Analytica Chimica Acta (v.429, #1)
Fluidics and sample handling in clinical chemical analysis by David D. Cunningham (1-18).
Newer clinical chemical instruments and devices feature advanced sample handling fluidic systems. This review presents descriptions of the technologies utilized in high throughput automated systems, remote point-of-care analyzers, non-automated test strip systems, blood glucose testing products, and automated nucleic acid based assay systems. The physical processes involved in the fluidic systems include simple diffusion, capillary action, positive pressure displacement, vacuum, magnetic forces, centrifugal motion, and electrochemical processes. Key issues and performance parameters are highlighted. Finally, the state-of-the-art in miniaturized fluidic components is presented with a summary of capillary electrophoresis chips and their application to clinically relevant targets.
Keywords: Fluidics; Sample handling; Clinical chemistry; Diagnostic chemistry; Point-of-care testing; Test strips; Nucleic acid analysis; Microchips;
Development of highly sensitive bioluminescent enzyme immunoassay with ultra-wide measurable range for thyroid-stimulating hormone using firefly luciferase by Yoshiaki Seto; Hiroshi Ohkuma; Susumu Takayasu; Toshihiko Iba; Atsuko Umeda; Katsushi Abe (19-26).
A bioluminescent enzyme immunoassay (BLEIA) of thyroid-stimulating hormone (TSH) using biotinylated firefly luciferase as a labelling enzyme for an antibody was developed. This immunoassay, of which method was one step sandwich method, had high sensitivity and very wide measurable range. The detection limit for TSH was found to be 0.0025 μU ml−1 and the measurable range for TSH was 0.0025–200 μU ml−1. The conjugate for this BLEIA was prepared by binding the antibody to the end of cross-linking reagent and streptavidin (SA)-biotinylated luciferase complex to the opposite end of the cross-linking reagent, respectively. The stability of the conjugate was excellent. It showed 70% conjugate activity at the storage of a week at 37°C and 100% at the storage of 6 months at 4°C.
Keywords: Bioluminescent enzyme immunoassay; Firefly luciferase; Thyroid-stimulating hormone; Ultra wide measurable range;
Serological determination of Helicobacter pylori infection using sandwiched and enzymatically amplified piezoelectric biosensor by Xiaodi Su; Sam F.Y Li (27-36).
A sandwiched and enzymatically amplified piezoelectric immunosensor is presented for Helicobacter pylori antibody detection. AT-cut quartz crystals (10 MHz) were used as reaction carrier, onto which recombinant H. pylori antigens were immobilized to capture the associated antibodies. Since the antibody titer in naturally infected patient sera is very low, direct patient serum incubation resulted in less significant positive signals when compared with the negative background. To increase the positive signal and reduce the negative background, serum incubation was followed by the application of anti-h (anti-human) IgG or anti-h IgG conjugates. The specific recognition of anti-h IgG to the bound IgG antibodies led to a sandwiched immunocomplex and the further frequency decrease. Since the binding of the secondary antibody were only observed when patient sera were H. pylori positive, the total frequency changes (caused by serum and secondary antibody) for the positives became more significant than those of the negatives. The resulting positive to negative ratio was increased to 4.1–5.0 depending on different conjugates used. When enzyme (HRP or AP) conjugated antibodies had been used, the sandwiched complexes were exposed to associated substrates. The enzymatically amplified deposition of the converted color products onto sensor surface led to significant enhancement in the sensitivity. By means of this strategy, human serum could be detected as positive in the dilution of 1/100.
Keywords: Piezoelectric quartz crystal; Biosensor; Sandwiched assay; Enzymatically amplified assay; Helicobacter pylori;
Detection of gentamicin in milk by immunoassay and flow injection analysis with electrochemical measurement by R.M Van Es; S.J. Setford; Y.J. Blankwater; D. Meijer (37-47).
A number of alternative approaches involving immunoassays coupled to flow-injection analysis and electrochemical detection have been evaluated with the aim of developing a simple, rapid field-based method for the quantification of gentamicin in milk to within regulatory guide-line levels. Anti-gentamicin antibodies were adsorbed onto nitrocellulose and polycarbonate solid-phase supports and positioned either in contact with flow-cell mounted electrodes or upstream of the electrode-cell assembly. Following the competitive immunoassay, analyte levels were quantified according to the extent of glucose oxidase enzyme label activity, measured electrochemically. The fully assembled flow-cell device (in situ method) was able to distinguish between 0 and 100 μg kg−1 gentamicin in milk is <10 min with a coefficient of variation value of 13.2% and with no sample pre-treatment. Performing the incubation step outside of the flow-cell prior to cell assembly and signal measurement required a 90 min incubation step and CV values of up to 38.7%. The in situ assay is simple to perform, automatable and by using cheap, mass-producible screen-printed electrodes, is amenable to the development of single-usage disposable measurement cartridges.
Keywords: Aminoglycoside; Electrochemistry; Flow-injection analysis; Gentamicin; Immunoassay; Milk; Screen-printed electrodes;
Monofluorinated polycyclic aromatic hydrocarbons in Shpol’skii spectroscopy by G. Luthe; J. Scharp; U.A.Th. Brinkman; C. Gooijer (49-54).
The basic requirement to achieve high resolution in Shpol’skii spectroscopy is that the guest molecules fit well in the crystalline host alkane matrix, a condition that is usually met by polycyclic aromatic hydrocarbons (PAHs). Despite the electronegativity of the fluorine atom, which might be thought to strongly reduce the compatibility of guest and host, high-quality spectra can be recorded also for monofluorinated polycyclic aromatic hydrocarbons (F-PAHs), as was shown for seven test analytes, viz. 1-fluoronaphthalene, 1-fluorophenanthrene, 3-fluorochrysene, 1-fluoropyrene, 2-fluorofluorene, 3-fluorofluoranthene and 9-flurobenzo[k]fluoranthene. Lamp-excited spectra were recorded in order to facilitate simultaneous excitation of PAHs and F-PAHs. The intensities and spectral qualities of the F-PAHs are similar to those of the parent compounds, but the 0–0 transitions are shifted from 0.5 to 7.3 nm in the fluorescence mode; that is, the spectra do not overlap. For a number of F-PAHs, both the fluorescence and the phosphorescence transitions can be utilized.
Keywords: Fluorescence; Polycyclic aromatic hydrocarbons; Phosphorescence; Monofluorinated polycyclic aromatic hydrocarbons;
Room-temperature phosphorescence fiber-optic instrumentation for simultaneous multiposition analysis of dissolved oxygen by Juan Dı́az-Garcı́a; José M Costa-Fernández; Nerea Bordel-Garcı́a; Alfredo Sanz-Medel (55-64).
The design and analytical characterization of a fiber-optic instrument for simultaneous multiposition water-dissolved oxygen monitoring by room-temperature phosphorescence (RTP) measurements is presented. The sensing principle is based on the RTP quenching by oxygen of the phosphorescent light emitted by the metal chelate formed by Al with 8-hydroxy-7-iodo-5-quinolinesulfonic acid (Al-ferron) trapped in a sol–gel solid support. Four RTP oxygen sensor flow-cells are assembled in order to measure the oxygen content in four different water streams. A xenon flash-lamp is used as the single excitation source for the four sensing regions, while multichannel phosphorescence detection is achieved by using a cooled intensified charge-coupled device (ICCD). Four bifurcated optical fibers are used to carry the light from the excitation source to the sensing active surface in the flow-cell and to bring the emitted RTP to the detector. The RTP light coming from each of the optical fibers was focused onto different sites (rows of pixels) of the ICCD. In this way, the RTP signals from each of the four sensing materials packed in the flow-cells can be differentiated and measured. Simultaneous dissolved oxygen monitoring in several water streams containing different dissolved oxygen levels is demonstrated by using the proposed instrumentation.Also, the relatively long phosphorescence lifetime of the materials used allow the use of the developed instrumentation to perform lifetime room-temperature phosphorescence measurements.
Keywords: Room-temperature phosphorescence (RTP); Simultaneous and multiposition analysis; Fiber-optic sensors; Intensified charge-coupled device (ICCD); Oxygen sensing;
Partition coefficients of pyrene to lake aquatic humic matter determined by fluorescence quenching and solubility enhancement by Juhani Peuravuori (65-73).
Partition coefficients for the binding affinities of pyrene to different humic materials were determined by fluorescence quenching and solubility enhancement techniques. Mathematical methods applied to separate a possible combination of static and dynamic quenching components, which may have contributed to the curvature of the Stern–Volmer plot, were unsuccessful. Fluorescence quenching tends to significantly overestimate the partition coefficient values. Physical–chemical characteristics of different humic materials have no significant effect on the dissimilarity of partition coefficients obtained by these two approaches. The utilization of fluorescence quenching for extensive systematic studies should therefore, be accepted with caution.
Keywords: Fluorescence quenching; Solubility enhancement; Partition coefficients; Aquatic humic substances;
Binding of pyrene on lake aquatic humic matter: the role of structural descriptors by Juhani Peuravuori (75-89).
Partition coefficients for the binding affinities of pyrene to 28 different humic materials were determined by the solubility enhancement method. The humic materials used in the present study were isolated by different methods from different environmental sources, and characterized by various methods including spectroscopic and pyrolysis (Py) approaches. Several structural descriptors were derived for testing the binding affinity of pyrene. A significant correlation was observed, supporting the earlier literature results, between the pyrene binding coefficient and the molecular weights, molar absorptivities at 280 nm, and aromaticity of the aquatic humic solutes. Also other structural descriptors, e.g. nitrogen and especially aliphatic moieties, have their own importance to the binding affinity of pyrene. The results confirm the fact that it is not meaningful to treat aquatic and terrestrial humic material as an integrated whole. The results also disclosed that the isolation and fractionation procedures of aquatic humic probes will strengthen, as a result of breaking hydrophobic bondings of aggregates between different humic solutes, the binding affinities of pyrene on different humic isolates compared with the original situation predominating in the water sample. One has to be very careful in the light of the forthcoming predictions what kind of humic probe has been adopted for representing aquatic humic matter (HM). A statistical approach for estimation of the most probable carbon distribution in a humus sample proved quite workable for the prediction of binding affinity of pyrene on aquatic humic materials.
Keywords: Solubility enhancement; Partition coefficients; Structural descriptors; Aquatic humic substances; Computational estimation;
Isolation of aucubin and catalpol from Melitaea cinxia larvae and quantification by micellar electrokinetic capillary chromatography by Johanna Suomi; Heli Sirén; Susanne K Wiedmer; Marja-Liisa Riekkola (91-99).
Two iridoid glycosides, aucubin and catalpol, were extracted from Melitaea cinxia larvae with hot, dilute aqueous SDS solution and then separated and quantified by micellar electrokinetic capillary chromatography. Solutions of pure aucubin and catalpol were used to optimise the separation. The extraction method was developed for a mixture of 80 larvae and single larvae were then analysed with the optimised procedure. The repeatabilities of both the pretreatment step and the determination were satisfactory.The study presents the first time that iridoid glycosides in Melitaea cinxia have been determined. Determinations were successfully made in butterfly larvae weighing only 1 mg. The total concentration of aucubin and catalpol in Melitaea cinxia varied between 0.1 and 3% of the dry mass of the larva.
Keywords: Aucubin; Catalpol; Micellar electrokinetic capillary chromatography; Extraction; Melitaea cinxia;
Measurement of parts per million levels of potassium hydroxide in polyether polyol streams by Sofia Galanis; Purnendu K. Dasgupta (101-110).
An automated reverse flow injection (FI) based continuous analyzer for KOH in polyols is presented. A solution of bromocresol green (BCG) in 2-propanol, with or without added mineral acid, is injected into the sample polyol stream in a single-line FI manifold maintained at 110°C (dispersion factor of 25 and 50 for a dilute aqueous stream at room temperature with 30 and 10 μl of solution injected) and the blue indicator dianion formed upon reaction with KOH is measured at 605 nm by a light emitting diode (LED) — fiber optic based absorbance detector. Injections of 10 μl of 10 mM BCG and 30 μl of 15 mM BCG, 50 mM HCl were respectively chosen to cover desired ranges of 0–20 and 20–120 mg KOH/kg polyol (ppm KOH). The absorbance signal is linear with KOH concentration (r 2=0.9949 in both cases). Measurements could be made in the present system every 4 min. Deliberate alteration of water content of the polyol stream in the range of interest was found to have no discernible effect on the measurement.
Keywords: Polyether polyols; KOH; Light emitting diode; Flow injection analysis;
Simultaneous spectrophotometric determination of fluoride and monofluorophosphate ions in toothpastes using a reversed flow injection manifold by Demetrius G Themelis; Paraskevas D Tzanavaras (111-116).
A new simple and rapid reagent injection (FI) method is reported for the simultaneous determination of fluoride and monofluorophsphate (MFP) ions in toothpastes, using a single calibration for both analytes. The method is based on the inhibitory effect of fluoride ions upon the Fe(III)-catalyzed oxidation of 2,4-diominophenol (DAP) by H2O2. Free fluoride ions form stable complexes with Fe(III), thus reducing its catalytic effect, while MFP does not inhibit the reaction under flow conditions. MFP ions are determined indirectly as fluoride ions after quantitative acid hydrolysis. The decrease in the absorbance of the oxidation product is monitored at 500 nm. The various chemical and physical variables of the FI system were optimized, and a study of interfering ions was also carried out.A linear calibration graph was obtained from 0 to 9 mg l−1 for F−. The relative standard deviation was very good (0.5% at 4.5 mg l−1 F−, n=12) and the 3σ detection limit was 0.09 mg l−1). The sampling rate was 120 injections h−1. The method was successfully applied to the simultaneous determination of F− and MFP in toothpastes.
Keywords: Reagent injection; Spectrophotometry; Fluoride; Monofluorophosphate; Toothpastes;
Silicate species in high pH solution molybdate, whose silica concentration is determined by colorimetry by Miho Tanakaa; Kazuya Takahashib (117-123).
The chemical species of silica, whose concentration was measured by colorimetry, were examined in alkaline hydroxide solutions. The total amount of SiO2 measured by colorimetry is in good agreement with that measured by weight loss on drying. The monomer (Si(OH)3O−), monomer-Na+ complex (Si (OH)2O2Na−), dimer (Si2(OH)5O2 −), dimer-Na+ complex (Si2(OH)4O3Na−), linear tetramer (Si4(OH)9O4 −), cyclic tetramer (Si4(OH)7O5 −) and cyclic tetramer-Na+ complex (Si4(OH)6O6Na−) were observed in NaOH and KOH solutions by fast atom bombardment-mass spectrometer. When the NaOH solutions containing silica were neutralized, they were found to contain the monomer-Na+ complex, dimer, dimer-Na+ complexes (Si2(OH)3O4Na2 −, Si2(OH)2O5Na3 −) in addition to dimer-Na+ complex (Si2(OH)4O3Na−), linear tetramer, cyclic tetramer and cyclic tetramer-Na+ complex. The solutions of silica in natural water and of silica at pH 7–12 contain monomer, monomer-Na+ complex, dimer, dimer-Na+ complexes, linear tetramer, cyclic tetramer and cyclic tetramer-Na+ complex of silica, as determined by colorimetry with ammonium molybdate.
Keywords: Silica species; Fast atom bombardment-mass spectrometer; Colorimetry;
Speciation analysis of antimony by high-performance liquid chromatography inductively coupled plasma mass spectrometry using ultrasonic nebulization by Michael Krachler; Hendrik Emons (125-133).
The two inorganic Sb species Sb(III) and Sb(V) as well as trimethlyantimony dichloride (TMSbCl2) were separated by anion exchange chromatography. HPLC eluents were directly aspirated into the plasma of an ICP-MS using ultrasonic nebulization (USN) with membrane desolvation. Leaching of Sb from glass bottles, especially pronounced under alkaline conditions, distinctly increased background 121Sb signals. The use of conventional HPLC syringes made of glass resulted in non reproducible blank signals for Sb(V). To overcome these problems, polyethylene bottles for the storage of mobile phases and plastic syringes for the injection of Sb compounds onto the analytical columns were employed. The analysis of Sb(III) standard solutions in the sub μg l−1 range prepared by dilution of stock standard solutions with high-purity water revealed that Sb(III) is easily oxidized to Sb(V) within a few hours. Deoxygenating the water for dilution of standards by flashing working standard solution with helium or using 1.25 mM EDTA — which served as mobile phase for the chromatographic separation — for the dilution of standards, no oxidation of Sb(III) was observed, at least for 8 h. USN parameters, such as the temperature of the heating area and of the desolvation area as well as the sweep gas flow rate were systematically optimized. Decreasing the USN heating temperature from the conventional settings of 140–80°C enhanced signal intensities by ∼25% and caused no condensation of the aspirated mobile phase. After optimization detection limits for all three Sb species in the low ng l−1 range could be established, namely 14 ng l−1 for Sb(III), 12 ng l−1 for Sb(V) and 9 ng l−1 for TMSbCl2.
Keywords: Antimony; Speciation; High-performance liquid chromatography; Inductively coupled plasma mass spectrometry; Ultrasonic nebulization;
On the transport of some metals into inductively coupled plasma during hydride generation process by P Pohl; W Zyrnicki (135-143).
Transport process of Ca, Cd, Co, Cr, Cu, Fe, Mn, Mg, Ni, Zn and Ti during continuous flow hydride generation in the system with phase separation was investigated using inductively coupled plasma atomic emission spectrometry. The effect of HCl and HNO3 (for concentrations from 0.01 to 5.0 mol l−1) and arsenic on transport efficiency of the metals into plasma was examined. It was observed that not only Cd but also Cu transport in a form of volatile species was significant. The present study also indicated the formation of Co, Cr, Fe and Ni volatile species during hydride generation reaction. Possibility of spectral interferences between Cd, Cu and Fe lines on the one side and As and Bi lines on the other side was analysed and discussed.
Keywords: Hydride generation; Transport of metals; Volatile species; Cd; Co; Cr; Cu; Fe; Ni; Atomic emission spectrometry; Inductively coupled plasma;
Determination of Si and Cu in Al target metal used in a semiconductor process by inductively coupled plasma-atomic emission spectrometry by H.Y Kim; H.B Lim (145-150).
In this work, a method for the determination of Cu and Si in Al target metal used in a semiconductor process was developed based on inductively coupled plasma-atomic emission spectrometry (ICP-AES). Wet acid digestion and high-pressure acid digestion, open and closed systems, respectively, were employed to dissolve the Al target metal containing 0.5% (w/w) Cu and >0.2% (w/w) Si. Recovery of 45% was obtained for Si, when hydrochloric acid was used for wet acid digestion. The recovery increased to 90% in high-pressure acid digestion, or wet acid digestion with the use of sulfuric acid instead of hydrochloric acid. This indicated that Si was lost during the wet acid digestion if hydrochloric acid was used.For application, three kinds of aluminum target metals containing more than 0.2% (w/w) Si, along with 0.5% (w/w) Cu, which are commonly used in semiconductor process, were analyzed with standard addition using ICP-AES.
Keywords: Determination of Si; Inductively coupled plasma-atomic emission spectrometry; Acid digestion; Al target metal;
Automatic identification of terpenoid skeletons through 13 C nuclear magnetic resonance data disfunctionalization by Marcelo J.P. Ferreira; Antônio J.C. Brant; Gilberto V. Rodrigues; Vicente P. Emerenciano (151-170).
The proposal of this paper is to present a procedure that utilizes 13 C NMR for terpenoid skeletons identification. By this reason, a novel program named REGRAS was developed for the specialist system SISTEMAT. This program carries out an analysis of the 13 C NMR data from a given compound and, from ranges of chemical shifts, recognizes the chemical functions existing on specific positions of carbon skeletons. At the end of this procedure, the program matches the types of carbon atoms obtained against a database, displaying as analysis results the likely skeletons of the questioned substance. The program REGRAS was tested on skeleton elucidation of 35 compounds from the most varied classes of terpenoids, exhibiting excellent results in skeleton prevision precesses.
Keywords: Terpenoids; Natural products; 13 C NMR; Skeletons elucidation; Expert system;
Data analysis in the chemical laboratory by Karel Kupka; Milan Meloun (171-183).
The regression model for a two-segments titration curve with a break-point at the end-point is analyzed. Both linear and nonlinear shapes of the titration curve segments are treated. An effective and simple method discriminates which of two segments is linear or curved. The point and interval estimates of the end-point are calculated by the nonlinear least squares of curve fitting technique. The nonlinear regression method is applied to any, linear or nonlinear, type of a two-segments titration curve without excluding any titration points to reach the most probable point estimate of the end-point together with its 100(1−α)% confidence interval. An accuracy and precision of the proposed end-point estimation is examined on several instrumental titrations. A sample program in S-Plus™ is provided.
Keywords: Two-segments titration curve; Break-point; Two lines intersection; End-point; Nonlinear least squares; Titration curve;