through further reaction with silanol groups of other hydrolyzed TEOS. Furthermore, it has been demonstrated the potential of these novel nanoparticls as Raman tags in ultrasensitive immunoassays for human IgG antigen with a detection limit of 4.9ng/ml.
Keywords: Surface-enhanced Raman scattering; Raman tags; Nanoparticles; Immunoassay
Electrochemical immuno-bioanalysis for carcinoma antigen 125 based on thionine and gold nanoparticles-modified carbon paste interface by Dianping Tang; Ruo Yuan; Yaqin Chai (pp. 158-165).
A novel electrochemical immunosensor for the determination of carcinoma antigen 125 (CA125) was developed by means of immobilizing CA125 antibody (anti-CA125) on gold nanoparticles (Au) and thionine (Thi)-modified carbon paste interface. To avoid the leak of hydrophilic gold nanoparticles and thionine from carbon paste interface, the Au–Thi-modified carbon paste electrodes (CPEs) were first treated in the mixture solution containing 10% HNO3 and 2.5% K2Cr2O7 for 1.5min at +1.5V to make the carbon surface with –COOH groups, which can react with –NH2 groups on the thionine molecule, in the meantime, gold nanoparticles were absorbed on the thionine surface. Subsequently, CA125 antibodies were assembled onto the surface of gold nanoparticles. The fabrication process of the immunosensor was characterized by fourier transform infrared spectroscopy (FTIR) and UV–vis absorption spectroscopy. The performance and factors influencing the performance of the immunosensor were studied in detail. A direct electrochemical immunoassay format was employed to detect CA125 antigen based on the current change before and after the antigen–antibody reaction. The current change was proportional to CA125 concentration ranging from 10 to 30U/ml with a detection limit of 1.8U/ml (at 3 δ). The immunosensors were used to analyze CA125 in human serum specimens. Analytical results of clinical samples show that the developed immunoassay has a promising alternative approach for detecting CA125 in the clinical diagnosis.
Keywords: Electrochemical immunoassay; Carbon paste interface; Carcinoma antigen 125; Gold nanoparticles; Thionine
A novel optical immunosensing system based on measuring surface enhanced light scattering signals of solid supports by Hua Wen Zhao; Cheng Zhi Huang; Yuan Fang Li (pp. 166-172).
An optical immunosensor based on measuring the surface enhanced light scattering (SELS) signals is developed by simultaneously scanning the excitation and the emission monochromators of a common spectrofluorometer. The formation of silanization on solid surface of glass slides and the binding of antibody with antigen in series have been confirmed by measuring the SELS signals. When 100μgml−1 goat anti-human IgG, goat anti-rabbit IgG and rabbit anti-human IgG are immobilized on the solid surface, human IgG and rabbit IgG in the range of 0.1–100μgml−1 and human IgG in the range of 0.1–10μgml−1 could be detected, and the lowest detectable concentration could, respectively, reach 10, 50 and 10ngml−1. This label-free assay is more rapid and simpler than those conventional immunoassay methods. Under optimal experimental conditions, the sensor based on SELS has a high specificity with corresponding antigen (Ag), and other co-existing proteins in solution, for example, such as bovine serum albumin (BSA) and human serum albumin (HSA) do not show interference effect. Regenerated simply by rinsing in carbamide solution, the sensor could achieve three assay cycles without great loss of sensitivity.
Keywords: Surface enhanced light scattering (SELS) signals; Immunosensor; Antigen (Ag); Antibody (Ab)
Determination of glycyrrhizin in Chinese prescriptions and biological samples by enzyme-linked immunosorbent assay by Hua-Jin Zeng; Bo-Yang Yu; Ji-Hua Liu; Nan Liu (pp. 173-178).
In the work described on this paper, a highly specific and sensitive enzyme-linked immunosorbent assay (ELISA) for the determination of glycyrrhizin (GL) was developed. To demonstrate the potential of the ELISA, a preliminary pharmacokinetic study of GL in rats and quantitative analysis of GL in several Chinese prescriptions were performed and compared with high-performance liquid chromatography (HPLC). The experimental data indicated that ELISA method exhibits more advantages over HPLC, such as lower detection limit, high specificity, low background and no requirement of sample pre-treatment, and more suitable for the determination of natural components in Chinese traditional medicines.
Keywords: Enzyme-linked immunosorbent assay (ELISA); Diammonium glycyrrhizinate; Glycyrrhizin; Polyclonal antiserum; Chinese prescriptions; Pharmacokinetics
Preparation of molecularly imprinted polymers with thiourea group for phosphate by Akimitsu Kugimiya; Hideo Takei (pp. 179-183).
Molecularly imprinted polymers selective for phosphate were prepared with the two types of functional monomers, 1-allyl-2-thiourea and N-methyl- N′-(4-vinylphenyl)-thiourea, and the binding abilities of the polymers were evaluated. Phenylphosphonic acid or diphenyl phosphate were used as the template molecules and the imprinted polymers prepared with 1-allyl-2-thiourea as functional monomer showed high binding ability to phosphate in aqueous media and nearly 90% of phosphate could be recovered. Also, the imprinted polymer prepared with N-methyl- N′-(4-vinylphenyl)-thiourea as functional monomer had a high binding ability and specific interaction with phosphate in acetonitrile solution and over 90% of phosphate-derivatives could be recovered selectively.
Keywords: Molecular imprinting; Phosphate; Thiourea group; Molecular recognition
Randomization tests to identify significant effects in experimental designs for robustness testing by B. Dejaegher; X. Capron; J. Smeyers-Verbeke; Y. Vander Heyden (pp. 184-200).
The screening designs applied in robustness tests are usually fractional factorial or Plackett–Burman designs. Different methods to identify significant factor effects estimated from experimental designs for robustness testing are described. In this paper, the use of randomization tests as a statistical interpretation method is examined and compared with both graphical (half-normal probability plot) and statistical methods, such as the estimation of error based on a priori considered negligible effects and the algorithm of Dong. It was found that all statistical methods usually gave similar results, i.e. the same effects are found to be significant. However, sometimes randomization tests indicate either less or more significant factor effects compared to the other methods, regardless the design size. Both randomization tests and the algorithm of Dong become unreliable when about 50% of the examined factors are significant. In such situation, it is advisable to perform an experimental design from which enough negligible effects can be estimated. The graphical interpretation method did not always succeed in indicating the correct number of significant effects.
Keywords: Experimental design; Half-normal probability plot; Dummy factor effect; Interaction effect; Algorithm of Dong; Randomization test
A study of the analytical behaviour of selected synthetic and naturally occurring coumarins using liquid chromatography, ion trap mass spectrometry, gas chromatography and polarography and the construction of an appropriate database for coumarin characterisation by W. Franklin Smyth; Venkataraman N. Ramachandran; Catherine J. Hack; Clare Joyce; Edmund O’Kane (pp. 201-210).
The aim of this paper is to provide analytical chemical information on a range of naturally occurring and synthetic coumarins. This analytical chemical information on liquid chromatography–electrospray ionisation-mass spectrometry (HPLC–ESI-MS), ion trap mass spectrometry (ESI-MS n), gas chromatography-flame ionisation detection (GLC-FID) and polarographic behaviour is then incorporated into a database which is of use in identifying unknown coumarins isolated from natural sources. This paper is also concerned with understanding the effect of functional groups in coumarins on their analytical chemical behaviour using the above techniques.
Keywords: Coumarins; Liquid chromatography–electrospray ionisation-mass spectrometry (HPLC–ESI-MS); Ion trap mass spectrometry (ESI-MS; n; ); Gas chromatography-flame ionisation detection (GLC-FID); Polarography; Database
Qualitative and quantitative analysis of iridoid glycosides in the flower buds of Lonicera species by capillary high performance liquid chromatography coupled with mass spectrometric detector by Yue Song; Song-Lin Li; Meng-Hua Wu; Hui-Jun Li; Ping Li (pp. 211-218).
A highly sensitive and specific method, based on capillary high performances liquid chromatography coupled with single quadrupole mass spectrometry using electrospray ionization (capillary HPLC–ESI/MS), is proposed for the identification and quantification of iridoid glycosides in the flower buds of five Lonicera species. A Zorbax SB-C18 (0.3mm×150mm, 5μm) capillary column and a gradient elution with methanol–acetonitrile–aqueous acetate acid were utilized. The most intensive electrospray ionisation signals were found in the negative ion spectra owing to CH3COO− adducts. Eight iridoid glycosides derived from the flower buds of Lonicera species were analyzed by mass spectrometry: sweroside (IG1), 7- O-ethyl sweroside (IG2), 7-epi vogeloside (IG3), secoxyloganin (IG4), secoxyloganin 7-butyl ester (IG5), dimethyl-secologanoside (IG6), centauroside (IG7), and loganin (IG8) using combined information on retention time, the molecular ion mass and fragment ion masses. Detection limits were lower than 1.9ng/mL in selected ion monitoring (SIM) mode and all calibration curves showed good linear regression ( r2>0.9938) within test ranges. The validated method was successfully applied to analyze eight iridoid glycosides in the flower buds of five Lonicera species and provided a new basis of assessment on quality of Flos Lonicerae.
Keywords: Capillary HPLC–ESI/MS; Quantification; Iridoid glycoside; Flos Lonicerae
Thermal quenching of fluorescence of freshwater, planktonic bacteria by S. Elliott; J.R. Lead; A. Baker (pp. 219-225).
This study aims to determine the thermal quenching properties of pure bacterial cultures as a means of aiding the development of fluorescence measurement in natural waters. The bacterium Pseudomonas aeruginosa was isolated from the urban River Tame, Birmingham, UK, and planktonic bacteria were grown in sterile, sealed glass jars, in 100mL of sterile growth media at 37°C for a maximum of 24h. Samples were taken at T=6h and at T=24h, and thermal fluorescence quenching measured at 5°C increments between 10 and 45°C over 30min. 3D excitation–emission matrix (EEM) plots were generated from the fluorescence analyses over time. It was found that the fluorescence of a microbial culture was significantly thermally quenched, but the results were dependent on the fluorophore type and the stage of the bacterial growth curve. Quenching was sometimes non-linear, presumably due to fluorophore production exceeding thermal quenching during the growth phase of the bacteria. Thermal quenching has the potential to allow us to confirm the importance of microbes in fluorescence signals by the non-linear response to increasing temperature, and to utilise the thermal fluorescence quenching properties of molecules to differentiate between fluorophores present during bacterial growth.
Keywords: Bacterial fluorescence; Thermal quenching; Pseudomonas aeruginosa; Planktonic bacteria; Temperature
A new electrochemiluminescent detection system equipped with an electrically controlled heating cylindrical microelectrode by Zhenyu Lin; Jianjun Sun; Jinhua Chen; Liang Guo; Guonan Chen (pp. 226-230).
A new electrochemiluminescent (ECL) detection system equipped with an electrically controlled heating cylindrical microelectrode (HME) was developed in this paper. The cylindrical microelectrode made of platinum wire (25μm in diameter, 6mm in long) was used as the working electrode of the ECL detection system, the temperature of the electrode could be controlled electrically. The Ru(bpy)32+-ECL and Ru(bpy)32+–C2O42−-ECL systems were used to evaluate this ECL detection system. The detection limit for oxalate was found to be 3.0×10−4mol/L when Te (temperature of the HME) was 22°C, and found to be 3.0×10−6mol/L at 80°C, which indicates that the detection limit can be improved greatly at higher Te, based on which, it is possible to establish a more sensitive method for measurement of ECL by using a heated microelectrode.
Keywords: Heated cylindrical microelectrode; Electrochemiluminescence; Ru(bpy); 3; 2+; Oxalate
Spectrophotometric flow-injection determination of total reducing sugars exploiting their alkaline degradation by Evandro R. Alves; Paula R. Fortes; Eduardo P. Borges; Elias A.G. Zagatto (pp. 231-235).
An automated procedure exploiting alkaline degradation of carbohydrates is proposed for spectrophotometric determination of total reducing sugars, TRS (hydrolyzed sucrose plus reducing sugars), in samples relevant to the sugar industry. An environmental friendly reagent is used instead of the commonly used toxic (e.g. arsenate) or expensive (e.g. enzymes) reagents. The analytical steps of acidic sucrose hydrolysis and sugar alkaline degradation are in-line accomplished at about 95°C. The formed colored solution is then spectrophotometrically monitored at 450nm.The proposed system handles 60 samples per hour, and yields precise results, relative standard deviation being estimated as <5% for a typical sample (1.24%, w/v, TRS). Baseline drift has been not verified during 8-h working periods, emphasizing the system ruggedness. A linear response is observed between 0.1 and 2.0% (w/v) TRS. Results are in agreement with HPIC.
Keywords: Total reducing sugars; Sucrose inversion; Spectrophotometry; Flow analysis; Sugar degradation
Elimination of interferences in determination of platinum and palladium in environmental samples by inductively coupled plasma mass spectrometry by Barbara A. Leśniewska; Beata Godlewska-Żyłkiewicz; Anna Ruszczyńska; Ewa Bulska; Adam Hulanicki (pp. 236-242).
Various approaches were evaluated in order to eliminate the spectral interferences noted when Pt and Pd has to be determined in environmental dust samples by ICP-MS. The chemical separation of Pt and Pd from the matrix components on ion-exchange resins was applied. The performance of cation-exchange resins (Dowex 50 WX-8, Dowex 50 WX-2, Dowex HCR-S, Varion KS, Cellex-P) for the separation of interfering ions was then examined. It was found that Dowex 50 WX-8 shows best performance. The effects of mass, mesh number of resin and concentration of Cl− ions on matrix separation were also studied. Another approach was to use the anion-exchange sorbent Cellex-T, which allows almost total retention of both analytes followed by their elution with 0.1molL−1 thiourea in 1molL−1 HCl. This procedure however can be used only for platinum determination by ICP-MS. The accuracy of proposed procedures was confirmed by the analysis of certified material BCR-723, and then it was used for determination Pt and Pd in samples of road dust.
Keywords: Cation-exchanger; Anion-exchanger; Sample pre-treatment; Matrix separation; Road and tunnel dust
Glassy carbon ceramic composite electrodes by Danzi Sun; Liande Zhu; Guoyi Zhu (pp. 243-247).
A new carbon composite electrode material, based on dispersing glassy carbon (GC) microparticles into methyltrimethoxysilane-derived sol, is described in the present paper. The resulting glassy carbon ceramic composite electrodes (GCCEs) combine the electrochemical properties of GC with the advantages of composite electrodes, and thus offer high electrochemical reactivity, low background current and are easy to prepare, modify and renew. The new material has a low double-layer capacitance and a wide potential window. Scanning electron microscopy (SEM) images indicate significant difference in the structure of GCCE and carbon ceramic composite electrode (CCE). The electrochemical properties and advantages of GCCE should find broad utility in electroanalysis.
Keywords: Glassy carbon (GC); Sol–gel; Composite electrode
Adsorptive stripping voltammetric measurements of trace beryllium at the mercury film electrode by Joseph Wang; Sompong Thongngamdee; Donglai Lu (pp. 248-252).
A highly sensitive adsorptive stripping voltammetric protocol for measuring trace beryllium, in which the preconcentration is achieved by adsorption of the beryllium–arsenazo-I complex at a preplated mercury-coated carbon-fiber electrode, is described. Optimal conditions were found to be a 0.05M ammonium buffer (pH 9.7) containing 5μM arsenazo-I, an accumulation potential of 0.0V (versus Ag/AgCl) and a square-wave voltammetric scan. The new procedure obviates the need for renewable mercury-drop electrodes used in early stripping protocols for beryllium. A linear response is observed over the 10–60μgl−1 concentration range (90s accumulation), along with a detection limit of 0.25μgl−1 beryllium (10min accumulation). A 15-s electrochemical cleaning enables the same mercury film to be used for a prolonged operation. High stability is thus indicated from the reproducible response of a 100μgl−1 beryllium solution ( n=60; RSD=3.3%) over a 2.5-h operation. Applicability to a seawater sample is illustrated. The attractive behavior of the new sensor holds great promise for on-site environmental and industrial monitoring of beryllium. Preliminary data in this direction using mercury-coated screen-printed electrodes are encouraging.
Keywords: Beryllium; Mercury film electrode; Arsenazo-I; Adsorptive stripping voltammetry
New potentiometric sensors for creatinine by M.A.F. Elmosallamy (pp. 253-257).
Three main types of creatinine potentiometric membrane sensors are described. They are based on the use of dibenzo-30-crown-10 (DB30C10) with potassium tetrakis( p-chlorophenyl)borate type (I), dibenzo-30-crown-10 alone type (II), and potassium tetrakis( p-chlorophenyl)borate alone type (III), incorporating in poly(vinyl chloride) matrix membrane plasticized with either o-nitrophenyl octyl ether or dioctylphthalate. The sensors are used for quantification of creatinine after soaking the membranes in 0.1M creatinine solution for 2 days. The sensors show almost the same potentiometric response characteristics. Sensor type (I) exhibits Nernstian responses over a concentration range of 5.0×10−5moll−1–1.0×10−2moll−1 creatinine with cationic slopes of 59.5±0.1 and 60±0.2mVdecade−1 and detection limits of 1.1×10−5moll−1 and 8×10−6moll−1 creatinine, over the pH range of 3.5–6.5 and 3.5–7.0, for o-NPOE and DOP solvent mediators, respectively. Sensor type (II) displays Nernstian responses over a concentration range of 6.0×10−5moll−1–1.0×10−2moll−1 creatinine with cationic slopes of 60.0±0.1 and 65.0±0.2mVdecade−1 and detection limits of 1.5×10−5moll−1 and 1.4×10−5moll−1 creatinine over the pH range of 2.6–6.2 and 2.5–6.0, for o-NPOE and DOP solvent mediators, respectively. Sensor type (III) shows Nernstian responses over a concentration range of 7.0×10−5moll−1–1.0×10−2moll−1 creatinine with cationic slopes of 60±0.1 and 62.0±0.2mVdecade−1 and detection limits of 2.7×10−5moll−1 and 2.0×10−5moll−1 creatinine over the pH range of 2.5–6.0, for o-NPOE and DOP solvent mediators, respectively. The response times of the sensors for 10−3moll−1 creatinine solution are instantaneous (4–10s). The sensors show long-term stability with life span of ∼6 months. The sensors are used for determination of serum creatinine of rats ( Rattus Norvigicus) with mean R.S.D. of 2.62%, and the results agreed well with the Jaffe kinetic method.
Keywords: Neutral carrier; Crown ether; Potentiometric sensors; Lipophilic additives; Creatinine determination; Real samples