Selective optical sensing of biothiols with Ellman's reagent: 5,5′-Dithio-bis(2-nitrobenzoic acid)-modified gold nanoparticles

Development of sensitive and selective methods of determination for biothiols is important because of their significant roles in biological systems. We present a new optical sensor using Ellman's reagent (DTNB)-adsorbed gold nanoparticles (Au-NPs) (DTNB-Au-NP) in a colloidal solution devised to selectively determine biologically important thiols (biothiols) from biological samples and pharmaceuticals. 5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB), a versatile water-soluble compound for quantitating free sulfhydryl groups in solution, was adsorbed through non-covalent interaction onto Au-NPs, and the absorbance changes associated with the formation of the yellow-colored 5-thio-2-nitrobenzoate (TNB²⁻) anion as a result of reaction with biothiols was measured at 410 nm. The sensor gave a linear response over a wide concentration range of standard biothiols comprising cysteine, glutathione, homocysteine, cysteamine, dihydrolipoic acid and 1,4-dithioerythritol. The calibration curves of individual biothiols were constructed, and their molar absorptivities and linear concentration ranges determined. The cysteine equivalent thiol content (CETC) values of various biothiols using the DTNB-Au-NP assay were comparable to those of the conventional DTNB assay, showing that the immobilized DTNB reagent retained its reactivity toward thiols. Common biological sample ingredients like amino acids, flavonoids, vitamins, and plasma antioxidants did not interfere with the proposed sensing method. This assay was validated through linearity, additivity, precision and recovery, demonstrating that the assay is reliable and robust. DTNB-adsorbed Au-NPs probes provided higher sensitivity (i.e., lower detection limits) in biothiol determination than conventional DTNB reagent. Under optimized conditions, cysteine (Cys) was quantified by the proposed assay, with a detection limit (LOD) of 0.57 μM and acceptable linearity ranging from 0.4 to 29.0 μM (r = 0.998).     

A fluorescence turn-on method for real-time monitoring of protease activity based on the electron transfer between a fluorophore labeled oligonucleotide and cytochrome c

A new continuous fluorescence turn-on assay for protease activity and inhibitor screening has been developed. A fluorophore labeled single stranded DNA (FAM-DNA) and cytochrome c (cyt c) were employed. The fluorescence of the FAM-DNA was efficiently quenched when binding to cyt c, through the electron transfer between the FAM fluorophore and the heme cofactor of cyt c. In the presence of a protease, such as trypsin, cyt c was digested into small peptide fragments. The FAM-DNA was released, which resulted in the recovery of the FAM fluorescence. The rate of the cyt c digestion could be reduced via the addition of an inhibitor. As a result, reduced degree of the fluorescence recovery was obtained. The limit of detection of our assay is 1 nM trypsin and the IC₅₀ values are 3.23 μg mL⁻¹ and 0.303 μg mL⁻¹ for the inhibitor from egg white and the inhibitor from soybean, respectively. Our method could be used for the sensing of protease activity for various biochemical applications, and for the screening of protease inhibitors as drugs for the treatment of various related diseases.     

Extremely sensitive sandwich assay of kanamycin using surface-enhanced Raman scattering of 2-mercaptobenzothiazole labeled gold@silver nanoparticles

Herein, we report the development of extremely sensitive sandwich assay of kanamycin using a combination of anti-kanamycin functionalized hybrid magnetic (Fe₃O₄) nanoparticles (MNPs) and 2-mercaptobenzothiazole labeled Au-core@Ag-shell nanoparticles as the recognition and surface-enhanced Raman scattering (SERS) substrate, respectively. The hybrid MNPs were first prepared via surface-mediated RAFT polymerization of N-acryloyl-l-glutamic acid in the presence of 2-(butylsulfanylcarbonylthiolsulfanyl) propionic acid-modified MNPs as a RAFT agent and then biofunctionalized with anti-kanamycin, which are both specific for kanamycin and can be collected via a simple magnet. After separating kanamycin from the sample matrix, they were sandwiched with the SERS substrate. According to our experimental results, the limit of detection (LOD) was determined to be 2 pg mL⁻¹, this value being about 3–7 times more than sensitive than the LOD of previously reported results, which can be explained by the higher SERS activity of silver coated gold nanoparticles. The analysis time took less than 10 min, including washing and optical detection steps. Furthermore, the sandwich assay was evaluated for investigating the kanamycin specificity on neomycin, gentamycin and streptomycin and detecting kanamycin in artificially contaminated milk.     

Sensitive and simple detection of Escherichia coli strain based on time-resolved fluorescence DNA hybridization assay

A two-probe tandem DNA hybridization assay based on time-resolved fluorescence was employed to detect Escherichia coli strain. The amino modified capture probe was covalently immobilized on the common glass slide surface. The Eu(TTA)₃(5-NH₂-phen) with the characteristics of long lifetime and intense luminescence was labeled with reporter probe. The original extracted DNA samples without the purification and amplification process were directly used in the hybridization assay. The concentration of capture probe, hybridization temperature, hybridization and washing time were optimized. The detection limit is about 1.49 × 10³ CFU mL⁻¹E. coli cells, which is comparable to the value of most microbiology methods. The proposed method has the advantages of easy operation, satisfactory sensitivity and specificity, which can provide a promising technique for monitoring the microorganisms.     

On-chip integrated hydrolysis, fluorescent labeling, and electrophoretic separation utilized for acetylcholinesterase assay

A sensitive on-chip acetylcholinesterase (AChE) assay that serves as a basis for the development of a fully integrated on-chip AChE-inhibitor detection assay is presented. The sequential steps required for the on-chip analysis process were integrated into a microchip. Transport and mixing of the reagents occurred by a combination of electroosmosis and electrophoresis using computer-controlled electrokinetic transport. AChE-catalyzed hydrolysis of acetylthiocholine to thiocholine was determined by on-chip reaction of thiocholine with eosinmaleimide, and the resulting thioether was electrophoretically separated and detected by laser-induced fluorescence (LIF). Enzyme-substrate mixing and reaction by confluent flow of reagents was compared with electrophoretically mediated microanalysis (EMMA), based on injection of an enzyme plug, and the utilization of differences in electrophoretic mobility as a driving force for efficient mixing and reaction. Both methods yielded similar results, however the EMMA-plug technique is preferable. The EMMA-plug technique was optimized for length and pushing time of enzyme plug, length of dyes mixture plug, acetylthiocholine concentration, and detector location. Detection of O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothiolate (VX) and paraoxon, two AChE inhibitors, was demonstrated by off-chip mixing of the inhibitor and AChE, followed by the on-chip AChE assay. Limit of detection of VX for 5.5 min incubation and of paraoxon for 8 min incubation was 4 × 10⁻¹⁰ and 4 × 10⁻⁷ M, respectively. Utilization of the AChE microchip assay for inhibition kinetics was demonstrated also by evaluation of the inhibitor-enzyme bimolecular reaction constant (k_i). The evaluated k_i values for VX and paraoxon for AChE from the electric eel were 3.5 × 10⁷ and 1.7 × 10⁵ M⁻¹ min⁻¹, respectively, conforming well to reported values obtained by bulk methods.     

A gold@silica core–shell nanoparticle-based surface-enhanced Raman scattering biosensor for label-free glucose detection

The gold nanostar@silica core–shell nanoparticles conjugated with glucose oxidase (GOx) enzyme molecules have been developed as the surface-enhanced Raman scattering (SERS) biosensor for label-free detection of glucose. The surface-immobilized GOx enzyme catalyzes the oxidation of glucose, producing hydrogen peroxide. Under laser excitation, the produced H₂O₂ molecules near the Au nanostar@silica nanoparticles generate a strong SERS signal, which is used to measure the glucose concentration. The SERS signal of nanostar@silica∼GOx nanoparticle-based sensing assay shows the dynamic response to the glucose concentration range from 25 μM to 25 mM in the aqueous solution with the limit of detection of 16 μM. The sensing assay does not show any interference when glucose co-exists with both ascorbic acid and uric acid. The sensor can be applied to a saliva sample.     

Generic simple enzyme immunoassay approach to avert small molecule immobilization problems on solid phases: Application to the determination of tobramycin in serum

Generic simple and sensitive universal enzyme immunoassay approach for the determination of small analytes has been developed to avert the problems associated with small molecule immobilization onto solid phases. The developed assay employed a heterogeneous non-competitive binding format. The assay used anti-analyte antibody coupled to polyacrylamide beads as a solid-phase and β-d-galactosidase enzyme-labeled analyte as a label. In this assay, the analyte in a sample was firstly incubated to react with an excess of the antibody-coupled beads, and then the unoccupied antibody binding sites were allowed to react with the enzyme-labeled analyte. Analyte bound to the antibody-coupled beads was separated by centrifugation, and the enzyme activity of the supernatant was measured spectrophotometrically at 420 nm, after reaction with 4-nitrophenyl-β-d-galactopyranoside as a substrate for the enzyme. The signal was directly proportional to the concentration of analyte in the sample. The optimum conditions for the developed assay were established and applied to the determination of tobramycin, as a representative example of the small analytes, in serum samples. The assay limit of detection was 10 ng mL⁻¹ and the effective working range at relative standard deviation of ≤10% was 40-800 ng mL⁻¹. The assay precisions were acceptable; the relative standard deviations were 4.36-5.17 and 5.62-7.40% for intra- and inter-assay precision, respectively. Analytical recovery of tobramycin spiked in serum ranged from 95.89 ± 4.25 to 103.45 ± 4.60%. The assay results correlated well with those obtained by high-performance liquid chromatography (r = 0.992). The assay described herein has great practical value in determination of small analytes because it is sensitive, rapid, and easy to perform in any laboratory. Although the assay was validated for tobramycin, however, it is also anticipated that the same methodology could be used for essentially any analyte for which a selective antibody exists, and an appropriate enzyme conjugate can be made.     

Development of a bienzyme reactor sensor system for the determination of ornithine

A bienzyme reactor sensor system with amperometric detection was developed for the determination of ornithine. The system based on the immobilized enzymes (ornithine carbamyl transferase and pyruvate oxidase) consisted of a buffer tank, a peristaltic pump, an enzyme reactor, an oxygen electrode and a recorder. Then, 0.1 M MOPS buffer, containing pyruvic acid (0.5 mM) and carbamyl phosphate (0.5 mM), was continuously transferred into the system at 35 °C. Phosphate ion was formed enzymatically by transformation of ornithine in the presence of carbamyl phosphate. Pyruvate oxidase is activated by the presence of phosphate. Therefore, ornithine was determined from the oxygen consumed upon oxidation of pyruvic acid catalyzed by pyruvate oxidase in the presence of phosphate ion. The limit of detection was 0.05 mM and the response was linear to 3 mM (R²=0.9905). The variation coefficients were 4.9 (n=15) and 3.9% (n=15) for 1.1 and 3.0 mM standard ornithine, respectively. Good comparative results (R²=0.9238) were observed between ornithine contents in prawn muscle determined by the proposed system and by the HPLC. One assay was completed within 4 min. The immobilized enzymes were stable for 2 months at 4 °C and more than 150 samples could be continually determined using this enzyme reactor.     

Surface-imprinted core–shell Au nanoparticles for selective detection of bisphenol A based on surface-enhanced Raman scattering

Surface-imprinted core–shell Au nanoparticles (AuNPs) were explored for the highly selective detection of bisphenol A (BPA) by surface-enhanced Raman scattering (SERS). A triethoxysilane-template complex (BPA-Si) was synthesized and then utilized to fabricate a molecularly imprinted polymer (MIP) layer on the AuNPs via a sol–gel process. The imprinted BPA molecules were removed by a simple thermal treatment to generated the imprint-removed material, MIP-ir-AuNPs, with the desired recognition sites that could selectively rebind the BPA molecules. The morphological and polymeric characteristics of MIP-ir-AuNPs were investigated by transmission electron microscopy and Fourier-transform infrared spectroscopy. The results demonstrated that the MIP-ir-AuNPs were fabricated with a 2 nm MIP shell layer within which abundant amine groups were generated. The rebinding kinetics study showed that the MIP-ir-AuNPs could reach the equilibrium adsorption for BPA within 10 min owning to the advantage of ultrathin core–shell nanostructure. Moreover, a linear relationship between SERS intensity and the concentration of BPA on the MIP-ir-AuNPs was observed in the range of 0.5–22.8 mg L⁻¹, with a detection limit of 0.12 mg L⁻¹ (blank ± 3 × s.d.). When applied to SERS detection, the developed surface-imprinted core–shell MIP-ir-AuNPs could recognize BPA and prevent interference from the structural analogues such as hexafluorobisphenol A (BPAF) and diethylstilbestrol (DES). These results revealed that the proposed method displayed significant potential utility in rapid and selective detection of BPA in real samples.     

Molecular beacons: a real-time polymerase chain reaction assay for detecting Escherichia coli from fresh produce and water

Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. The development of a real-time polymerase chain reaction (PCR) assay to detect the presence of Escherichia coli using these fluorogenic reporter molecules is reported. MBs were designed to recognize a 19-bp region of the uid A gene, coding for an enzyme β-glucuronidase. The specificity of the MB-based PCR assay was evaluated for various E. coli strains as well as bacteria species that are present in nature. The capability of the assay to detect E. coli in drinking water and produce was demonstrated. Positive detection of E. coli was demonstrated when >10¹ CFU mL⁻¹ (colony forming unit) was present in the water samples and fresh produce after 18 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli in food and environmental samples.     

Enzyme enhanced quantitative determination of multiple DNA targets based on capillary electrophoresis

In the current paper, enzyme enhanced simultaneous quantitative determination of multiple DNA targets based on capillary electrophoresis (CE) was described. We used three biotin-modified DNA probes, which reacted with avidin-conjugated horseradish peroxidase (avidin-HRP) conjugate to obtain the HRP labeled probes, to hybridize with three corresponding targets. The resulting mixture containing double-strand DNA (dsDNA)-HRP, excess single-strand DNA (ssDNA)-HRP and remaining avidin-HRP was separated by capillary electrophoresis, and then the system of HRP catalyzing H₂O₂/o-aminophenol (OAP) reaction was adopted. The catalytic product was detected with electrochemical detection. With this protocol, the limits of quantification for the hybridization assay of 21-, 39- and 80-mer DNA fragments were of 1.2 × 10⁻¹¹, 2.4 × 10⁻¹¹ and 3.0 × 10⁻¹¹ M, respectively. The multiplex assay also provided good specificity without any cross-reaction.     

Development of a liquid chromatography-based screening methodology for proteolytic enzyme activity

A new methodology for the detection and isolation of serine proteases in complex mixtures has been developed. It combines the characterization of crude samples by electrospray tandem mass spectrometry (ESI-MS/MS) in a multi-substrate assay and the differentiated sensitive detection of the responsible enzymes by means of liquid chromatography hyphenated online to biochemical detection (BCD). First, active samples are identified in the multi-substrate assay monitoring the conversion of eight substrates in multiple reaction monitoring in parallel within 60 s. Hereby, the product patterns are investigated and the suitable peptide as substrate for BCD analysis is selected. Subsequently, the active proteases are identified online in the continuous-flow reactor serving as BCD after non-denaturing separation by size-exclusion chromatography and ion-exchange chromatography. For BCD, the selected para-nitroaniline (pNA) labeled peptide is added post-column and is cleaved by eluting proteases under release of the coloured pNA in a reaction coil (reaction time 5 min). The method was optimized and the figures of merit were characterized with trypsin and chymotrypsin serving as the model proteases. For trypsin, a limit of detection in LC–BCD of 0.1 U/mL corresponding to an injected amount of 0.4 ng protein (∼18 fmol) was observed. The BCD signal remained linear for an injected enzyme concentration of 0.3–10 U/mL (1.3–42 ng enzyme). The method was applied to the characterization of the crude venom of the pit viper Bothrops moojeni and the extracellular protease of the pathogenic amoeba Acanthamoeba castellanii. In the two samples, fractions with proteolytic activity potentially interfering with the blood coagulation cascade were identified. The described methodology represents a tool for serine protease screening in complex mixtures by a fast ESI-MS/MS identification of active samples followed by the separation and isolation of active sample constituents in LC–BCD.     

Electrochemical detection of glucose from whole blood using paper-based microfluidic devices

Electrochemical paper-based analytical devices (ePADs) with integrated plasma isolation for determination of glucose from whole blood samples have been developed. A dumbbell shaped ePAD containing two blood separation zones (VF2 membranes) with a middle detection zone was fabricated using the wax dipping method. The dumbbell shaped device was designed to separate plasma while generating homogeneous flow to the middle detection zone of the ePAD. The proposed ePADs work with whole blood samples with 24–60% hematocrit without dilution, and the plasma was completely separated within 4 min. Glucose in isolated plasma separated was detected using glucose oxidase immobilized on the middle of the paper device. The hydrogen peroxide generated from the reaction between glucose and the enzyme pass through to a Prussian blue modified screen printed electrode (PB-SPEs). The currents measured using chronoamperometry at the optimal detection potential for H₂O₂ (−0.1 V versus Ag/AgCl reference electrode) were proportional to glucose concentrations in the whole blood. The linear range for glucose assay was in the range 0–33.1 mM (r² = 0.987). The coefficients of variation (CVs) of currents were 6.5%, 9.0% and 8.0% when assay whole blood sample containing glucose concentration at 3.4, 6.3, and 15.6 mM, respectively. Because each sample displayed intra-individual variation of electrochemical signal, glucose assay in whole blood samples were measured using the standard addition method. Results demonstrate that the ePAD glucose assay was not significantly different from the spectrophotometric method (p = 0.376, paired sample t-test, n = 10).     

Spectrophotometric determination of ascorbic acid by the modified CUPRAC method with extractive separation of flavonoids-La(III) complexes

The proposed method for ascorbic acid: AA (Vitamin C) determination is based on the oxidation of AA to dehydroascorbic acid with the CUPRAC reagent of total antioxidant capacity assay, i.e., Cu(II)-neocuproine (Nc), in ammonium acetate-containing medium at pH 7, where the absorbance of the formed bis(Nc)-copper(I) chelate is measured at 450 nm. The flavonoids (essentially flavones and flavonols) normally interfering with the CUPRAC procedure were separated with preliminary extraction as their La(III) chelates into ethylacetate (EtAc). The Cu(I)-Nc chelate responsible for color development was formed immediately with AA oxidation. Beer's law was obeyed between 8.0 × 10⁻⁶ and 8.0 × 10⁻⁵ M concentration range, with the equation of the linear calibration curve: A_450 nm = 1.60 × 10⁴C (mol dm⁻³) − 0.0596. The relative standard deviation (R.S.D.) in the analysis of N = 45 synthetic mixtures containing 1.25 × 10⁻² mM AA with flavonoids was 5.3%. The Cu(II)-Nc reagent is a lower redox-potential and therefore more selective oxidant than the Fe(III)-1,10-phenanthroline reagent conventionally used for the same assay. This feature makes the proposed method superior for real samples such as fruit juices containing weak reductants such as citrate, oxalate and tartarate that may otherwise produce positive errors in the Fe(III)-phen method when equilibrium is achieved. The developed method was applied to some commercial fruit juices and pharmaceutical preparations containing Vitamin C + bioflavonoids. The findings of the developed method for fruit juices and pharmaceuticals were statistically alike with those of HPLC. The proposed spectrophotometric method was practical, low-cost, rapid, and could reliably assay AA in the presence of flavonoids without enzymatic procedures open to interferences by enzyme inhibitors.     

Using a photochemical method and chitosan to prepare surface-enhanced Raman scattering–active silver nanoparticles

In this paper, we report a new strategy for the preparation of surface-enhanced Raman scattering (SERS)-active silver nanoparticles (Ag NPs), using a photochemical method and the presence of chitosan (Ch). First, Ag substrates were subjected to electrochemical oxidation/reduction cycles (ORCs) in deoxygenated aqueous solutions containing 0.1 M HNO₃ and 1 g L⁻¹ Ch (pH 6.9, adjusted by adding 1 M NaOH), resulting in Ag⁺–Ch complexes. These substrates were then irradiated with UV light at various wavelengths to yield the SERS-active Ag NPs. A stronger SERS effect was observed on the SERS-active Ag NPs prepared by using UV irradiation at 310 nm. The pH of the solution and the presence of Ch during the preparation process both affected the resulting SERS activities.     

Monitoring inorganic mercury and methylmercury species with liquid chromatography-piezoelectric detection

A piezoelectric detection system coupled with a liquid chromatographic system is developed for the speciation of inorganic mercury(II) and methylmercury. Piezoelectric detection has been demonstrated to be a very sensitive detection system for total mercury determination when a gold-coated piezoelectric quartz crystal was used. The analytical features of this detection unit make it very suitable to be used as a detector coupled with a liquid chromatographic system for monitoring mercury species. After separation by a chromatography column (Spherisorb ODS-2, 5 μm,  mm i.d.), mercury species are liberated and reduced, by using stannous chloride, and are detected as an amalgam on the gold-coated piezoelectric quartz crystal, the sensor subsequently was regenerated by passing a peroxydisulfate solution. This detector exhibits good sensitivity, it allows the determination of mercury at sub-ppb concentration levels (0.30-1.20 μg l⁻¹). The precision, expressed as relative standard deviation, was ±2.7% (n=11) for a 0.5 μg l⁻¹ total mercury concentration. The proposed method is free of interferences and it allowed the determination of inorganic mercury and methylmercury species in natural waters.     

Simultaneous enzymatic kinetic determination of pesticides, carbaryl and phoxim, with the aid of chemometrics

A sensitive and selective enzymatic kinetic method for the simultaneous determination of mixtures of carbaryl and phoxim pesticides was researched and developed. It was based on the inhibitory effect of the pesticides on acetylcholinesterase (AChE), and the use of 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB) as a chromogenic reagent for the thiocholine iodide (TChI) released from the acetylthiocholine iodide (ATChI) substrate. The DTNB-thiocholine reaction was investigated by a spectrophotometric-kinetic approach. The complex rate equation for the formation of the chromogenic product, P, was solved under certain experimental conditions, which enabled the absorbance (A_P, at λ_max = 412 nm) from the mixtures of the two pesticide inhibitors to be directly related to their concentrations provided the absorbance additivity was followed. The spectra were measured for mixtures of carbaryl and phoxim at different concentrations, and at t = 904 s, T = 35 °C, pH = 7.5, c_ATChI = 0.14, and c_AChE = 0.10 mg mL⁻¹. The detection limits of the enzymatic kinetic spectrophotometric procedures for the determination of the carbaryl and phoxim were 4.7 and 0.59 μg L⁻¹, respectively.Calibration models for chemometrics methods, such as principal component regression (PCR), partial least squares (PLS) and radial basis function-artificial neural network (RBF-ANN) were constructed and verified with synthetic samples of the mixtures of the two pesticides. The best performing model was based on the RBF-ANN method yielding at approximately 10 ppb analyte concentrations, %RPE_T (carbaryl = 5.2; phoxim = 6.5), %Recovery (approx.105%) and %RPE_T (6.5). Various spiked town-water samples produced recoveries in the range of 98.8-103% for each pesticide.     

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.     

A novel analytical system involving hydride generation and gold-coated W-coil trapping atomic absorption spectrometry for selenium determination at ng l level

A novel analytical technique was developed where gaseous hydrogen selenide formed by sodium tetrahydroborate reduction is transported to and trapped on a resistively heated gold-coated W-coil atom trap for in situ preconcentration. Gold coating on W-coil was prepared by using an organic solution of Au. The atom trap is held at 165 °C during the collection stage and is heated up to 675 °C for revolatilization; analyte species formed are transported to an externally heated quartz T-tube where the atomization takes place and the transient signal is obtained. The carrier gas consisted of 112.5 ml min^− 1 Ar with 75 ml min^− 1 H₂ during the collection step and 112.5 ml min^− 1 Ar with 450 ml min^− 1 H₂ in the revolatilization step. The half width of the transient signal obtained is less than 0.5 s. The RSD for the measurements was found to be 3.9% (n = 11) for 0.10 µg l^− 1 Se using peak height measurements.     

Silica gel modified with lumogallion for aluminum determination by spectroscopic methods

Simple, easy to use and selective method of Al(III) sorption-spectroscopic (SS) determination was proposed. For this purpose, silica modified with tridecyloctadecylammonium chloride(SGII) using adsorption technique and glass slide modified with thin silica-poly(dimethyldiallyl-ammonium chloride) (SGI) composite film obtained by sol-gel technique were worked out. It was shown that lumogallion (LG) easily absorbs on SGI and SGII. Obtained sorbents SGIII and SGIV, respectively, were used for aluminum(III) determination by diffuse reflectance and spectrophotometric methods. The ranges of determination were (mg L⁻¹): (0.08-0.54), s_r ≤ 0.13, n = 4 for SGIII and (0.05-2.0), s_r ≤ 0.11, n = 4 for SGIV. The detection limits (blank + 3σ) for aluminum were 70 and 30 μg L⁻¹ using SGIII and SGIV, respectively, where σ is the standard deviation of blank estimation. The accuracy of the developed spectrophotometric method was examined by the determination of standard addition of aluminum in alcohol-free beverages. The relative error did not exceed 9%. SGIII can be regenerated by 0.05 M EDTANa₂H₂ solution and reused. SGIV was shown to be perspective for determination of aluminum in solution in the range of 0.01-0.13 mg L⁻¹ by solid phase luminescent technique.