Determination of aminoglycoside antibiotics using an on-chip microfluidic device with chemiluminescence detection

We describe an on-chip microflow injection (μFI) approach for the determination of aminoglycoside antibiotics using chemiluminescence (CL) detection. The method is based on the inhibition of the Cu(II)-catalyzed CL reaction of luminol and hydrogen peroxide by the aminoglycosides due to the formation of a complex between the antibiotic and Cu(II). The main features of the method include small sample volumes and a fast response. Syringe pumps were used to insert the sample and the reagents into the microfluidic device. CL was collected using a fiber optic bundle connected to a luminescence detector. All instrumental, hydrodynamic and chemical variables involved in the system were optimized using neomycin as the aminoglycoside model. Inhibition is proportional to the concentration of the antibiotics. The dynamic ranges of the calibration graphs obtained for neomycin, streptomycin and amikacin are 0.3–3.3, 0.9–13.7, and 0.8–8.5?μmol?L^?1, and the detection limits are 0.09, 0.28 and 0.24?μmol?L^?1, respectively. The precision of the methods, expressed as relative standard deviation, is in the range from 0.8 to 5.0?%. The method was successfully applied to the determination of neomycin in water samples, with recoveries ranging from 80 to 120?%.

In vitro monitoring of picogram levels of risperidone in human urine via luminollysozyme flow injection chemiluminescence

The anti-schizophrenic drug risperidone (RSP) exerts an inhibitory effect on the chemiluminescence (CL) of the luminol-lysozyme system. This finding forms the basis for a sensitive flow injection method for its determination at picogram levels. RSP binds to Trp62 in the lysozyme, and this leads to a conformational change upon which the CL of the system is quenched. The decrease in CL is proportional to the logarithm of the concentration of RSP, and the calibration graph is linear in the range from 0.1 pg?mL^?1 to 1.0 ng?mL^?1, with relative standard deviations of <5.0%, and a detection limit of 0.05 pg?mL^?1 (3σ). At a flow rate of 2.0 mL?min^?1, the whole process including sampling and washing is completed within 20 s. The method was successfully applied to monitoring RSP in human urine after incorporation of 2 mg of RSP, with a total excretion of 16.6% within 8.5 h.

Selective light-triggered chemiluminescence between fluorescent dyes and luminol, and its analytical application

We report herein a novel chemiluminescence (CL) phenomenon triggered by light irradiation when a fluorescent dye, for example hematoporphyrin, fluorescein, eosin, or methylene blue is present in the luminol solution. A possible mechanism is proposed for the photoinduced chemiluminescence (PICL) reaction. Compared with reported methods for CL triggering, for example flow-injection, static reagent injection, and the electrochemical technique, the proposed in-situ PICL method presented has three advantages. First, the method is more selective, because the PICL signal of the target fluorescent dyes is initiated by excitation at a selective wavelength only. Second, the space and time resolution of the PICL method are better. Last, and most important, compared with injecting a reagent or inserting a electrode into the CL system to initiate the CL reaction, with the in-situ PICL method there is no physical interference with the target detecting system. All these advantages of the PICL method indicate it has many potential applications in the analytical sciences. The proposed method was applied to analysis of urine containing adrenaline. The linear range for adrenaline is 2.0?×?10^?10–1.0?×?10^?7 g mL^?1 and the detection limit is 6.0?×?10^?11 g mL^?1.

Detection of trace nitrite in waters using a QDs-based chemiluminescence analysis system

In the present work, a novel flow-injection chemiluminescence method based on CdTe quantum dots (QDs) was developed for the determination of nitrite. Weak chemiluminescence (CL) signals were observed from a CdTe QDs–H₂O₂ system under basic conditions. The addition of a trace amount of hemoglobin (Hb) caused the CL from the CdTe QDs–H₂O₂ system to increase substantially. In the presence of nitrite, the ferrous Hb reacted with the nitrate to form ferric Hb and NO. The NO then bound to ferrous Hb to generate iron nitrosyl Hb. As a result, the CL signal from the CdTe QDs–H₂O₂–Hb system was quenched. Thus, a flow-injection CL analytical system for the determination of trace nitrite was established. Under optimum conditions, there was a good linear relationship between CL intensity and the concentration of nitrite in the range 1.0?×?10^?9 to 8.0?×?10^?7 mol L^?1 (R ²?=?0.9957). The limit of detection for nitrite using this system was 3.0?×?10^?10 mol L^?1 (S/N?=?3). This method was successfully applied to detect nitrite in water samples.

Development of a lateral flow fluorescent microsphere immunoassay for the determination of sulfamethazine in milk

The fluorescent microsphere has been increasingly used as detecting label in immunoassay because of its stable configuration, high fluorescence intensity, and photostability. In this paper, we developed a novel lateral flow fluorescent microsphere immunoassay (FMIA) for the determination of sulfamethazine (SMZ) in milk in a quantitative manner with high sensitivity, selectivity, and rapidity. A monoclonal antibody to SMZ was covalently conjugated with the carboxylate-modified fluorescent microsphere, which is polystyrene with a diameter of 200 nm. Quantitative detection of SMZ in milk was accomplished by recording the fluorescence intensity of microspheres captured on the test line after the milk samples were diluted five times. Under optimal conditions, the FMIA displays a rapid response for SMZ with a limit of detection of as low as 0.025 ng mL^?1 in buffer and 0.11 μg L^?1 in milk samples. The FMIA was then successfully applied on spiked milk samples and the recoveries ranged from 101.1 to 113.6 % in the inter-batch assay with coefficient of variations of 6.0 to 14.3 %. We demonstrate here that the fluorescent microsphere-based lateral flow immunoassay (LFIA) is capable of rapid, sensitive, and quantitative detection of SMZ in milk.

Novel chemiluminescent assay for staphylococcal enterotoxin B

An enzyme-linked immunosorbent assay, a horseradish peroxidase-catalyzed fluorogenic reaction, and chemiluminescence (CL) analysis have been combined to develop a sandwich ELISA for Staphylococcal enterotoxin B (SEB) using monoclonal antibodies for different epitopes of SEB. The enzyme catalyzed reaction of 3-(4-hydroxyphenyl propionate) with the urea complex of hydrogen peroxide produced a fluorescent dimer which was detected by chemiluminescence analysis. The CL response to SEB is linear in the range from 6.0 to 564?pg?mL^?1 (r?=?0.9993), and the detection limit is 3.3?pg?mL^?1 (S/N?=?3). Intra- and interassay coefficients of variation are <7.0% at three concentrations (24, 96 and 384?pg?mL^?1). The method was applied to the analysis of SEB in serum, lake water and milk samples. The results compared well with those obtained by conventional ELISAs.

Determination of formaldehyde based on the enhancement of the chemiluminescence produced by CdTe quantum dots and hydrogen peroxide

Water-soluble cadmium telluride quantum dots (CdTe QDs) capped with glutathione (GSH) display chemiluminescence (CL) emission on reaction with hydrogen peroxide (H₂O₂) in strongly alkaline medium. It is found that the CL is strongly enhanced on addition of formaldehyde in aqueous solution. A flow injection system was developed, and it is shown that there is good linearity between CL intensity and the concentration of formaldehyde in the 0.06–3.0 μg L^?1 range. The limit of detection is as low as 10 ng L^?1. The method was successfully applied to the determination of formaldehyde in indoor air after adsorption into an aqueous phase. The recoveries for the real samples range from 97 % to 102.5 %, and the relative standard deviation is <3.8 % for intra- and inter-assay precision.

Portable chemiluminescence multiplex biosensor for quantitative detection of three B19 DNA genotypes

A miniaturized multiplex biosensor exploiting a microfluidic oligonucleotide array and chemiluminescence (CL) lensless imaging detection has been developed for parvovirus B19 genotyping. The portable device consists of a reaction chip, comprising a glass slide arrayed with three B19 genotype-specific probes and coupled with a polydimethylsiloxane microfluidic layer, and a charge-coupled device camera modified for lensless CL imaging. Immobilized probes were used in DNA hybridization reactions with biotin-labeled targets, and then hybrids were measured by means of an avidin-horseradish peroxidase (HRP) conjugate and CL detection. All hybridization assay procedures have been optimized to be performed at room temperature through the microfluidic elements of the reaction chip, with sample and reagents delivery via capillary force exploiting adsorbent pads to drive fluids along the microchannels. The biosensor enabled multiplex detection of all B19 genotypes, with detectability down to 80 pmol?L^?1 for all B19 genotype oligonucleotides and 650 pmol?L^?1 for the amplified product of B19 genotype 1, which is comparable with that obtained in traditional PCR-ELISA formats and with notably shorter assay time (30 min vs. 2 h). The specificity of the assay has been evaluated by performing DNA–DNA hybridization reactions among sequences with different degrees of homology, and no cross hybridizations among B19 genotypes have been observed. The clinical applicability has been demonstrated by assaying amplified products obtained from B19 reference serum samples, with results completely consistent with the reference PCR-ELISA method. The next crucial step will be integration in the biosensor of a miniaturized PCR system for DNA amplification and for heat treatment of amplified products.

Molecularly imprinted dispersive solid-phase microextraction for determination of sulfamethazine by capillary electrophoresis

We have developed a rapid, selective and efficient method for dispersive solid-phase microextraction (DSPME) using microbeads of a molecularly imprinted polymer (MIP). It enables the pre-concentration of sulfamethazine and sample clean-up prior to capillary electrophoresis with UV detection. The microbeads were synthesized via precipitation polymerization using sulfamethazine, methacrylic acid and ethylene glycol dimethacrylate (EGDMA) as the template molecule, the functional monomer and the cross-linking monomer, respectively. Characterization by SEM displayed the high uniformity and dispersibility of the MIP microbeads. The adsorption and desorption of sulfamethazine and the parameters for CE were optimized to result in a limit of detection of 1.1?μg?L^?1, which is 373-fold lower than that of direct CE detection. The equilibration time of extraction was reduced to 5?min, and the selectivity of the microbeads was significantly improved compared to the non-imprinted polymer. The method was successfully applied to the determination of trace sulfamethazine in several milk samples, with recoveries in the range of 89?% to 110?%.

Development of chemiluminescent assays for the quantitative detection and imaging of 5-bromo-2′deoxyuridine-labeled DNA in parvovirus B19-infected cells

Incorporation of exogenous analogues is a widely used method to evaluate DNA synthesis in cultured cells exposed to exogenous factors such as infectious agents. Herein, two new quantitative methodologies exploiting ultrasensitive chemiluminescence (CL) detection of 5-bromo-2′-deoxyuridine (BrdU) have been developed: a CL microscope imaging assay to evaluate BrdU labelling at single-cell level and a CL dot-blot assay to measure the amounts of DNA produced in the course of an in vitro infection of proliferating cells. The assays have been optimized on UT7/EpoS1 cells cultured in presence of different concentrations of BrdU (from 3 to 100 μM) and used to monitor parvovirus B19 (B19) life cycle in infected cells. The CL microscope imaging assay provided a detailed localization of BrdU-labelled nuclei allowing to count positive cells and measure their related CL intensity signals. The CL dot-blot assay, coupled with a B19 capture procedure performed with a specific peptide nucleic acid probe, has been designed to discriminate and selectively quantify cellular and viral BrdU-labelled genomes. Quantitative evaluation of BrdU-labelled B19 DNA has been achieved by means of a CL calibration curve. The high detectability, down to 2?×?10⁶ B19 genome copies, and the linear range extending up to 5?×?10⁸ copies make the method suitable to evaluate the amounts of B19 DNA produced throughout a replicative viral cycle.

Quantum dot induced phototransformation of 2,4-dichlorophenol, and its subsequent chemiluminescence reaction

We have studied the CdTe quantum dot-induced phototransformation of 2,4-dichlorophenol (2,4-DCP) and its subsequent chemiluminescence (CL) reaction. Quantum dots (QDs) of different size and capped with thioglycolic acid were prepared and characterized by molecular spectroscopy, X-ray diffraction and transmission electron microscopy. In the presence of QDs, 2,4-DCP is photochemically transformed into a long-living light emitting precursor which can react with N-bromosuccinimide to produce CL with peak wavelengths at 475 and 550 nm. The formation of singlet oxygen during the phototransformation process was confirmed by the enhancement effect of deuterium oxide on the CL reaction and the change in the UV spectrum of a chemical trap. The CL intensity is linearly related to the concentration of 2,4-DCP in the range from 0.36 to 36 μmol L^?1, and the detection limit (at 3σ) is 0.13 μmol L^?1.

Mixed immunoassay design for multiple chemical residues detection

In this research, a mixed immunoassay design for multiple chemical residues detection based on combined reverse competitive enzyme-linked immunosorbent assay (ELISA) procedure was developed. This method integrated two reverse ELISA reactions in one assay by labeling horseradish peroxidase to deoxynivalenol (DON) and orbifloxacin. Within this method, IC50 of the two mAbs for each analyte we produced ranged from 23?～?68 ng?mL^?1 for DONs and 4.1?～?49 ng?mL^?1 for quinolones (QNs). The limit of detection measured by IC10 was achieved at 0.45–1.3 ng?mL^?1 for DONs and 0.59–6.9 ng?mL^?1 for QNs, which was lower than the maximum residue levels. Recoveries in negative samples spiked at concentrations of 100, 200, and 500 ng?mL^?1 ranged from 91.3 to 102.2 % for DONs and 88.7–98.05 % for QNs with relative standard deviation less than 9.88 and 12.67 %. The results demonstrated that this developed immunoassay was suitable for screening of low molecular weight contaminants.

A portable electrochemical magnetoimmunosensor for detection of sulfonamide antimicrobials in honey

A new electrochemical magnetoimmunosensor (EMIS) has been developed for the screening of residues of sulfonamide antimicrobials in honey samples. The immunosensor is able to detect up to ten different sulfonamide congeners at levels below the action points established in some European countries (25 μg kg^?1) after a hydrolysis step in which the sulfonamides are released from the corresponding conjugates formed in samples of this type. In spite of the complexity of the sample after the hydrolysis procedure, the EMIS could perform quantitative measurements, directly in these samples, without any additional sample cleanup or extraction step. For example, sulfapyridine, used as a reference, can be detected in hydrolyzed honey with a limit of detection (IC₉₀) of 0.1?±?0.03 μg kg^?1. Considering that the use of antibiotics for bee treatment is prohibited in the European Union, the immunosensor presented here could be an excellent screening tool. Moreover, several samples can be processed in parallel, which facilitates the analysis, reducing the necessity to use more costly confirmatory methods for just screening. As a proof of concept, a set of blind honey samples (spiked and incurred) were analyzed and the results were compared with those obtained by high-performance liquid chromatography–tandem mass spectrometry, demonstrating the potential of the EMIS as a screening tool.

Chemiluminescent determination of the activity of caspase-3 using a specific peptide substrate and magnetic beads

We report on a new scheme for the determination of the activity of caspase-3 using a specific peptide labeled with N-(4-aminobutyl)-N-ethylisoluminol (ABEI) as a chemiluminescent (CL) probe and on the development of magnetic separation technology. Firstly, the ABEI-labeled and biotinylated peptide was prepared and conjugated to streptavidin-coated magnetic beads (MBs) to form f-MBs (functionalized magnetic beads). The f-MBs contain a site (DEVD, Asp-Glu-Val-Asp) that is cleaved by caspase-3. Upon cleavage, the terminal residue attached to ABEI can dissociate from the f-MBs and can be used for CL detection. CL intensity is linearly related to the concentration of caspase-3 in the range 1.0 to 600 ng mL^?1, with a detection limit of 0.3 ng mL^?1. The relative standard deviation of the assay is 3.6 % at a level of 50 ng mL^?1 of caspase-3 (for n?=?11). The CL assay has been applied to the determination of caspase-3 in Jurkat cell extract with recoveries between 96.6 % and 106.1 % (n?=?5).

Multiplex bead-array competitive immunoassay for simultaneous detection of three pesticides in vegetables

We report on a multiplex bead-based competitive immunoassay using suspension array technology for the simultaneous detection of the pesticides triazophos, carbofuran and chlorpyrifos. Three hapten-protein conjugates were covalently bound to carboxylated fluorescent microspheres to serve as probes. The amount of conjugates and antibodies were optimized. The new multi-analyte assay has dynamic ranges of 0.02–50 ng?mL^?1, 0.5–500 ng?mL^?1 and 1.0–1000 ng?mL^?1 for triazophos, carbofuran and chlorpyrifos, respectively, and the detection limits are 0.024, 0.93 and 1.68 ng?mL^?1. This new multiplex assay is superior to the traditional ELISA in possessing a wider detection range, better reproducibility and the feature of multi-target detection. Cross-reactivity studies indicated that the bead-array method is highly selective for the three target pesticides, and that individual analyses have no significant influence between each other, also without cross-reactions from other structurally related pesticides. The method was applied to analyze vegetables spiked with the three pesticides, and the recoveries were in ranges of 78.5–112.1 %, 72.2–120.2 % and 70.2–112.8 %, respectively, with mean coefficients of variation of <15 %.

Aptamer based test stripe for ultrasensitive detection of mercury(II) using a phenylene-ethynylene reagent on nanoporous silver as a chemiluminescence reagent

We describe a paper-based chemiluminescence (CL) test for the determination of mercury(II) ion. A single-stranded DNA aptamer was first covalently immobilized via its amino groups to the hydroxy groups on the surface of cellulosic paper. The aptamer probes can capture Hg(II) ions due to their specific interaction with thymine. The CL reagent (a caboxylated phenylene-ethynylene referred to as P-acid) was immobilized on nanoporous silver (NPS@P-acid) and used a CL label on the aptamer. The stripe is then contacted with a sample containing Hg(II) ions and CL is induced by the addition of permanganate. CL intensity depends on the concentration of Hg(II) because Hg(II) increases the quantity of the P-acid-conjugated aptamer. The highly active surface of the NPS@P-acid composites results in an 8-fold higher CL intensity compared to the use of pure P-acid. This enables Hg(II) ion to be quantified in the 20 nM to 0.5 μM concentration range, with a limit of detection as low as 1 pM. This CL aptasensor is deemed to represent a promising tool for simple, rapid, and sensitive detection of Hg(II).

Biotin-avidin-conjugated metal sulfide nanoclusters for simultaneous electrochemical immunoassay of tetracycline and chloramphenicol

We report on a protocol for a simultaneous competitive immunoassay for tetracycline (TC) and chloramphenicol (CAP) on the same sensing interface. Conjugates of TC and of CAP with bovine serum albumin were first co-immobilized on a glassy carbon electrode modified with gold nanoparticles. In parallel, monoclonal anti-TC and anti-CAP antibodies were conjugated onto CdS and PbS nanoclusters, respectively. In a typical assay, the immobilized haptens and the added target analytes competed for binding to the corresponding antibodies on the nanoclusters. Subsequently, Cd(II) and Pb(II) ions are released from the surface of the corresponding nanoclusters by treatment with acid and then were detected by square wave anodic stripping voltammetry. The currents at the peak potentials for Cd(II) and Pb(II) were used as the sensor signal for TC and CAP, respectively. This multiplex immunoassay enables the simultaneous determination of TC and CAP in a single run with dynamic ranges from 0.01 to 50 ng mL^?1 for both analytes. The detection limits for TC and for CAP are 7.5 pg mL^?1 and 5.4 pg mL^?1, respectively. No obvious nonspecific adsorption and cross-reactivity was observed in a series of analyses. Intra-assay and inter-assay coefficients of variation were less than 10 %. The method was evaluated by analyzing TC and CAP in spiked samples of milk and honey. The recoveries range from 88 % to 107 % for TC, and from 91 % to 119 % for CAP.

Silicon-hybrid carbon dots strongly enhance the chemiluminescence of luminol

We report on a 4-min microwave pyrolytic method for the preparation of fluorescent and water-soluble silicon-hybrid carbon dots (C-dots) with high fluorescent quantum yield. The material is prepared by preheating aminopropyltriethoxysilane and ethylene diamine tetraacetic acid for 1 min, then adding a mixture of poly(ethylene glycol) and glycerin to the solution and heating for another 3 min. It is found that the hybrid carbon dots strongly enhance the chemiluminescence (CL) of the luminol/N-bromosuccinimide system. A study on the enhancement mechanism via CL, fluorescence and electron paramagnetic resonance spectroscopy showed that the effect most probably is due to electrostatic interaction between the C-dots and the luminol anion which facilitates electron transfer from luminol anion to the N-bromosuccinimide oxidant. CL intensity is linearly related to the concentration of the C-dots in the range between 1.25 and 20 μg mL^?1. The detection limit is 0.6 μg mL^?1 (at an S/N of 3).

L-Argininamide biosensor based on S1 nuclease hydrolysis signal amplification

A sensitive method is presented for the detection of L-argininamide. It is based on the amplification of the hydrolysis of S1 nuclease of single-stranded regions of an aptamer-target complex. The S1 nuclease, which is sequence-independent, is used to “recycle” target molecules, thus leading to strongly enhanced chemiluminescence (CL). L-Argininamide was chosen as model analyte. The DNA aptamer and its complementary DNA were labeled with the CL reagent N-(4-aminobutyl)-N-ethylisoluminol (ABEI). The DNA complementary to the aptamer was labeled with ABEI and immobilized on magnetic beads (MBs) coated with gold. The aptamer was also labeled with ABEI and self-assembled on the MBs. A duplex was formed due to hybridization between the DNA aptamer and the DNA complementary to the aptamer. In the presence of the target L-argininamide, a stem-loop aptamer structure is formed which subsequently denatures the duplex. This switch from a duplex structure to a stem-loop structure causes the formation of single-stranded regions both in the target-aptamer and in the single-stranded DNA on the MBs. The nuclease hydrolyzes the single-stranded regions and single-stranded DNA. Ultimately, L-argininamide is released which then interacts with another aptamer on the MB, thereby leading to one more L-argininamide. This autocatalytic cycle can generate substantial quantities of ABEI which then can be sensitively determined by the diperiodatonickelate-isoniazide reaction system. L-argininamide can be detected in the concentration range from 3.0?×?10^?4 to 3.0?×?10^?7 M, and the limit of detection is 1.0?×?10^?7 M.

Prussian blue-doped nanogold microspheres for enzyme-free electrocatalytic immunoassay of p53 protein

We report on a new enzyme-free electrochemical immunoassay for the sensitive detection of the p53 protein (p53; a model analyte) by using a screen-printed carbon electrode modified with monoclonal mouse anti-human p53 antibody tagged with gold nanoparticles. First, nanogold microspheres doped with Prussian Blue were synthesized by a reverse micelle method. The resulting microspheres were used to label polyclonal anti-p53 antibody which then was applied in a sandwich immunoassay in pH 6.5 buffer solution using the Prussian Blue in the particles as the redox-active reporter. The electrochemical signal of the immunosensor is shown to increase with the concentration of the analyte (p53 protein) in the range from 0.5 to 80 U mL^?1, with a detection limit of 0.1 U mL^?1. No non-specific adsorption was observed. Coefficients of variation for intra-assay and inter-assay were below 8.5 % and 11.5 %, respectively. In addition, the method was applied to the analysis of 15 human serum samples, and a good relationship was found between the new immunoassay and the referenced electro-chemiluminescence method.