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papers from the latest issue:
Stability-indicating capillary zone electrophoresis method for the assessment of recombinant human granulocyte-macrophage colony-stimulating factor and its correlation with reversed-phase liquid chromatography method and bioassay
25 May 2012,
10:06:28
Publication year:
2012
Source:Talanta, Volume 94
Sergio Luiz Dalmora, Cairo dos Santos Butzge, Francine Trevisan Machado, Maurício Elesbão Walter, Maria Elisabeth de Ávila Dalmora, Ricardo Bizogne Souto
A stability-indicating capillary zone electrophoresis (CZE) method was validated for the analysis of granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using leuprorelin acetate (LA), as internal standard (IS). A fused-silica capillary (75μm i.d.; effective length, 72cm) was used at 25°C; the applied voltage was 12kV. The background electrolyte solution consisted of 50mM di-sodium hydrogen phosphate solution at pH 8.8. Injections were performed using a pressure mode at 50mbar for 9s, with detection by photodiode array detector set at 200nm. Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The method was linear over the concentration range of 2.5–200μgmL−1 (r 2 =0.9995) and the limit of detection (LOD) and limit of quantitation (LOQ) were 0.79μgmL−1 and 2.5μgmL−1, respectively. The accuracy was 99.14% with bias lower than 1.40%. The method was applied to the quantitative analysis of biopharmaceutical formulations, and the results were correlated to those of a validated reversed-phase LC method (RP-LC), and an in vitro bioassay, showing non-significant differences (p >0.05).
Source:Talanta, Volume 94
Sergio Luiz Dalmora, Cairo dos Santos Butzge, Francine Trevisan Machado, Maurício Elesbão Walter, Maria Elisabeth de Ávila Dalmora, Ricardo Bizogne Souto
A stability-indicating capillary zone electrophoresis (CZE) method was validated for the analysis of granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using leuprorelin acetate (LA), as internal standard (IS). A fused-silica capillary (75μm i.d.; effective length, 72cm) was used at 25°C; the applied voltage was 12kV. The background electrolyte solution consisted of 50mM di-sodium hydrogen phosphate solution at pH 8.8. Injections were performed using a pressure mode at 50mbar for 9s, with detection by photodiode array detector set at 200nm. Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The method was linear over the concentration range of 2.5–200μgmL−1 (r 2 =0.9995) and the limit of detection (LOD) and limit of quantitation (LOQ) were 0.79μgmL−1 and 2.5μgmL−1, respectively. The accuracy was 99.14% with bias lower than 1.40%. The method was applied to the quantitative analysis of biopharmaceutical formulations, and the results were correlated to those of a validated reversed-phase LC method (RP-LC), and an in vitro bioassay, showing non-significant differences (p >0.05).
Highlights
► Stability-indicating capillary zone electrophoresis method for rhGM-CSF. ► Correlation between physicochemical methods and bioassay for rhGM-CSF. ► Content/potency assessment of rhGM-CSF in biotechnology-derived product.
25 May 2012,
10:06:28
Publication year:
2012
Source:Talanta, Volume 94
Bojidarka B. Ivanova, Michael Spiteller
The paper presented with qualitative and quantitative analysis of alkaloids in solid-state, using the excitations within the THz-spectroscopic region of 300–30cm−1 (9.00–0.9THz). Series of nine plant flavonoids (FLs) and their mixtures were analyzed both qualitatively and quantitatively. For first time in the literature was reported the quantitative analysis of alkaloids and their mixtures within the THz-region using the solid-state Raman spectroscopy, and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The calibration and validation, concentration limit of detection (LOD), limit of quantification (LOQ), and linearity limit (LL) were obtained. The chemometric nonlinear and linear approaches for analysis and interpretation of the quantities were applied. The results obtained were compared with a parallel QA, using the calibrated and validated HPLC electrospray ionization (ESI) mass spectrometric method, electronic absorption (EAs) and CD spectroscopy. The metrology, including accuracy, measurement repeatability (intra-serial precision condition of measurement), measurement precision, trueness of the measurement, and reproducibility of the measurement, measurement bias and errors of the measurements were discussed.
Source:Talanta, Volume 94
Bojidarka B. Ivanova, Michael Spiteller
The paper presented with qualitative and quantitative analysis of alkaloids in solid-state, using the excitations within the THz-spectroscopic region of 300–30cm−1 (9.00–0.9THz). Series of nine plant flavonoids (FLs) and their mixtures were analyzed both qualitatively and quantitatively. For first time in the literature was reported the quantitative analysis of alkaloids and their mixtures within the THz-region using the solid-state Raman spectroscopy, and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The calibration and validation, concentration limit of detection (LOD), limit of quantification (LOQ), and linearity limit (LL) were obtained. The chemometric nonlinear and linear approaches for analysis and interpretation of the quantities were applied. The results obtained were compared with a parallel QA, using the calibrated and validated HPLC electrospray ionization (ESI) mass spectrometric method, electronic absorption (EAs) and CD spectroscopy. The metrology, including accuracy, measurement repeatability (intra-serial precision condition of measurement), measurement precision, trueness of the measurement, and reproducibility of the measurement, measurement bias and errors of the measurements were discussed.
Highlights
► Forensic chemistry. ► Raman spectroscopy. ► Mass spectrometry. ► Natural products. ► Alkaloids.An LC method for the analysis of phosphatidylcholine hydrolysis products and its application to the monitoring of the acyl migration process
25 May 2012,
10:06:28
Publication year:
2012
Source:Talanta, Volume 94
Grzegorz Kiełbowicz, Damian Smuga, Witold Gładkowski, Anna Chojnacka, Czesław Wawrzeńczyk
An assay for quantitative analysis of phosphatidylcholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and its hydrolysis products: 1-hydroxy-2-palmitoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, sn-glycero-3-phosphocholine and palmitic acid using high-performance liquid chromatography with charge aerosol detector (CAD) was developed. The separation of the compounds of interest was achieved on a reversed-phase/hydrophilic interaction stationary phase with a mobile phase consisting of acetonitrile:methanol:10mM ammonium acetate solution. The method was applied to control the acyl migration process of LPC regioisomers in the most common solvents used in the synthesis or modification of PC.
Source:Talanta, Volume 94
Grzegorz Kiełbowicz, Damian Smuga, Witold Gładkowski, Anna Chojnacka, Czesław Wawrzeńczyk
An assay for quantitative analysis of phosphatidylcholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and its hydrolysis products: 1-hydroxy-2-palmitoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, sn-glycero-3-phosphocholine and palmitic acid using high-performance liquid chromatography with charge aerosol detector (CAD) was developed. The separation of the compounds of interest was achieved on a reversed-phase/hydrophilic interaction stationary phase with a mobile phase consisting of acetonitrile:methanol:10mM ammonium acetate solution. The method was applied to control the acyl migration process of LPC regioisomers in the most common solvents used in the synthesis or modification of PC.
Highlights
► Phosphatidylcholine (PC) hydrolysis product formation is a serious problem especially in the enzyme-catalyzed acyl exchange of PC. ► Quantitative HPLC analysis of PC and its hydrolysis products is presented. ► The method was applied to examine the stability of the lysophosphatidylcholine (LPC) isomers in 16 different solvents. ► 1-Palmitoyl LPC isomer is more stable than 2-palmitoyl LPC isomer. ► The acyl migration and hydrolysis processes are prefer at alkaline pH.A Pt layer/Pt disk electrode configuration to evaluate respiration and alkaline phosphatase activities of mouse embryoid bodies
25 May 2012,
10:06:28
Publication year:
2012
Source:Talanta, Volume 94
Raquel Obregon, Yoshiko Horiguchi, Toshiharu Arai, Shihomi Abe, Yuanshu Zhou, RyosukeTakahashi, Akiko Hisada, Kosuke Ino, Hitoshi Shiku, Tomokazu Matsue
A Pt layer/Pt disk electrode configuration was used as a scanning electrochemical microscopy (SECM) probe. The glass seal part of the insulator was covered with a Pt layer to form an exposed pseudo reference electrode. In a HEPES-based medium at pH 7.5, the half-wave potential (E 1/2) for [Fe(CN)6]4− oxidation and O2 reduction measured versus the internal Pt pseudo reference was shifted by about −0.2V, compared with the E 1/2 measured versus the external Ag/AgCl reference electrode. The shape and the current of the cyclic voltammograms (CVs) did not change notably over time, indicating that the Pt layer is sufficiently stable to be used as an integrated pseudo reference for voltammetric measurements. To demonstrate the suitability for SECM applications, the Pt/Pt probe configuration was used for measuring the oxygen consumption and the alkaline phosphatase (ALP) activity of a single mouse embryoid body (mEB). Ten individual mEB samples were characterized to monitor the oxygen concentration profile. Oxygen reduction currents were monitored at −0.7V versus the Pt pseudo reference and compared with those monitored at −0.5V versus Ag/AgCl. The respiration rate of mEBs becomes greater with increasing cultivation dates. We have plotted the oxygen consumption rate (F(O2)) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. The linearity of the plot was excellent (coefficient of determination R 2 =0.90). The slope of the least squares method was 1. In a 1.0mM p-aminophenylphospate (PAPP) HEPES buffer (pH 9.5) solution, APL activity of mEBs can be characterized, to monitor the p-aminophenol (PAP) oxidation current. ALP catalyzes the hydrolysis of PAPP to PAP. The E 1/2 for PAP oxidation measured versus the Pt layer was not shifted, compared with the E 1/2 versus Ag/AgCl. The mEB samples were characterized to monitor the PAP concentration profile. PAP oxidation currents were monitored at +0.3V versus the Pt layer and compared with those monitored at +0.3V versus Ag/AgCl. We have plotted the PAP production rate (F(PAP)) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. In this case, the linearity of the plot became slightly scattered, but it was found to be possible to evaluate ALP activities of mEB samples utilizing the Pt/Pt probe configuration. This type of probe is very useful because it is not necessary to insert a reference electrode into the measuring solution to obtain an electrical connection, and thus electrochemical measurement in a small volume becomes much easier.
Source:Talanta, Volume 94
Raquel Obregon, Yoshiko Horiguchi, Toshiharu Arai, Shihomi Abe, Yuanshu Zhou, RyosukeTakahashi, Akiko Hisada, Kosuke Ino, Hitoshi Shiku, Tomokazu Matsue
A Pt layer/Pt disk electrode configuration was used as a scanning electrochemical microscopy (SECM) probe. The glass seal part of the insulator was covered with a Pt layer to form an exposed pseudo reference electrode. In a HEPES-based medium at pH 7.5, the half-wave potential (E 1/2) for [Fe(CN)6]4− oxidation and O2 reduction measured versus the internal Pt pseudo reference was shifted by about −0.2V, compared with the E 1/2 measured versus the external Ag/AgCl reference electrode. The shape and the current of the cyclic voltammograms (CVs) did not change notably over time, indicating that the Pt layer is sufficiently stable to be used as an integrated pseudo reference for voltammetric measurements. To demonstrate the suitability for SECM applications, the Pt/Pt probe configuration was used for measuring the oxygen consumption and the alkaline phosphatase (ALP) activity of a single mouse embryoid body (mEB). Ten individual mEB samples were characterized to monitor the oxygen concentration profile. Oxygen reduction currents were monitored at −0.7V versus the Pt pseudo reference and compared with those monitored at −0.5V versus Ag/AgCl. The respiration rate of mEBs becomes greater with increasing cultivation dates. We have plotted the oxygen consumption rate (F(O2)) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. The linearity of the plot was excellent (coefficient of determination R 2 =0.90). The slope of the least squares method was 1. In a 1.0mM p-aminophenylphospate (PAPP) HEPES buffer (pH 9.5) solution, APL activity of mEBs can be characterized, to monitor the p-aminophenol (PAP) oxidation current. ALP catalyzes the hydrolysis of PAPP to PAP. The E 1/2 for PAP oxidation measured versus the Pt layer was not shifted, compared with the E 1/2 versus Ag/AgCl. The mEB samples were characterized to monitor the PAP concentration profile. PAP oxidation currents were monitored at +0.3V versus the Pt layer and compared with those monitored at +0.3V versus Ag/AgCl. We have plotted the PAP production rate (F(PAP)) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. In this case, the linearity of the plot became slightly scattered, but it was found to be possible to evaluate ALP activities of mEB samples utilizing the Pt/Pt probe configuration. This type of probe is very useful because it is not necessary to insert a reference electrode into the measuring solution to obtain an electrical connection, and thus electrochemical measurement in a small volume becomes much easier.
Graphical abstract
Graphical abstract Highlights
► An integrated probe was fabricated, with working/pseudo reference electrode. ► We measured oxygen consumption and alkaline phosphatase activity of a single mouse embryoid body. ► The consumption pattern observed agreed well with the conventional SECM method.A simple and selective fluorometric assay for dopamine using a calcein blue–Fe2+ complex fluorophore
25 May 2012,
10:06:28
Publication year:
2012
Source:Talanta, Volume 94
Daisuke Seto, Tomoharu Maki, Nobuaki Soh, Koji Nakano, Ryoichi Ishimatsu, Toshihiko Imato
A novel fluorimetric assay for dopamine using calcein blue (CB) complexed with Fe2+ ion as a chemical sensor is described. The fluorescence arising from CB of the CB–Fe2+ complex is quenched by the Fe2+ ion. When dopamine is added to a solution of the CB–Fe2+ complex, a dopamine–Fe2+ complex is formed as the result of a ligand exchange reaction between CB and dopamine which permits the fluorescence from CB to be recovered. The fluorescence intensity at the wavelength of 440nm (at the excitation wavelength of 340nm) was found to be proportional to the concentration of the dopamine added to the CB–Fe2+ complex solution, which permits dopamine to be quantitatively determined. The selectivity for dopamine in the presence of other catecholamines and related compounds was good. The calibration curve for dopamine, determined using experimental data was successfully simulated based on the equilibrium of the ligand exchange reaction between CB and dopamine. The working range is from 50μM to 1mM and the limit of detection and limit of quantization are ca 10μM and 50μM, respectively. The assay is simple and economical, compared with conventional methods such as an enzyme-linked immunosorbent assay (ELISA).
Source:Talanta, Volume 94
Daisuke Seto, Tomoharu Maki, Nobuaki Soh, Koji Nakano, Ryoichi Ishimatsu, Toshihiko Imato
A novel fluorimetric assay for dopamine using calcein blue (CB) complexed with Fe2+ ion as a chemical sensor is described. The fluorescence arising from CB of the CB–Fe2+ complex is quenched by the Fe2+ ion. When dopamine is added to a solution of the CB–Fe2+ complex, a dopamine–Fe2+ complex is formed as the result of a ligand exchange reaction between CB and dopamine which permits the fluorescence from CB to be recovered. The fluorescence intensity at the wavelength of 440nm (at the excitation wavelength of 340nm) was found to be proportional to the concentration of the dopamine added to the CB–Fe2+ complex solution, which permits dopamine to be quantitatively determined. The selectivity for dopamine in the presence of other catecholamines and related compounds was good. The calibration curve for dopamine, determined using experimental data was successfully simulated based on the equilibrium of the ligand exchange reaction between CB and dopamine. The working range is from 50μM to 1mM and the limit of detection and limit of quantization are ca 10μM and 50μM, respectively. The assay is simple and economical, compared with conventional methods such as an enzyme-linked immunosorbent assay (ELISA).
Highlights
► A fluorimetric determination of dopamine using calcein blue (CB) complexed with Fe2+ ion based on ligand exchange mechanism is the first report. ► The method is simple just mixed the CB–Fe2+ complex solution with a dopamine solution. ► The method is with good selectivity to dopamine.Simultaneous analysis of organochlorinated pesticides (OCPs) and polychlorinated biphenyls (PCBs) from marine samples using automated pressurized liquid extraction (PLE) and Power Prep™ clean-up
25 May 2012,
10:06:28
Publication year:
2012
Source:Talanta, Volume 94
Murad I.H. Helaleh, Amal Al-Rashdan, A. Ibtisam
An automated pressurized liquid extraction (PLE) method followed by Power Prep™ clean-up was developed for organochlorinated pesticide (OCP) and polychlorinated biphenyl (PCB) analysis in environmental marine samples of fish, squid, bivalves, shells, octopus and shrimp. OCPs and PCBs were simultaneously determined in a single chromatographic run using gas chromatography–mass spectrometry-negative chemical ionization (GC–MS-NCI). About 5g of each biological marine sample was mixed with anhydrous sodium sulphate and placed in the extraction cell of the PLE system. PLE is controlled by means of a PC using DMS 6000 software. Purification of the extract was accomplished using automated Power Prep™ clean-up with a pre-packed disposable silica column (6g) supplied by Fluid Management Systems (FMS). All OCPs and PCBs were eluted from the silica column using two types of solvent: 80mL of hexane and a 50mL mixture of hexane and dichloromethane (1:1). A wide variety of fish and shellfish were collected from the fish market and analyzed using this method. The total PCB concentrations were 2.53, 0.25, 0.24, 0.24, 0.17 and 1.38ngg−1 (w/w) for fish, squid, bivalves, shells, octopus and shrimp, respectively, and the corresponding total OCP concentrations were 30.47, 2.86, 0.92, 10.72, 5.13 and 18.39ngg−1 (w/w). Lipids were removed using an SX-3 Bio-Beads gel permeation chromatography (GPC) column. Analytical criteria such as recovery, reproducibility and repeatability were evaluated through a range of biological matrices.
Source:Talanta, Volume 94
Murad I.H. Helaleh, Amal Al-Rashdan, A. Ibtisam
An automated pressurized liquid extraction (PLE) method followed by Power Prep™ clean-up was developed for organochlorinated pesticide (OCP) and polychlorinated biphenyl (PCB) analysis in environmental marine samples of fish, squid, bivalves, shells, octopus and shrimp. OCPs and PCBs were simultaneously determined in a single chromatographic run using gas chromatography–mass spectrometry-negative chemical ionization (GC–MS-NCI). About 5g of each biological marine sample was mixed with anhydrous sodium sulphate and placed in the extraction cell of the PLE system. PLE is controlled by means of a PC using DMS 6000 software. Purification of the extract was accomplished using automated Power Prep™ clean-up with a pre-packed disposable silica column (6g) supplied by Fluid Management Systems (FMS). All OCPs and PCBs were eluted from the silica column using two types of solvent: 80mL of hexane and a 50mL mixture of hexane and dichloromethane (1:1). A wide variety of fish and shellfish were collected from the fish market and analyzed using this method. The total PCB concentrations were 2.53, 0.25, 0.24, 0.24, 0.17 and 1.38ngg−1 (w/w) for fish, squid, bivalves, shells, octopus and shrimp, respectively, and the corresponding total OCP concentrations were 30.47, 2.86, 0.92, 10.72, 5.13 and 18.39ngg−1 (w/w). Lipids were removed using an SX-3 Bio-Beads gel permeation chromatography (GPC) column. Analytical criteria such as recovery, reproducibility and repeatability were evaluated through a range of biological matrices.
Determination of metal impurities in advanced lead zirconate titanate ceramics by axial view mode inductively coupled plasma optical emission spectrometry
25 May 2012,
10:06:28
Publication year:
2012
Source:Talanta, Volume 94
M.E. Villanueva Tagle, M.T. Larrea Marín, O. Martin Gavilán, M.D. Durruthy Rodríguez, F. Calderón Piñar, M.S. Pomares Alfonso
An inductively coupled plasma optical emission spectrometry quantification method for the determination of Al, Ca, Cr Cu, Fe, Mn, Mg, Ni, Zn, Ba, K, In and Co in lead zirconate-titanate (PZT) ceramics, modified with strontium and chromium, was developed. Total digestion of ceramics was achieved with a HNO3, H2O2 and HF (ac) mixture by using a microwave furnace. The sensitivity of the net signal intensity respect to radiofrequency power (P) and nebulizer argon flow (F N) variations was strongly dependent of the total excitation energy of line (TEE). For lines with TEE near metastable atoms and ions of argon, an increment of the sensitivities to P and F N variation was observed. At robust plasma conditions the matrix effect was reduced for all matrices and analytes considered. The precision of analysis ranged from 3 to 13%, whereas the analytes recoveries in the spiked samples varied, mostly, from 90 to 110%. The detection limits of studied elements were from 0.004 to 10mgkg−1.
Source:Talanta, Volume 94
M.E. Villanueva Tagle, M.T. Larrea Marín, O. Martin Gavilán, M.D. Durruthy Rodríguez, F. Calderón Piñar, M.S. Pomares Alfonso
An inductively coupled plasma optical emission spectrometry quantification method for the determination of Al, Ca, Cr Cu, Fe, Mn, Mg, Ni, Zn, Ba, K, In and Co in lead zirconate-titanate (PZT) ceramics, modified with strontium and chromium, was developed. Total digestion of ceramics was achieved with a HNO3, H2O2 and HF (ac) mixture by using a microwave furnace. The sensitivity of the net signal intensity respect to radiofrequency power (P) and nebulizer argon flow (F N) variations was strongly dependent of the total excitation energy of line (TEE). For lines with TEE near metastable atoms and ions of argon, an increment of the sensitivities to P and F N variation was observed. At robust plasma conditions the matrix effect was reduced for all matrices and analytes considered. The precision of analysis ranged from 3 to 13%, whereas the analytes recoveries in the spiked samples varied, mostly, from 90 to 110%. The detection limits of studied elements were from 0.004 to 10mgkg−1.
Highlights
► ICP-OES method has been developed for analysis of PZT ceramics. ► Influence of operating parameters on SBR, net signal intensity and matrix effect was studied. ► Some lines with an anomalous behaviour were observed. ► The precision of analysis ranged from 3 to 13%. ► The accuracy was from 90 to 110%.An integrated electrochemical device based on immunochromatographic test strip and enzyme labels for sensitive detection of disease-related biomarkers
25 May 2012,
10:06:28
Publication year:
2012
Source:Talanta, Volume 94
Zhe-Xiang Zou, Jun Wang, Hua Wang, Yao-Qun Li, Yuehe Lin
A novel electrochemical biosensing device that integrates an immunochromatographic test strip and a screen-printed electrode (SPE) connected to a portable electrochemical analyzer was presented for rapid, sensitive, and quantitative detection of disease-related biomarker in human blood samples. The principle of the sensor is based on sandwich immunoreactions between a biomarker and a pair of its antibodies on the test strip, followed by highly sensitive square-wave voltammetry (SWV) detection. Horseradish peroxidase (HRP) was used as a signal reporter for electrochemical readout. Hepatitis B surface antigen (HBsAg) was employed as a model protein biomarker to demonstrate the analytical performance of the sensor in this study. Some critical parameters governing the performance of the sensor were investigated in detail. Under optimal conditions, this sensor was capable of detecting a minimum of 0.3ngmL−1 (S/N=3) HBsAg with a wide linear concentration range from 1 to 500ngmL−1. The sensor was further utilized to detect HBsAg spiked in human plasma with an average recovery of 91.3%. In comparison, a colorimetric immunochromatographic test strip assay (ITSA) was also conducted. The result shows that the SWV detection in the electrochemical sensor is much more sensitive for the quantitative determination of HBsAg than the colorimetric detection, indicating that such a sensor is a promising platform for rapid and sensitive point-of-care testing/screening of disease-related biomarkers in a large population.
Source:Talanta, Volume 94
Zhe-Xiang Zou, Jun Wang, Hua Wang, Yao-Qun Li, Yuehe Lin
A novel electrochemical biosensing device that integrates an immunochromatographic test strip and a screen-printed electrode (SPE) connected to a portable electrochemical analyzer was presented for rapid, sensitive, and quantitative detection of disease-related biomarker in human blood samples. The principle of the sensor is based on sandwich immunoreactions between a biomarker and a pair of its antibodies on the test strip, followed by highly sensitive square-wave voltammetry (SWV) detection. Horseradish peroxidase (HRP) was used as a signal reporter for electrochemical readout. Hepatitis B surface antigen (HBsAg) was employed as a model protein biomarker to demonstrate the analytical performance of the sensor in this study. Some critical parameters governing the performance of the sensor were investigated in detail. Under optimal conditions, this sensor was capable of detecting a minimum of 0.3ngmL−1 (S/N=3) HBsAg with a wide linear concentration range from 1 to 500ngmL−1. The sensor was further utilized to detect HBsAg spiked in human plasma with an average recovery of 91.3%. In comparison, a colorimetric immunochromatographic test strip assay (ITSA) was also conducted. The result shows that the SWV detection in the electrochemical sensor is much more sensitive for the quantitative determination of HBsAg than the colorimetric detection, indicating that such a sensor is a promising platform for rapid and sensitive point-of-care testing/screening of disease-related biomarkers in a large population.
Highlights
► A test strip based biosensor for rapid screening of HBsAg is proposed. ► Enzymatic electrochemical measurement allows sensitive and quantitative detection. ► The enzyme label simplifies the fabrication of test strip and detection procedure.A rational route to the development of a competitive capillary electrophoresis immunoassay: Assessment of the variables affecting the performances of a competitive capillary electrophoresis immunoassay for human serum albumin
25 May 2012,
10:06:28
Publication year:
2012
Source:Talanta, Volume 94
Cristina Giovannoli, Claudio Baggiani, Cinzia Passini, Flavia Biagioli, Laura Anfossi, Gianfranco Giraudi
Affinity capillary electrophoresis is a powerful analytical tool to extract quantitative information about the binding properties of different interacting systems. The use of LIF detection makes the technique suitable for screening strong binding interactions. The non-equilibrium electrophoretic separations of pre-equilibrated mixtures of ligand and receptor are generally used for such strong molecular interactions allowing the assessment of capillary electrophoresis immunoassays, mostly in competitive formats. As the analytical performances of the assay strongly depend on the preservation of the binding properties during the separation, a rational route to assay development has to be followed to get the best conditions. The paper describes the steps followed to set-up a competitive immunoassay for human serum albumin (HSA) by using a labeled protein (HSA-FITC) and an anti-HSA polyclonal antiserum. A labeling degree of around 1 of the HSA-FITC conjugates is needed to get narrow electrophoretic peak while the titration curve is used to define the optimal antiserum dilution. An antiserum-labeled protein affinity constant of 1.34×107 M−1 was measures in the selected separation conditions. Furthermore, in order to maximize the assay competition between the labeled and unlabelled HSA a short pre-incubation step of the antiserum with the unlabelled HSA (the analyte) was introduced to promote a sharp increase in assay sensitivity.
Source:Talanta, Volume 94
Cristina Giovannoli, Claudio Baggiani, Cinzia Passini, Flavia Biagioli, Laura Anfossi, Gianfranco Giraudi
Affinity capillary electrophoresis is a powerful analytical tool to extract quantitative information about the binding properties of different interacting systems. The use of LIF detection makes the technique suitable for screening strong binding interactions. The non-equilibrium electrophoretic separations of pre-equilibrated mixtures of ligand and receptor are generally used for such strong molecular interactions allowing the assessment of capillary electrophoresis immunoassays, mostly in competitive formats. As the analytical performances of the assay strongly depend on the preservation of the binding properties during the separation, a rational route to assay development has to be followed to get the best conditions. The paper describes the steps followed to set-up a competitive immunoassay for human serum albumin (HSA) by using a labeled protein (HSA-FITC) and an anti-HSA polyclonal antiserum. A labeling degree of around 1 of the HSA-FITC conjugates is needed to get narrow electrophoretic peak while the titration curve is used to define the optimal antiserum dilution. An antiserum-labeled protein affinity constant of 1.34×107 M−1 was measures in the selected separation conditions. Furthermore, in order to maximize the assay competition between the labeled and unlabelled HSA a short pre-incubation step of the antiserum with the unlabelled HSA (the analyte) was introduced to promote a sharp increase in assay sensitivity.
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