Intensified biochip system using chemiluminescence for the detection of Bacillus globigii spores

This paper reports the first intensified biochip system for chemiluminescence detection and the feasibility of using this system for the analysis of biological warfare agents is demonstrated. An enzyme-linked immunosorbent assay targeting Bacillus globigii spores, a surrogate species for Bacillus anthracis, using a chemiluminescent alkaline phosphatase substrate is combined with a compact intensified biochip detection system. The enzymatic amplification was found to be an attractive method for detection of low spore concentrations when combined with the intensified biochip device. This system was capable of detecting approximately 1 × 10⁵ Bacillus globigii spores. Moreover, the chemiluminescence method, combined with the self-contained biochip design, allows for a simple, compact system that does not require laser excitation and is readily adaptable to field use. Figure Schematic diagram of the miniature biochip detection system     

Online phototransformation–flow injection chemiluminescence determination of triclosan

A highly selective and sensitive chemiluminescence method for the determination of triclosan is proposed. The method is based on the phototransformation of triclosan to a light-emitting precursor in the presence of fluorescein in alkaline medium and the chemiluminescence reaction is then triggered by strong base or oxidants such as N-bromosuccinimide. Based on this reaction an online phototransformation–flow injection manifold was developed, in which the photoreactor comprises a 150-cm-long × 0.8-mm-i.d. piece of PTFE tubing coiled around a 25-W fluorescent lamp, and the phototransformed products were then injected into a carrier stream of borate buffer. After mixing with the oxidant stream the produced light was detected by a photomultiplier. A wide calibration range from 8.0 × 10⁻⁸ to 1.0 × 10⁻⁴ mol L⁻¹ was obtained under the optimized conditions, and the detection limit was as low as 5.0 × 10⁻⁸ mol L⁻¹. The whole process of analysis, including the online phototransformation and subsequent chemiluminescence detection, could be completed in 6 min. Most of the foreign substances tested showed high tolerance levels, and the proposed method was directly applied to the determination of triclosan in toothpaste samples without any pre-separation procedure. Figure Schematic representation of the phototransformation of triclosan and subsequent chemiluminescence reaction     

Chemiluminescent imaging analysis of interferon alpha in serum samples

Enzyme-linked immunosorbent assay (ELISA), horseradish peroxidase (HRP)-catalyzed fluorescent reaction, and oxalate chemiluminescence imaging analysis have been combined to develop a sensitive, simple, and rapid method for analysis of interferon alpha (α-IFN) in human serum samples. A typical “sandwich type” immunoassay was used. Reaction of o-phenylenediamine (OPD) with hydrogen peroxide (H₂O₂), catalyzed by HRP, produced 2,3-diaminophenazine (PDA), which was detected by chemiluminescence imaging analysis with the bis(2,4,6-trichlorophenyl)oxalate (TCPO)–H₂O₂–glyoxaline–PDA chemiluminescent system. The TCPO chemiluminescent imaging system is more sensitive and the chemiluminescence quantum yield is at least five times higher than for the luminol–H₂O₂–HRP–PIP (p-iodophenol) chemiluminescent imaging system. The results showed there was a very good linear correlation between response and amount of α-IFN in the range 1.3–156.0 pg mL⁻¹ (R = 0.9991) and the detection limit was 0.8 pg mL⁻¹ (S/N=3). The relative standard deviation (n = 9) was 4.7%. The proposed method has been used for successful analysis of the amount of α-IFN in human serum. The results obtained compared well with those obtained by conventional colorimetric ELISA and luminol chemiluminescent ELISA. Figure Procedures of the proposed method     

Chemiluminescence assay for quinones based on generation of reactive oxygen species through the redox cycle of quinone

A sensitive and selective chemiluminescence assay for the determination of quinones was developed. The method was based on generation of reactive oxygen species through the redox reaction between quinone and dithiothreitol as reductant, and then the generated reactive oxygen was detected by luminol chemiluminescence. The chemiluminescence was intense, long-lived, and proportional to quinone concentration. It is concluded that superoxide anion was involved in the proposed chemiluminescence reaction because the chemiluminescence intensity was decreased only in the presence of superoxide dismutase. Among the tested quinones, the chemiluminescence was observed from 9,10-phenanthrenequinone, 1,2-naphthoquinone, and 1,4-naphthoquinone, whereas it was not observed from 9,10-anthraquinone and 1,4-benzoquinone. The chemiluminescence property was greatly different according to the structure of quinones. The chemiluminescence was also observed for biologically important quinones such as ubiquinone. Therefore, a simple and rapid assay for ubiquinone in pharmaceutical preparation was developed based on the proposed chemiluminescence reaction. The detection limit (blank + 3SD) of ubiquinone was 0.05 μM (9 ng/assay) with an analysis time of 30 s per sample. The developed assay allowed the direct determination of ubiquinone in pharmaceutical preparation without any purification procedure. Figure Chemiluminescence generated through the redox cycle of quinone     

Aptamer-based label-free method for hemin recognition and DNA assay by capillary electrophoresis with chemiluminescence detection

An aptamer-based label-free approach to hemin recognition and DNA assay using capillary electrophoresis with chemiluminescence detection is introduced here. Two guanine-rich DNA aptamers were used as the recognition element and target DNA, respectively. In the presence of potassium ions, the two aptamers folded into the G-quartet structures, binding hemin with high specificity and affinity. Based on the G-quartet–hemin interactions, the ligand molecule was specifically recognized with a K _d ≈ 73 nM, and the target DNA could be detected at 0.1 μM. In phosphate buffer of pH 11.0, hemin catalyzed the H₂O₂-mediated oxidation of luminol to generate strong chemiluminescence signal; thus the target molecule itself served as an indicator for the molecule–aptamer interaction, which made the labeling and/or modification of aptamers or target molecules unnecessary. This label-free method for molecular recognition and DNA detection is therefore simple, easy, and effective. Figure A label-free approach to aptamer-based hemin recognition and DNA detection is introduced, which gives great potential for using a small molecule itself as the indicator for molecular recognition and DNA detection thereby avoiding any labeling or modification step     

Photofragmentation of nitro-based explosives with chemiluminescence detection

A simple, fast, reliable, sensitive and potentially portable explosive detection device was developed employing laser photofragmentation (PF) followed by heterogeneous chemiluminescence (CL) detection. The PF process involves the release of NO_{x(x = 1,2)} moieties from explosive compounds such as TNT, RDX, and PETN through a stepwise excitation–dissociation process using a 193 nm ArF laser. The NO_{x(x = 1,2)} produced upon PF is subsequently detected by its CL reaction with basic luminol solution. The intensity of the CL signal was detected by a thermoelectrically cooled photomultiplier tube with high quantum efficiency and negligible dark current counts. The system was able to detect trace amounts of explosives in various forms in real time under ambient conditions. Detection limits of 3 ppbv for PETN, 2 ppbv for RDX, and 34 ppbv for TNT were obtained. It was also demonstrated that the presence of PETN residue within the range of 61 to 186 ng/cm² can be detected at a given signal-to-background ratio of 10 using a few microjoules of laser energy. The technique also demonstrated its potential for the direct analysis of trace explosive in soil. An LOD range of 0.5–4.3 ppm for PETN was established, which is comparable to currently available techniques. Figure Photofragmentation–chemiluminescence detector     

Determination of vancomycin in human plasma using high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the quantification of vancomycin in human plasma was developed and validated. The method includes an extraction of vancomycin by deproteinization with acetonitrile. The analyses were carried out at 258 nm as the emission wavelength while exciting at 225 nm on a reversed-phase column (30 cm × 4 mm i.d. × 10 μm Waters Associates μBondapak C18) using a mobile phase composed of methanol and phosphate buffer at pH 6.3. Vancomycin was quantitatively recovered from human plasma samples (>96%) with high values of precision. The separation was completed within 27 min. The calibration curve was linear over the range from 5 to 1,000 ng/mL with the detection and quantification limits of 2 ng/mL and 5 ng/mL, respectively. This method is suitable for the routine assay of plasma samples. Figure The effect of the deproteinization solvent on the signal of the interference peak at retention time of 15.0 min. The peak which interferes with the peaks of Erythromycin and Vancomycin has been disappeared by using 2 mL acetonitrile as the deproteinization solvent.     

Flow injection chemiluminescence immunoassay for 17β-estradiol using an immunoaffinity column

A new flow injection chemiluminescent immunoassay was developed for the detection of 17β-estradiol (E₂). The method uses p–iodophenol (PIP) as enhancer and is based on a solid-phase immunoassay format in which an E₂–OVA immobilized immunoaffinity column inserted in the flow system is used to trap unbound horseradish peroxidase (HRP)-labeled anti-E₂ antibody after an off-line incubation of E₂ with HRP-labeled anti-E₂ antibody. The trapped enzyme conjugate was detected by injecting substrates to produce an enhanced chemiluminescence (CL) response. The linear range for E₂ was 10.0–1,000.0 ng mL⁻¹ with a correlation coefficient of 0.996 and a detection limit of 3.0 ng mL⁻¹. The sampling and chemiluminescence detection time for one sample was 400 s after a pre-incubation procedure of 30 min. Serum samples detected by this method were in good agreement with the results obtained by EIA with E₂–biotin.      

Investigating the post-chemiluminescence behavior of phenothiazine medications in the luminol–potassium ferricyanide system: molecular imprinting–post-chemiluminescence method for the determination of chlorpromazine hydrochloride

A new post-chemiluminescence (PCL) phenomenon was observed when phenothiazine medications were injected into the reaction mixture after the chemiluminescence (CL) reaction of luminol and potassium ferricyanide had finished. A possible reaction mechanism was proposed based on studies of the kinetic characteristics of the CL, CL spectra, fluorescence spectra, and on other experiments. The feasibility of determining various phenothiazine medications by utilizing these PCL reactions was examined. A molecular imprinting–post-chemiluminescence (MI-PCL) method was established for the determination of chlorpromazine hydrochloride using a chlorpromazine hydrochloride-imprinted polymer (MIP) as the recognition material. The method displayed high selectivity and high sensitivity. The linear range of the method was 1.0×10⁻⁸∼1.0×10⁻⁶, with a linear correlation coefficient of 0.9985. The detection limit was 3×10⁻⁹ g/ml chlorpromazine hydrochloride, and the relative standard deviation for a 1.0×10⁻⁷ g/ml chlorpromazine hydrochloride solution was 4.0% (n=11). The method has been applied to the determination of chlorpromazine hydrochloride in urine and animal drinking water with satisfactory results.      

Small-volume fiber-optic evanescent-wave absorption sensor for nitrite determination

A novel small-volume fiber-optic evanescent-wave absorption sensor based on the Griess–Ilosvay reaction has been developed and evaluated for nitrite determination. The sensor was constructed by inserting a decladded optical fiber into a transparent capillary to form an annular column microchannel. The Evanescent wave (EW) field produced on the optical fiber core surface penetrated into the surrounding medium and interacted with the azo dye, which was generated by the reaction of nitrite and nitrite-sensitive reagents. The detector was designed to be parallel to the axis of the optical fiber. The defined absorbance was linear with the concentration of nitrite in the range from 0.05 to 10 mg L⁻¹, and the detection limit was 0.02 mg L⁻¹ (3σ) with the relative standard deviation (RSD) of 2.6% (n = 8). The present sensor was successfully used to determine nitrite in real samples of mineral water, tap water, rain water, and seawater. The results were consistent with the data obtained by standard spectrophotometric method, showing potential of the proposed sensor for practical application.      

Development of a multichannel flow-through chemiluminescence microarray chip for parallel calibration and detection of pathogenic bacteria

Pathogen detection is important for health and safety reasons. Several outbreaks all over the world have shown the need for rapid, qualitative, quantitative, and, particularly, multianalyte detection systems. Hence, a multichannel flow-through chemiluminescence microarray chip for parallel detection of pathogenic bacteria was developed. The disposable chip made of acrylonitrile–butadiene–styrene (ABS) copolymer was devised as a support for a multiplexed sandwich immunoassay. Calibration and measurement was possible in one experiment, because the developed chip contains six parallel flow-through microchannels. Polyclonal antibodies against the pathogenic bacteria Escherichia coli O157:H7, Salmonella typhimurium, and Legionella pneumophila were immobilized on the chip by microcontact printing in order to use them as specific receptors. Detection of the captured bacteria was carried out by use of specific detection antibodies labelled with biotin and horseradish peroxidase (HRP)–streptavidine conjugates. The enzyme HRP generates chemiluminescence after adding luminol and hydrogen peroxide. This signal was observed by use of a sensitive CCD camera. The limits of detection are 1.8 × 10⁴ cells mL⁻¹ for E. coli O157:H7, 7.9 × 10⁴ cells mL⁻¹ for L. pneumophila, and 2.0 × 10⁷ cells mL⁻¹ for S. typhimurium. The overall assay time for measurement and calibration is 18 min, enabling very fast analysis.      

Fluorescence-detecting cationic surfactants using luminescent CdTe quantum dots as probes

A novel fluorescence quenching method for the determination of cationic surfactants (CS), specifically cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), and cetylpyridinium chloride (CPC), has been developed using water-soluble luminescent CdTe quantum dots (QDs) modified with thioglycolic acid (TGA). The possible interference from heavy and transition metals (HTM) has been efficiently eliminated through simple sample treatment with mercapto cotton made in-house. Under optimum conditions, the extent of fluorescence quenching of CdTe QDs is linearly proportional to the concentration of CS from 2.0 × 10⁻⁷ to 7.0 × 10⁻⁶ mol L⁻¹ with a detection limit of 5.0 × 10⁻⁸ mol L⁻¹. The relative standard deviation for 1.0 × 10⁻⁶ mol L⁻¹ CTAB is 2.5% (n = 6). The proposed method exhibits high sensitivity and selectivity and furthermore avoided the use of toxic organic solvents and tedious solvent extraction procedures. It has been applied to the determination of trace CS in natural river water and commodity samples with satisfactory results. Potential interference from heavy and transition metals is eliminated during photoluminescence detection of CS through simple sample pre-treatment with mercapto cotton     

Simultaneous determination of uric acid and ascorbic acid at a ferrocenium–thioglycollate modified electrode

Self-assembled monolayers (SAMS) of chemisorbed thioglycollate on a gold electrode surface have been used as a base interface for the electrostatic adsorption of ferrocenium ion. Electrochemical impedance spectra (EIS) and cyclic voltammetry (CV) were used to evaluate the electrochemical properties of the supramolecular film. The bare gold electrode failed to distinguish the oxidation peaks of ascorbic acid (AA) and uric acid (UA) in phosphate buffer solution (PBS, pH 7.0), while the ferricinium–thioglycollate modified electrode could separate them efficiently. In differiential pulse voltammetric measurements, the prepared gold electrode could separate AA and UA signals, allowing the simultaneous determination of AA and UA. Under optimal conditions and within the linear range of 1.0 × 10⁻⁶ to 5.0 × 10⁻⁴ M, the detection limits of AA and UA achieved were 2.0 × 10⁻⁷ and 1.0 × 10⁻⁷ M, respectively. The applicability of the prepared electrode was demonstrated by measuring AA and UA in human urine without any pretreatment. Figure Fabrication process for the modified electrode     

Determination of glucosinolates in traditional Chinese herbs by high-performance liquid chromatography and electrospray ionization mass spectrometry

A reversed-phase HPLC method has been developed for determination of twelve intact glucosinolates—glucoiberin, glucocheirolin, progoitrin, sinigrin, epiprogoitrin, glucoraphenin, sinalbin, gluconapin, glucosibarin, glucotropaeolin, glucoerucin, and gluconasturtiin—in ten traditional Chinese plants. The samples were extracted with methanol and the extracts were cleaned on an activated Florisil column. A mobile phase gradient prepared from methanol and 30 mmol L⁻¹ ammonium acetate at pH 5.0 enabled baseline separation of the glucosinolates. Glucosinolate detection was confirmed by quadrupole time-of-flight tandem mass spectrometric analysis in negative-ionization mode. Detection limits ranged from 0.06 to 0.36 μg g⁻¹ when 5 g of dried plant was analyzed. Recoveries of the glucosinolates were better than 85% and precision (relative standard derivation, n = 3) ranged from 5.3 to 14.6%. Analysis of the glucosinolates provided scientific evidence enabling differentiation of three pairs of easily confused plants. Figure Glucosinolates Analysis for the Differentiation of Easily-Confusing Herbs     

Surface modification of amine-functionalised graphite for preparation of cobalt hexacyanoferrate (CoHCF)-modified electrode: an amperometric sensor for determination of butylated hydroxyanisole (BHA)

A cobalt hexacyanoferrate (CoHCF)-modified graphite paraffin wax composite electrode was prepared by a new approach. An amine-functionalised graphite powder was used for the fabrication of the electrode. A functionalised graphite paraffin wax composite electrode was prepared and the surface of the electrode was modified with a thin film of CoHCF. Various parameters that influence the electrochemical behaviour of the modified electrode were studied by varying the background electrolytes, scan rates and pH. The modified electrode showed good electrocatalytic activity towards the oxidation of butylated hydroxyanisole (BHA) under optimal conditions and showed a linear response over the range from 7.9 × 10⁻⁷ to 1.9 × 10⁻⁴ M of BHA with a correlation coefficient of 0.9988. The limit of detection was 1.9 × 10⁻⁷ M. Electrocatalytic oxidation of BHA was effective at the modified electrode at a significantly reduced potential and at a broader pH range. The utility of the modified electrode as an amperometric sensor for the determination of BHA in flow systems was evaluated by carrying out hydrodynamic and chronoamperometric experiments. The modified electrode showed very good stability and a longer shelf life. The modified electrode was applied for the determination of BHA in spiked samples of chewing gum and edible sunflower oil. The advantage of this method is the ease of electrode fabrication, good stability, longer shelf life, low cost and its diverse application for BHA determination. Figure Cyclic Voltammogram of () CoHCF modified electrode, () in presence of 1.9 x 10⁻⁵ M of BHA and () bare electrode, () in the presence of 1.9 x 10⁻⁵ M of BHA in 1.0 M NaCl, pH 7.0     

Determination of diquat by flow injection–chemiluminescence

A simple, economic, sensitive and rapid method for the determination of the pesticide diquat was described. This new method was based on the coupling of flow injection analysis methodology and direct chemiluminescent detection; to the authors’ knowledge, this approach had not been used up to now with this pesticide. It was based on its oxidation with ferricyanide in alkaline medium; significant improvements in the analytical signal were achieved by using high temperatures and quinine as sensitiser. Its high throughput (144 h⁻¹), together with its low limit of detection (2 ng mL⁻¹), achieved without need of preconcentration steps, permitted the reliable quantification of diquat over the linear range of (0.01–0.6) μg mL⁻¹ in samples from different origins (river, tap, mineral and ground waters), even in the presence of a 40-fold concentration of paraquat, a pesticide commonly present in the commercial formulations of diquat. Figure Quartz luminometer cell     

A microfluidic system for evaluation of antioxidant capacity based on a peroxyoxalate chemiluminescence assay

A microfluidic system incorporating chemiluminescence detection is reported as a new tool for measuring antioxidant capacity. The detection is based on a peroxyoxalate chemiluminescence (PO-CL) assay with 9,10-bis-(phenylethynyl)anthracene (BPEA) as the fluorescent probe and hydrogen peroxide as the oxidant. Antioxidant plugs injected into the hydrogen peroxide stream result in inhibition of the CL emission which can be quantified and correlated with antioxidant capacity. The PO-CL assay is performed in 800-μm-wide and 800-μm-deep microchannels on a poly(dimethylsiloxane) (PDMS) microchip. Controlled injection of the antioxidant plugs is performed through an injection valve. Of the plant-food based antioxidants tested, β-carotene was found to be the most efficient hydrogen peroxide scavenger (SA _HP of 3.27 × 10⁻³ μmol⁻¹ L), followed by α-tocopherol (SA _HP of 2.36 × 10⁻³ μmol⁻¹ L) and quercetin (SA _HP of 0.31 × 10⁻³ μmol⁻¹ L). Although the method is inherently simple and rapid, excellent analytical performance is afforded in terms of sensitivity, dynamic range, and precision, with RSD values typically below 1.5%. We expect our microfluidic devices to be used for in-the-field antioxidant capacity screening of plant-sourced food and pharmaceutical supplements. Figure Assembled PDMS microchip sandwiched between two glass plates with the top plate containing capillary reservoirs     

Electrochemical deposition of silicate–cetyltrimethylammonium bromide nanocomposite film on glassy carbon electrode for sensing of methyl parathion

A novel electrochemical sensor for methyl parathion based on silicate– cetyltrimethylammonium bromide nanocomposite film has been fabricated by electro-assisted deposition onto glassy carbon electrode in one-step via an electrochemical modulation of pH at the electrode/solution interface to promote controlled gelification of tetraethylorthosilicate sol, and was characterized with scanning electron microscopy, X-ray diffraction, and electrochemical impedance spectroscopy. The electrochemical sensing of methyl parathion on the film-modified electrode was investigated applying cyclic voltammetry and square wave voltammetry. Compared to the unmodified electrode, the shapes of the redox peaks were improved and the peak currents significantly increased. Experimental parameters such as deposition time, pH value, and accumulation conditions have been optimized. A linear relationship between the peak current and methyl parathion concentration was obtained in the range from 1.0 × 10⁻⁷ to 1.0 × 10⁻⁴ mol L⁻¹ with a detection limit of 1.04 × 10 ⁻⁸ mol L⁻¹ (S/N = 3) after accumulation at 0 V for 120 s. The film electrode shows great promise for determination of methyl parathion in real samples.      

Usefulness of cyclodextrin media for the determination of α-cypermethrin by photochemically induced fluorescence: analytical applications to natural waters

The photochemically induced fluorescence (PIF) spectral properties of α-cypermethrin in organic solvents (hexane, dichloromethane, acetonitrile, ethanol) and in cyclodextrin aqueous solutions (β-CD and 2-hydroxypropyl-β-CD, 2-HP-β-CD) were investigated. The photolysis kinetics of α-cypermethrin were evaluated in the various media. The PIF signal was found to be significantly enhanced in the CD media relative to the organic solvents. The stoichiometry and the formation constants of the α-cypermethrin inclusion complexes formed with the CDs were determined. The analytical performances of the PIF method were improved in the presence of HP-β-CD relative to the other media, and a CD-enhanced PIF analytical method was developed. The limits of detection and limits of quantification ranged, respectively, between 6 and 98 ng/mL and between 24 and 343 ng/mL, depending on the medium. Application to the analysis of tap water and Senegal natural water samples collected close to agricultural areas and spiked with α-cypermethrin yielded satisfactory recoveries going from about 77% to 98%. An interference study of foreign species, including pesticides and inorganic ions likely to be present in natural waters, was also carried out. Figure Photolysis reaction of α-cypermethrin in presence of HP-β-CD     

Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine

A multianalyte lateral-flow immunochromatographic technique using colloidal gold-labeled polyclonal antibodies was developed for the rapid simultaneous detection of clenbuterol and ractopamine. The assay procedure could be accomplished within 5 min, and the results of this qualitative one-step assay were evaluated visually according to whether test lines appeared or not. When applied to the swine urines, the detection limit and the half maximal inhibitory concentration (IC₅₀) of the test strip under an optical density scanner were calculated to be 0.1 ± 0.01 ng mL⁻¹ and 0.1 ± 0.01 ng mL⁻¹, 0.56 ± 0.08 ng mL⁻¹, and 0.71 ± 0.06 ng mL⁻¹, respectively, the cut-off levels with the naked eye of 1 ng mL⁻¹ and 1 ng mL⁻¹ for clenbuterol and ractopamine were observed. Parallel analysis of swine urine samples with clenbuterol and ractopamine showed comparable results obtained from the multianalyte lateral-flow test strip and GC-MS. Therefore, the described multianalyte lateral-flow test strip can be used as a reliable, rapid, and cost-effective on-site screening technique for the simultaneous determination of clenbuterol and ractopamine residues in swine urine.