Amperometric magnetoimmunoassay for the direct detection of tumor necrosis factor alpha biomarker in human serum

An amperometric immunoassay for the determination of tumor necrosis factor alpha (TNFα) protein biomarker in human serum based on the use of magnetic microbeads (MBs) and disposable screen-printed carbon electrodes (SPCEs) has been developed. The specifically modified microbeads were magnetically captured on the working electrode surface and the amperometric responses were measured at −0.20 V (vs. Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as electron transfer mediator and H₂O₂ as the enzyme substrate. After a thorough optimization of the assay, extremely low limits of detection were achieved: 2.0 pg mL⁻¹ (36 fM) and 5.8 pg mL⁻¹ (105 fM) for standard solutions and spiked human serum, respectively. The simplicity, robustness and this clinically interesting LOD proved the developed TNFα immunoassay as a good contender for real clinical application.     

A novel multicommuted fluorimetric optosensor for determination of resveratrol in beer

Resveratrol (3,5,4′-trihydroxystilbene) is a phytoalexin that plays a central role in the human diet because of its antioxidant, anticarcinogenic and antimutagenic properties. This paper shows the development of a multicommuted optosensing device for the determination of resveratrol in beer. The method is based on the measurement of the fluorescence (277/382 nm, λ_ex/λ_em) of the photoproduct on-line generated by UV-irradiation of resveratrol. The fluorescent photoproduct is monitored once it is retained on a solid support (Sephadex QAE A-25) in the detection area, which improves both sensitivity and selectivity. The sample was delipidated with toluene and cyclohexane and resveratrol was extracted by solid-phase extraction (SPE) on C₁₈ cartridges, using methanol as eluent. This pre-treatment allowed recovering about an 82% resveratrol and removing its 3-O-β-d-glycoside (piceid) and other interfering substances present in beer. The method provides a detection limit (DL) of 1.0 ng mL⁻¹ and a linear dynamic range (LDR) of 3.3-100 ng mL⁻¹. It was satisfactorily applied to the determination of resveratrol in top- and bottom-fermented beers by standard addition calibration. Resveratrol concentrations in the analysed samples varied from 4.1 to 14.1 ng mL⁻¹. This is the first proposed spectroscopic method for determination of resveratrol in beer.     

A reversed-phase high performance liquid chromatography coupled with resonance Rayleigh scattering detection for the determination of four tetracycline antibiotics

A new reversed-phase high performance liquid chromatography with resonance Rayleigh scattering detection (HPLC-RRS) was developed for simultaneous separation and determination of four tetracycline antibiotics (TCs). A good chromatographic separation among the compounds was achieved using a Synergi Fusion-RP column (150 mm × 4.6 mm; 4 μm) and a mobile phase consisting of methanol-acetonitrile-oxalic acid (5 mM) at the flow rate of 0.8 mL min⁻¹. Column temperature was 30 °C. The RRS signal was detected at λ_ex = λ_em = 370 nm. The recoveries of sample added standard ranged from 95.3% to 103.5%, and the relative standard deviation was below 2.79%. A detection limit of 2.12-5.12 μg mL⁻¹ was reached and a linear range was found between peak height and concentration in the range of 10.36-518.0 μg mL⁻¹ for oxytetracycline (OTC), 12.11-605.5 μg mL⁻¹ for tetracycline (TC), 11.79-589.5 μg mL⁻¹ for chlortetracycline (CTC) and 10.32-516.0 μg mL⁻¹ for doxycycline (DC). The linear regression coefficients were all above 0.999. The method has been applied successfully to the determination of OTC, TC, CTC, DC in pharmaceutical formulations and in honey. The method was simple, rapid and showed a better linear relation and high repeatability.     

Double electrochemical covalent coupling method based on click chemistry and diazonium chemistry for the fabrication of sensitive amperometric immunosensor

A double electrochemical covalent coupling method based on click chemistry and diazonium chemistry for the fabrication of sensitive amperometric immunosensor was developed. As a proof-of-concept, a designed alkyne functionalized human IgG was used as a capture antibody and a HRP-labeled rabbit anti-goat IgG was used as signal antibody for the determination of the anti-human IgG using the sandwich model. The immunosensor was fabricated by electrochemically grafting a phenylazide on the surface of a glassy carbon electrode, and then, by coupling the alkyne functionalized human IgG with the phenylazide group through an electro-click chemistry in the presence of Cu(II). The amperometric measurement for the determination of the anti-human IgG was performed after the fabricated immunosensor was incubated with the target anti-human IgG and then with the HRP-labeled anti-goat IgG at −0.25 V in 0.10 M PBS (pH 7.0) containing 0.1 mM hydroquinone and 2.0 mM H₂O₂. The results showed that the increased current was linear with the logarithm of the concentration of the anti-human IgG in the range from 1.0 × 10⁻¹⁰ g mL⁻¹ to 1.0 × 10⁻⁸ g mL⁻¹ with a detection limit of 3 × 10⁻¹¹ g mL⁻¹. Furthermore, the feasibility of the double electrochemical covalent coupling method proposed in this work for fabricating the amperometric immunosensor array was explored. This work demonstrates that the double electrochemical covalent coupling method is a promising approach for the fabrication of the immunosensor and immunosensor array.     

Amperometric detection of Enterobacteriaceae in river water by measuring β-galactosidase activity at interdigitated microelectrode arrays

Two simple methodologies are compared for the detection of faecal contamination in water using amperometry at gold interdigitated microelectrodes. They rely on the detection of β-galactosidase (β-gal) by redox cycling amperometry of the p-aminophenol (PAP) produced by the enzyme from the 4-aminophenyl β-d-galactopyranoside (PAPG) substrate. The use of phages as specific agents for the release of the bacteria-enclosed enzyme allowed the detection of 6 × 10⁵ CFU mL⁻¹Escherichia coli in 2 h without any pre-enrichment or preconcentration steps. Better limits of detection were achieved for the second strategy in the absence of phages. In this case, bacteria were enriched in the presence of both β-d-1-thiogalactopyranoside (IPTG) and substrate but in the absence of phages. Under such experimental conditions, 5 × 10⁴ CFU mL⁻¹E. coli could be detected after 2 h of incubation, while 7 h of incubation were enough to detect down to 10 CFU mL⁻¹ in river water samples. This represents a straightforward one-step method for the detection of faecal contamination that can be conducted in a single working day with minimal sample manipulation by the user.     

Production and characterization of a broad-specificity polyclonal antibody for O,O-diethyl organophosphorus pesticides and a quantitative structure-activity relationship study of antibody recognition

Polyclonal antibody (PAb) with broad-specificity for O,O-diethyl organophosphorus pesticides (OPs) against a generic hapten, 4-(diethoxyphosphorothioyloxy)benzoic acid, was produced. The obtained PAb showed high sensitivity to seven commonly used O,O-diethyl OPs in a competitive indirect enzyme-linked immunosorbent assay (ciELISA) using a heterologous coating antigen, 4-(3-(diethoxyphosphorothioyloxy)phenylamino)-4-oxobutanoic acid. The 50% inhibition value (IC₅₀) was 348 ng mL⁻¹ for parathion, 13 ng mL⁻¹ for coumaphos, 22 ng mL⁻¹ for quinalphos, 35 ng mL⁻¹ for triazophos, 751 ng mL⁻¹ for phorate, 850 ng mL⁻¹ for dichlofenthion, and 1301 ng mL⁻¹ for phoxim. The limit of detection (LOD) met the ideal detection criteria of all the seven OP residues. A quantitative structure-activity relationship (QSAR) model was constructed to study the mechanism of antibody recognition using multiple linear regression analysis. The results indicated that the frontier-orbital energies (energy of the highest occupied molecular orbital, E_HOMO, and energy of the lowest unoccupied molecular orbital, E_LUMO) and hydrophobicity (log of the octanol/water partition coefficient, log P) were mainly responsible for the antibody recognition. The linear equation was log(IC₅₀) = −63.274E_HOMO + 15.985E_LUMO + 0.556 log P − 25.015, with a determination coefficient (r²) of 0.908.     

Simultaneous screening analysis of multiple beta-blockers in urine by gas chromatography-mass spectrometry in selected ion monitoring mode

A rapid screening procedure is described for the simultaneous determination of 13 β-blockers in urine at the range of 0.010-1.0 μg mL⁻¹. The procedure involves N-ethoxycarbonyl (EOC) derivatization of β-blockers in urine sample, followed by extraction and further conversion to trimethylsilyl (TMS) derivatives for the analysis by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode (GC-SIM-MS). The characteristic fragment ions at m/z 260 and m/z 144, and [M − 15]⁺ ions permitted sensitive and selective detection of most of the β-blockers in the presence of co-extracted urinary amino alcohols at much higher levels. The whole procedure of EOC/TMS derivatization with subsequent GC-SIM-MS analysis was linear (r ≥ 0.9988). The LODs were varied from 0.03 to 2.7 ng mL⁻¹. The ranges of precision (%relative standard deviation) and accuracy (%relative error) of the overall procedure at two different added amounts (0.02 and 0.5 μg mL⁻¹) in urine matrix varied from 1.3 to 9.4 and from −9.6 to 9.7, respectively. The recoveries were measured to be ranged from 90.4 to 109.7%.     

Determination of beta-lactam antibiotics in milk using micro-flow chemiluminescence system with on-line solid phase extraction

In this paper, a chemiluminescence (CL) micro-flow system combined with on-line solid phase extraction (SPE) is presented for determination of β-lactam antibiotics (penicillin, cefradine, cefadroxil, cefalexin) in milk. It is based on the enhancement effect of β-lactam antibiotics on the luminol-K₃Fe(CN)₆ CL system. The micro-flow system was fabricated from two polymethyl methacrylate (PMMA) plates (50 mm × 40 mm × 5 mm) with the microchannels of 200 μm wide and 150 μm deep. C₁₈-modified silica gel was packed into the microchannel (length: 10 mm; width: 1 mm; depth: 500 μm) to serve as SPE device. Extraction and preconcentration of the analytes were carried out using on-line SPE micro-flow system and the selectivity of CL detection was improved. The detection limits were 0.5 μg mL⁻¹ of penicillin, 0.04 μg mL⁻¹ of cefradine, 0.08 μg mL⁻¹ of cefadroxil and 0.1 μg mL⁻¹ of cefalexin. The proposed method was also applied to analyze the β-lactam antibiotics in milk. Experimental results were in good agreement with those obtained by high performance liquid chromatography (HPLC) method with UV detection.     

Amperometric determination of bonded glucose with an MnO(2) and glucose oxidase bulk-modified screen-printed electrode using flow-injection analysis

A screen-printed amperometric biosensor based on carbon ink double bulk-modified with MnO₂ as a mediator and glucose oxidase as a biocomponent was investigated for its ability to serve as a detector for bonded glucose in different compounds, such as cellobiose, saccharose, (-)-4-nitrophenyl-β-d-glucopyranoside, as well as in beer samples by flow-injection analysis (FIA). The biosensor could be operated under physiological conditions (0.1 M phosphate buffer, pH 7.5) and exhibited good reproducibility and stability. Bonded glucose was released with glucosidase in solution, and the free glucose was detected with the modified screen-printed electrode (SPE). The release of glucose by the aid of glucosidase from cellobiose, saccharose and (-)-4-nitrophenyl-β-d-glucopyranoside in solution showed that stoichiometric quantities of free glucose could be monitored in all three cases.The linear range of the amperometric response of the biosensor in the FIA-mode flow rate 0.2 mL min⁻¹, injection volume 0.25 mL, operation potential 0.48 V versus Ag/AgCl) extends from 11 to 13,900 μmol L⁻¹ glucose in free form. The limit of detection (3σ) is 1 μmol L⁻¹ glucose. A concentration of 100 μmol L⁻¹ yields a relative standard deviation of approximately 7% with five injections. These values correspond to the same concentrations of bonded glucose supposed that it is liberated quantitatively (incubation for 2 h with glucosidase).Bonded glucose could be determined in beer samples using the same assay. The results corresponded very well with the reference procedure.     

Novel reductive–reductive mode electrochemical detection of Rohypnol following liquid chromatography and its determination in coffee

Rohypnol (flunitrazepam) has been successfully determined in coffee by high performance liquid chromatography dual electrode detection (LC-DED) in the dual reductive mode. Initial studies were performed to optimise the chromatographic conditions and these were found to be 50% acetonitrile, 50% 50 mM pH 2.0 phosphate buffer at a flow rate of 0.75 mL min⁻¹, employing a Hypersil C₁₈, 5 μm, 250 mm × 4.6 mm column. Cyclic voltammetric studies were made to ascertain the redox behaviour of Rohypnol at a glassy carbon electrode over the pH range 2–12. Hydrodynamic voltammetry was used to optimise the applied potential at the generator and detector cells; these were identified to be −2.4 V and +0.8 V for the redox mode and −2.4 V and −0.1 V for the dual reductive mode respectively. A linear range of 0.5–100 μg mL⁻¹, with a detection limit of 20 ng mL⁻¹ was obtained for the dual reductive mode. Further studies were then performed to identify the optimum conditions required for the LC-DED determination of Rohypnol in beverage samples. A convenient and rapid method for the determination of Rohypnol in beverage samples was developed using a simple sample pre-treatment procedure. A recovery of 95.5% was achieved for a sample of white coffee fortified at 9.6 μg mL⁻¹ Rohypnol.     

Investigation on enhanced chemiluminescence reaction systems with bis(hydrogenperiodato) argentate(III) complex anion for fluoroquinolones synthetic antibiotics

A novel chemiluminescence (CL) reaction system with bis(hydrogenperiodato) argentate(III) complex anion (Ag(III) complex, [Ag(HIO₆)₂]⁵⁻), for the first time, is developed for the determination of lomefloxacin (LMFX), enrofloxacin (ENLX) and pefloxacin (PFLX). The possible CL emission mechanism was discussed by comparing the fluorescence emission with CL spectra. The CL conditions of [Ag(HIO₆)₂]⁵⁻-H₂SO₄-LMFX/ENLX/PFLX systems were investigated and optimized. Under the optimized experimental conditions, the CL intensity is proportional to the concentration of the drugs in the range 0.2994-36.80 × 10⁻⁷ g mL⁻¹ for LMFX, 4.00-30.0 × 10⁻⁷ g mL⁻¹ for ENLX and 1.54-27.64 × 10⁻⁷ g mL⁻¹ for PFLX. The limit of detection (s/n = 3) was 9.1 × 10⁻⁹ g mL⁻¹ for LMFX, 3.1 × 10⁻⁹ g mL⁻¹ for ENLX and 4.4 × 10⁻⁹ g mL⁻¹ for PFLX. The recovery of LMFX, ENLX and PELX from the spiked pharmaceutical preparations was in the range of 92.3-105% with the RSDs of 0.5-2.7%. For urine, serum and milk samples the recoveries of the three drugs were in the range of 85.1-107% for LMFX with the RSDs of 2.3-3.4%. 80.2-112% for ENLX with the RSDs of 1.4-2.8%, and 87.8-114% for PFLX with the RSDs of 1.6-2.7%. The proposed method was applied successfully to the determination of these compounds in real samples.     

Determination of the antidepressant paroxetine and its three main metabolites in human plasma by liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method has been developed for the determination in human plasma of the specific serotonin reuptake inhibitor (SSRI) antidepressant paroxetine and its three main metabolites (M1, M2, M3). Fluorescence detection was used, exciting at λ = 294 nm and monitoring emission at λ = 330 nm for paroxetine (λ_exc = 280 nm, λ_em = 330 nm for M1 and M2; λ_exc = 268 nm, λ_em = 290 nm for M3). Separation was obtained on a reversed-phase C18 column using a mobile phase composed of 66.7% aqueous phosphate at pH 2.5 and 33.3% acetonitrile. Imipramine (λ_exc = 252 nm, λ_em = 390 nm) was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with C8 cartridges (50 mg, 1 mL). The calibration curves were linear over a working range of 2.5-100 ng mL⁻¹ for paroxetine and of 5-100 ng mL⁻¹ for all metabolites. The limit of detection (LOD) was 1.2 ng mL⁻¹ for PRX and 2.0 ng mL⁻¹ for the metabolites. The method was applied with success to plasma samples from depressed patients undergoing treatment with paroxetine. Hence, the method seems to be suitable for the therapeutic drug monitoring of paroxetine and its main metabolites in depressed patients’ plasma.     

Nanostructured progesterone immunosensor using a tyrosinase-colloidal gold-graphite-Teflon biosensor as amperometric transducer

A novel progesterone immunosensor using a colloidal gold-graphite-Teflon-tyrosinase composite biosensor as amperometric transducer is reported. A sequential competitive configuration between the analyte and progesterone labelled with alkaline phosphatase (AP) was used. Phenyl phosphate was employed as the AP-substrate and the enzyme reaction product, phenol, was oxidized by tyrosinase to o-quinone, which is subsequently reduced at −0.1 V at the biocomposite electrode. Variables such as the concentration of phenyl phosphate, the amount of antibody attached to the electrode surface, immersion time in a 2% BSA solution, working pH and incubation times in progesterone and AP conjugate were optimized. A linear calibration graph for progesterone was obtained between 0 and 40 ng mL⁻¹ with a slope value of −82.3 nA ng⁻¹ mL, and a detection limit of 0.43 ng mL⁻¹. The time needed to reach the steady-state current from the addition of phenyl phosphate was 30-40 s. These analytical characteristics improve substantially those reported for other progesterone immunosensors. A lifetime of 14 days with no need to apply any regeneration procedure was also achieved. The usefulness of the immunosensor was evaluated by determining progesterone in milk samples spiked with the analyte at 5.0 and 1.5 ng mL⁻¹ concentration levels. Following a very simple procedure, involving only sample dilution, mean recoveries (n = 7) of 98 ± 3% and 99 ± 3%, respectively, were obtained.     

Construction of label-free electrochemical immunosensor on mesoporous carbon nanospheres for breast cancer susceptibility gene

In this contribution, mesoporous carbon nanospheres (MCN) were used to fabricate a label-free electrochemical immunosensor for breast cancer susceptibility gene (BRCAl). The detection platform was constructed by conjugation of anti-BRCA1 on glassy carbon electrodes which were modified by mesoporous carbon nanospheres–toluidine blue nanocomposite (MCN–TB)/room temperature ionic-liquid (RTIL) composited film. TB was adsorbed onto MCN and acted as a redox probe. The electroactivity of TB was greatly enhanced in the presence of MCN. The good conductivity of MCN and BMIM·BF₄ could promote the electron transfer and thus enhance the detection sensitivity. Moreover, the large surface area of MCN and the protein-binding properties of BMIM·BF₄ could greatly increase the antibody loading. The specific antibody–antigen immunoreaction on the electrode surface resulted in a decrease of amperometric signal of the electrode. Under optimized conditions, the amperometric signal decreased linearly with BRCAl concentration in the range of 0.01–15 ng mL⁻¹ with a low detection limit of 3.97 pg mL⁻¹. The immunosensor exhibits high sensitivity, good selectivity and stability.     

Development of an immunosensor for the detection of testosterone in bovine urine

In the presented work, a disposable immunosensor for the detection of testosterone, an endogenous steroid hormone, in bovine urine has been developed using screen-printed electrodes (SPEs). Due to concerns over the use of steroid hormones as growth promoters, the EU prohibits their use in food producing animals. Consequently, rigorous screening procedures have been implemented in all member states to detect the illegal administration of such compounds. Competitive immunoassays were developed, initially by enzyme linked immunosorbent assay (ELISA), and subsequently transferred to an electrochemical immunosensor format using disposable screen-printed carbon electrodes. Horseradish peroxidase (HRP) was the enzyme label of choice and chronoamperometric detection was carried out using a tetramethylbenzidine/hydrogen peroxide (TMB/H₂O₂) substrate system, at +100 mV. The EC₅₀ values obtained for the assay in buffer and urine gave relatively comparable results, 710 pg mL⁻¹ and 960 pg mL⁻¹, respectively. The linear range obtained for the assay in buffer extended from 0.03 ng mL⁻¹ to 40 ng mL⁻¹; while that in urine ranged from 0.03 ng mL⁻¹ to 1.6 ng mL⁻¹. The corresponding limits of detection (LOD) in buffer and urine were 26 pg mL⁻¹ and 1.8 pg mL⁻¹. Cross reactivity profiles of the antibody have been examined, with notable cross reactivities with 19-nortestosterone (11.6%) and boldenone (9.86%). Precision studies for the sensor demonstrated adequate reproducibility (CV < 13%, n = 3) and repeatability (CV < 9%, n = 3). Recovery data obtained showed good agreement between spiking studies and known concentrations of analyte. Sensors showed stability for 4 days at +4 °C. A sensitive, highly specific, inexpensive, disposable immunosensor, showing excellent overall performance for the detection of testosterone in bovine urine, has been developed.     

Modification of polypyrrole nanowires array with platinum nanoparticles and glucose oxidase for fabrication of a novel glucose biosensor

A novel glucose biosensor, based on the modification of well-aligned polypyrrole nanowires array (PPyNWA) with Pt nanoparticles (PtNPs) and subsequent surface adsorption of glucose oxidase (GOx), is described. The distinct differences in the electrochemical properties of PPyNWA–GOx, PPyNWA–PtNPs, and PPyNWA–PtNPs–GOx electrodes were revealed by cyclic voltammetry. In particular, the results obtained for PPyNWA–PtNPs–GOx biosensor showed evidence of direct electron transfer due mainly to modification with PtNPs. Optimum fabrication of the PPyNWA–PtNPs–GOx biosensor for both potentiometric and amperometric detection of glucose were achieved with 0.2 M pyrrole, applied current density of 0.1 mA cm⁻², polymerization time of 600 s, cyclic deposition of PtNPs from −200 mV to 200 mV, scan rate of 50 mV s⁻¹, and 20 cycles. A sensitivity of 40.5 mV/decade and a linear range of 10 μM to 1000 μM (R² = 0.9936) were achieved for potentiometric detection, while for amperometric detection a sensitivity of 34.7 μA cm⁻² mM⁻¹ at an applied potential of 700 mV and a linear range of 0.1–9 mM (R² = 0.9977) were achieved. In terms of achievable detection limit, potentiometric detection achieved 5.6 μM of glucose, while amperometric detection achieved 27.7 μM.     

Simultaneous determination of salbutamol sulphate, bromhexine hydrochloride and etofylline in pharmaceutical formulations with the use of four rapid derivative spectrophotometric methods

Four simple, rapid, accurate, precise, reliable and economical spectrophotometric methods have been proposed for simultaneous determination of salbutamol sulphate (SS), bromhexine hydrochloride (BH) and etofylline (ET) in pure and commercial formulations without any prior separation or purification. They were first derivative zero crossing spectrophotometry (method 1), simultaneous equation method (method 2), derivative ratio spectra zero crossing method (method 3) and double divisor ratio spectra derivative method (method 4). The ranges for SS, BH and ET were found to be 1-35 μg mL⁻¹, 4-40 μg mL⁻¹ and 5-80 μg mL⁻¹. For methods 1 and 2, the values of limit of detection (LOD) were 0.2314 μg mL⁻¹, 0.4865 μg mL⁻¹ and 0.2766 μg mL⁻¹ and the values of limit of quantitation (LOQ) were 0.7712 μg mL⁻¹, 1.6217 μg mL⁻¹ and 0.9221 μg mL⁻¹ for SS, BH and ET, respectively. For method 3, LOD values were 0.3297 μg mL⁻¹, 0.2784 μg mL⁻¹ and 0.7906 μg mL⁻¹ and LOQ values were 0.9325 μg mL⁻¹, 0.9282 μg mL⁻¹ and 2.6352 μg mL⁻¹ for SS, BH and ET, respectively. For method 4, LOD values were 0.3161 μg mL⁻¹, 0.2495 μg mL⁻¹ and 0.2064 μg mL⁻¹ and LOQ values were 0.9869 μg mL⁻¹, 0.8317 μg mL⁻¹ and 0.6879 μg mL⁻¹ for SS, BH and ET. The precision values were less then 2% R.S.D. for all four methods. The common excipients and additives did not interfere in their determinations. The results obtained by the proposed methods have been statistically compared by means of Student t-test and by the variance ratio F-test.     

Facile, sensitive and selective fluorescence turn-on detection of HSA/BSA in aqueous solution utilizing 2,4-dihydroxyl-3-iodo salicylaldehyde azine

A novel fluorescence turn-on detection method of human serum albumin (HSA) and bovine serum albumin (BSA) in aqueous solution is investigated using 2,4-dihydroxyl-3-iodo salicylaldehyde azine (DISA). Upon the addition of DISA to HSA/BSA solution, a fluorescence turn-on effect at 529 nm can be observed with a large stokes shift of ∼129 nm based on hydrophobic binding-mode between protein and dye. Under the optimal condition, the linear ranges of fluorescence intensity for HSA and BSA are 0.1-30 μg mL⁻¹ with the relative correlation coefficient of R² = 0.991 (n = 10) and 0.3-50 μg mL⁻¹ with R² = 0.997 (n = 10); and the detection limits for HSA and BSA based on IUPAC (C_DL = 3S_b/m) are 20 ng mL⁻¹ and 50 ng mL⁻¹, respectively.     

Flame atomic absorption spectrometry for the determination of trace amount of rhodium after separation and preconcentration onto modified multiwalled carbon nanotubes as a new solid sorbent

The present article reports on the application of modified multiwalled carbon nanotubes (MMWCNTs) as a new, easily prepared and stable solid sorbent for the preconcentration of trace rhodium ion in aqueous solution. Rhodium ions were complexed with 1-(2-pyridylazo)-2-naphthol (PAN) in the pH range of 3.2-4.7 and then the formed Rh-PAN complex was adsorbed on the oxidized MWCNTs. The adsorbed complex was eluted from MWCNTs sorbent with 5.0 mL of N,N-dimethylformamide (DMF). The rhodium in eluted solution was determined by flame atomic absorption spectrometry (FAAS). Linear range for the determination of rhodium was maintained between 0.16 ng mL⁻¹ and 25.0 μg mL⁻¹ in initial solution. Relative standard deviation for the 10 replicated determination of 4.0 μg mL⁻¹ of rhodium was ±0.97%. Detection limit was 0.010 ng mL⁻¹ in initial solution (3S_bl, n = 10) and preconcentration factor was 120. Sensitivity for 1% absorbance of rhodium (III) was 0.112 μg mL⁻¹. The sorption capacity of oxidized MWCNTs for Rh (III) was 6.6 mg g⁻¹. The effects of the experimental parameters, including the sample pH, flow rates of sample and eluent solution, eluent type, breakthrough volume and interference ions were studied for the preconcentration of Rh³⁺. The proposed method was successfully applied to the extraction and determination of rhodium in different samples.     

Disposable amperometric magnetoimmunosensor for the sensitive detection of the cardiac biomarker amino-terminal pro-B-type natriuretic peptide in human serum

A novel amperometric magnetoimmunosensor using an indirect competitive format is developed for the sensitive detection of the amino-terminal pro-B-type natriuretic peptide (NT-proBNP). The immunosensor design involves the covalent immobilization of the antigen onto carboxylic-modified magnetic beads (HOOC-MBs) activated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), and further incubation in a mixture solution containing variable concentrations of the antigen and a fixed concentration of an HRP-labeled detection antibody. Accordingly, the target NT-proBNP in the sample and that immobilized on the MBs compete for binding to a fixed amount of the specific HRP-labeled secondary antibody. The immunoconjugate-bearing MBs are captured by a magnet placed under the surface of a disposable gold screen-printed electrode (Au/SPE). The amperometric responses measured at –0.10 V (vs. a Ag pseudo-reference electrode), upon addition of 3,3′,5,5′-tetramethylbenzidine (TMB) as electron transfer mediator and H₂O₂ as the enzyme substrate, are used to monitor the affinity reaction. The developed magnetoimmunosensor provides attractive analytical characteristics in 10-times diluted human serum samples, exhibiting a linear range of clinical usefulness (0.12–42.9 ng mL⁻¹) and a detection limit of 0.02 ng mL⁻¹, which can be used in clinical diagnosis of chronic heart failure in the elderly and for classifying patients at risk of death after heart transplantation. The magnetoimmunosensor was successfully applied to the analysis of spiked human serum samples.