Chemiluminescence multichannel immunosensor for biodetection

An improved portable detector for biological compounds, the chemiluminescence multichannel immunosensor (CL-MADAG), has been developed and characterised. The device is based on a capillary ELISA technique in combination with a miniaturised fluidics system and uses chemiluminescence as the detection principle. The fluidics system construction allows three chemiluminescence immunoassays to be performed simultaneously within three fused silica capillaries (FSC). The CL-MADAG was characterised in a series of experiments with staphylococcal enterotoxin B (SEB) as a model toxin, the bacterial phage virus M13 as a virus simulant, and a pathogenic strain of Escherichia coli as simulant for bacteria. It was shown that the CL-MADAG can assay liquid samples for these substances within 24 min. The detection limits were 5 ng/ml for SEB, 10⁵ cfu/ml for E. coli O157:H7 and 10⁷ pfu/ml for M13.     

Development of a highly sensitive inhibition immunoassay for microcystin-LR

The development of a rapid one-step antigen-immobilized inhibition ELISA for microcystin-LR is described. For microplate coating a microcystin-biotin conjugate was synthesized. Using the commercially available monoclonal antibody MC10E7 in our newly established assay, IC₅₀ values of 0.045 μg l⁻¹ have been achieved. The detection limit for microcystin-LR was 4 ng l⁻¹. Considering the guidelines proposed by the world health organization (WHO) for microcystin-LR in drinking water (1 μg l⁻¹) the sensitivity of our test is more than sufficient. The period of assay processing could successfully be shortened to about 3 h without any loss in sensitivity. The suitability of the newly developed assay was evaluated with microcystin-LR spiked environmental water samples. Recovery rates for microcystin-LR between 60 and 165% were obtained in the linear range of the test format. The antigen-immobilized test format provides a highly reproducible, easy, and fast to perform detection system for microcystin allowing an internal retrospective quality control of the assay.     

Sensitive and rapid chemiluminescence enzyme immunoassay for microcystin-LR in water samples

A highly sensitive, specific, simple, and rapid chemiluminescence enzyme immunoassay (CLEIA) was developed for the determination of microcystin-LR (MC-LR). Several physicochemical parameters such as the chemiluminescent assay mediums, the dilution ratio of MC-LR-OVA conjugate, monoclonal antibody concentration, and peroxidase labeled antibody concentration were studied and optimized. Under optimum conditions, calibration curve obtained for MC-LR had detection limits of 0.032 ± 0.003 μg L⁻¹, the 50% inhibition concentration (IC₅₀) was 0.20 ± 0.02 μg L⁻¹ and the quantitative detection range was 0.062-0.65 μg L⁻¹. The proposed methods was successfully applied to the monitoring of MC-LR in spiked water samples without significant effect of the matrix, and the recovery of MC-LR added to water samples at different concentrations ranged from 80% to 115% with the coefficients of variation (CVs) less than 9%. The LOD attained from the calibration curves and the results obtained for the real samples demonstrate the potential use of CLEIA as a screening tool for the analysis of MC-LR in environmental samples.     

Determination of clenbuterol by capillary electrophoresis immunoassay with chemiluminescence detection

A competitive immunoassay for clenbuterol (CLB) based on capillary electrophoresis with chemiluminescence (CL) detection was established. The method was based on the competitive reaction of horseradish peroxidase (HRP)-labeled CLB (CLB-HRP) and free CLB with anti-CLB antiserum. The factors affecting the electrophoresis and CL detection were systematically investigated with HRP as a model sample. Under the optimal conditions, the tracer CLB-HRP and the immunoassay complex were separated, and the linear range and the detection limit (S/N = 3) for CLB were 5.0-40 nmol l⁻¹ and 1.2 nmol l⁻¹, respectively. The proposed method has been applied satisfactorily in the analysis of urine sample.     

Analysis of Follicle-Stimulating Hormone by CE with Chemiluminescence Detection Based on Competitive Immunoassay

A competitive immunoassay and capillary electrophoresis with enhanced chemiluminescence detection have been used for determination of follicle-stimulating hormone (FSH) in human serum. The method is based on the competitive immunochemical reaction of FSH and horseradish peroxidase (HRP)-labeled FSH with anti-FSH, CE separation of antibody-bound and free HRP-labeled FSH, then chemiluminescence detection. The detection limit depends on the stability of the immune complex, which depends on analysis time and detector design, and on the chemiluminescence enhancer used. A unique chemiluminescence detector without dead volume or diluent effects was therefore used, and sodium tetraphenylboron was selected as the optimum enhancer. As a result sensitivity was substantially improved. Free HRP-labeled FSH and the immune complex could be separated within 15 min in alkaline borate buffer by use of a potential of 15 kV. Under the optimum conditions a calibration plot for FSH was established in the concentration range 0–100 mIU mL⁻¹; the detection limit was 0.06 mIU mL⁻¹. The concentration sensitivity achieved was 30 times better than that of ELISA.     

Amorphous carbon nanoparticle used as novel resonance energy transfer acceptor for chemiluminescent immunoassay of transferrin

Amorphous carbon nanoparticles (ACNPs) showing highly efficient quenching of chemiluminescence (CL) were prepared from candle soot with a very simple protocol. The prepared ACNP was employed as the novel energy acceptor for a chemiluminescence resonance energy transfer (CRET)-based immunoassay. In this work, ACNP was linked with transferrin (TRF), and horseradish peroxidase (HRP) was conjugated to TRF antibody (HRP–anti-TRF). The immunoreaction rendered the distance between the ACNP acceptor and the HRP-catalyzed CL emitter to be short enough for CRET occurring. In the presence of TRF, this antigen competed with ACNP–TRF for HRP–anti-TRF, thus led to the decreased occurrence of CRET. A linear range of 20–400 ng mL⁻¹ and a limit of detection of 20 ng mL⁻¹ were obtained in this immunoassay. The proposed method was successfully applied for detection of TRF levels in human sera, and the results were in good agreement with ELISA method. Moreover, the ACNPs show higher energy transfer efficiency than other conventional nano-scaled energy acceptors such as graphene oxide in CRET assay. It is anticipated that this approach can be developed for determination of other analytes with low cost, simple manipulation and high specificity.     

A rapid sandwich immunoassay for human fetuin A using agarose-3-aminopropyltriethoxysilane modified microtiter plate

A rapid sandwich immunoassay (IA) with enhanced signal response for human fetuin A (HFA) was developed by modifying the surface of a KOH-treated polystyrene microtiter plate (MTP) with agarose and 3-aminopropyltriethoxysilane (APTES). The agarose-APTES complex binds covalently to the hydroxyl moiety of the MTP plate to serve as a binding platform for bioconjugation of EDC-activated anti-HFA antibody (Ab) via carbodiimide coupling. The one-step kinetics-based sandwich enzyme-linked immunosorbent assay (ELISA) enabled the detection of HFA in 30 min with a limit of detection (LOD) and a linear range of 0.02 ng mL⁻¹ and 1–243 ng mL⁻¹, respectively. It detected HFA spiked in diluted human whole blood and serum, and HFA in ethylenediaminetetraacetic acid (EDTA)-plasma of patients with high precision similar to that of conventional ELISA. The anti-HFA Ab-bound agarose-functionalized MTPs retained their functional activity after 6 weeks of storage in 0.1 M PBS, pH 7.4 at 4 °C.     

Immunochemical method for sulfasalazine determination in human plasma

Sulfasalazine is an antibiotic used in the treatment of inflammatory bowel diseases. For the assessment of sulfasalazine in several biological matrices, an Enzyme-Linked Immunosorbent Assay (ELISA) method based on polyclonal antibodies was developed and characterized.The immunoassay showed a high sensitivity (IC₅₀ = 0.51 ng mL⁻¹) and specificity, a detection limit of 0.02 ng mL⁻¹ and a dynamic range of 0.06-3.75 ng mL⁻¹ (80-20% inhibition). The immunoassay performed well when it was applied to spiked plasma samples (from 0.5 to 2.0 ng mL⁻¹) previously cleaned up by protein precipitation with methanol. Recoveries ranged from 83 to 119%, with a mean value of 99% (CV = 13%).Since sulfasalazine remaining of a treatment reaches the systemic circulation in unchanged form, the immunoassay can be applied to the determination of this pharmaceutical in human plasma in order to facilitate the control of the patients through the application of personal doses.     

A monoclonal antibody-based time-resolved fluoroimmunoassay for chloramphenicol in shrimp and chicken muscle

A time-resolved fluoroimmunoassay (TR-FIA) for determination of chloramphenicol (CAP) in shrimp and chicken muscle was developed. The method was based on a direct competitive immunoassay using europium-labeled anti-CAP monoclonal antibody (MAb) and CAP-ovalbumin as coated antigen. The limit of detection was 0.05 ng g⁻¹ and limit of quantification was 0.1 ng g⁻¹. Recoveries ranged from 101.2 to 112.5% for shrimp and 104.9 to 115.3% for chicken muscle at spiked levels of 0.1-5 ng g⁻¹, with intra-assay and inter-assay variations 8.7-14.6 and 9.6-17.8%, respectively. The results obtained by the TR-FIA and ELISA correlated well. The established TR-FIA was validated for the determination of incurred shrimp samples and confirmed by gas chromatography with microcell electron capture detector (GC-μECD).     

Analysis of antioxidants using a capillary electrophoresis with chemiluminescence detection system

We developed a capillary electrophoresis with chemiluminescence detection system using 2-methyl-6-p-methoxyphenylethynylimidazopyrazinone as a chemiluminescence reagent for determination of antioxidants of superoxide anions. 2-Methyl-6-p-methoxyphenylethynylimidazopyrazinone reacted with superoxide anions generated through the reaction of hypoxanthine and xanthine oxidase, and then emitted chemiluminescence. Suppression of the chemiluminescence in the presence of antioxidants for superoxide anions was introduced as a detection principle for antioxidants into the capillary electrophoresis with chemiluminescence detection system. After optimizing the analytical conditions, various antioxidants, such as superoxide dismutase, nitroblue tetrazolium, ascorbic acid, and catechin, were subjected to the present system. They gave negative peaks due to the quenching effect; the detection limits of superoxide dismutase, nitroblue tetrazolium, ascorbic acid, and catechin were 1, 100, 100, and 10 μM, respectively (S/N = 2). A model sample consisting of superoxide dismutase and nitroblue tetrazolium was satisfactorily separated and detected within ca. 10 min. We also applied the present system to analysis of catechin in green tea as a real sample.     

Detection of anti-tetanus toxoid antibody on modified polyacrylonitrile fibers

Accurate determination of concentration of immunoglobulin (IgG) to tetanus toxoid is important in order to evaluate the immunogenicity of tetanus toxoid vaccines, immune competence in individual patients and to measure the prevalence of immunity in populations. Surface modified polyacrylonitrile (PAN) fibers were evaluated as a matrix to develop highly sensitive method for the detection of anti-tetanus antibody in a sandwich ELISA format. In the proposed method tetanus toxoid immobilized on modified PAN fibers was used to detect anti-tetanus antibody (raised in horse hence represented as horse anti-tetanus toxoid or HAT-Ab) with horse raddish peroxidase enzyme conjugated with Rabbit anti-Horse IgG (RAH-HRP) as the label within 2.5 h. A sigmoidal pattern for the detection of different concentration of antibody ranging from 1.0 to 0.0001 IU mL⁻¹ was validated. The immunoassay recorded a very high sensitivity as concentration as low as 0.0005 IU mL⁻¹ of HAT-Ab was detected. The intra- and inter-assay precision for 3 parallel measurements of 0.01 and for 0.001 IU mL⁻¹ of antibody varied from 5.4% to 11% and 5.7% to 20% respectively. PAN fibers were also used to qualitatively access the presence of different level of anti-tetanus antibody spiked in human blood. Seroepidemiological studies to measure the immunity against tetanus were conducted with twenty-five human beings belonging to various age groups using modified PAN-ELISA. The sensitivity, specificity and the reproducibility of the developed immunoassay indicate the potential application of modified PAN fibers in the field of immunodiagnostics.     

An ultrasensitive chemiluminescence immunosensor for PSA based on the enzyme encapsulated liposome

A highly sensitive chemiluminescence immunosensor for the detection of prostate-specific antigen (PSA) was developed based on a novel amplification procedure with the application of enzyme encapsulated liposome. Horseradish peroxidase (HRP) encapsulated and antibody-modified liposome acts as the carrier of a large number of markers and specific recognition label for the amplified detection of PSA. In the detection of PSA, the analyte was first bound to the specific capture antibody immobilized on the microwell plates, and then sandwiched by the antibody-modified liposomes encapsulating HRP. The encapsulated markers, HRP molecules were released by the lysis of the specifically bound liposomes in the microwell with Triton X-100 solution. Then, the analyte PSA could be determined via the chemiluminescence signal of HRP-catalyzed luminol/peroxide/enhancer system. The “sandwich-type” immunoassay provides the amplification route for the PSA detection in ultratrace levels. The CL emission intensity exhibits dynamic correlation to PSA concentration in the range from 0.74 pg/ml to 0.74 μg/ml with readily achievable detection limit of 0.7 pg/ml.     

Competitive magnetic immunoassay for protein detection in thin channels

Functional magnetic nanoparticles are prepared and characterized for protein detection in a magnetic separation channel. This detection method is based on a competitive immunoassay of magnetic separation in thin channels using functional magnetic nanoparticles. We used protein A–IgG complex to demonstrate the feasibility. Free IgG and fixed number of IgG-labeled microparticles were used to compete for limited sites of protein A on the magnetic nanoparticles. Several experimental parameters were investigated for protein detection. The deposited percentages of IgG-labeled microparticles at various concentrations of free IgG were determined and used as a reference plot. The IgG concentration in a sample was deduced and determined based on the reference plot using the deposited percentage of IgG-labeled microparticles from the sample. The linear range of IgG detection was from 5.0 × 10⁻⁸ to 1.0 × 10⁻¹¹ M. The detection limit was 3.69 × 10⁻¹² M. The running time was less than 10 min. Selectivities were higher than 92% and the relative errors were less than 7%. The IgG concentration of serum was determined to be 3.6 mg ml⁻¹. This measurement differed by 8.3% from the ELISA measurement. The recoveries of IgG spiked in serum were found to be higher than 94%. This method can provide simple, fast, and selective analysis for protein detection and other immunoassay-related applications.     

Development of an automatic multi-channel ink-jet ejection chemiluminescence system and its application to the determination of horseradish peroxidase

In this work, an automatic multi-channel ink-jet for chemiluminescence (CL) analysis was developed. The four-channel ink-jet device was controlled by a home-made circuit. Differing from the classic flow injection CL, the whole procedure for CL analysis was automatically completed on a hydrophobic glass side. CL reaction of luminal and hydrogen peroxide for the determination of horseradish peroxidase (HRP) was selected as an application to automatic CL analysis platform. All solutions delivered by different channels were precisely ejected to the same position of the glass slide for the CL analysis. The consumption of reaction solution was reduced to nanoliter level. The whole CL analysis could be completed in less than 4 min, which was benefited from the prompt solution mixing in small size of droplet. The CL intensity increased linearly with HRP concentration in the range from 0.01 to 0.5 μg mL⁻¹. The limit of detection (LOD) (S/N = 3) was 0.005 μg mL⁻¹. Finally, the automatic CL system could also be used for the detection of HRP in HRP–protein conjugates, which showed its practical application in immunoassay.     

Comparative study of three immunoassays based on monoclonal antibodies for detection of the pesticide parathion-methyl in real samples

Three immunoassay systems: indirect, direct competitive enzyme-linked immunosorbent assay (IC-ELISA and DC-ELISA), fluorescence polarization immunoassay (FPIA) based on monoclonal antibodies for the detection of parathion-methyl (PM) were developed and optimized. Several PM derivatives (haptens) were conjugated to proteins and fluoresceinthiocarbamyl ethylenediamine (EDF) to obtain immunogens and competitors. The influence of immunogen and competitor structures on the assay performance was investigated. IC-ELISA was the most sensitive of all techniques developed, with a detection limit of 0.08 ng ml⁻¹, but assay time was the longest (3.5 h per 96-well microtitre plate). DC-ELISA was easier to perform and quicker (1.5 h per 96-well microtitre plate) but less sensitive than IC-ELISA (detection limit was 0.5 ng ml⁻¹). FPIA was the fastest and simplest (7 min per 10 samples) but the least sensitive (detection limit was 15 ng ml⁻¹) technique. The methods were characterized by high specificity and reproducibility. The cross-reactivity for parathion-ethyl was around 30-40% for IC-ELISA and FPIA, but significantly higher (125%) for DC-ELISA. The immunoassays were applied to the analysis of PM residues in different food and environmental matrices. Methanol extracts of vegetable, fruit and soil samples were used for the analysis. Recoveries for most spiked samples averaged between 85 and 110%. The methods developed can be used for screening of food and environmental samples for PM residues without complicated clean-up.     

Application of a polyaniline based ammonium sensor for the amperometric immunoassay of a urease conjugated Tal 1 protein

An amperometric immunoassay was developed by coupling the enzyme-linked immunosorbent assay (ELISA) microtiter-plate system with a polyaniline-perfluorosulfonated ionomer composite (PA/NF) electrode incorporated flow injection analysis (FIA) system and used for the analysis of Tal 1 protein, found in leukemic T cell. Rabbit polyclonal antibody (pAb) against Tal 1 and urease-pAb were used, respectively as the captured protein and enzyme labeled conjugate for sandwich immunoassay of Tal 1. Characteristics of the PA/NF electrode such as reproducibility, stability and sensitivity were studied. The detection limits of the PA/NF electrode for NH₄⁺ and urease were found to be 5 μM and 0.05 nM, respectively. Assay conditions such as the amount of pAb needed for coating the plate, the concentration of urease-pAb conjugate appropriate for the immunoreaction and the incubation time for urea to react with the bound urease-pAb in the microtiter-wells were also studied. A detection limit as lower as 0.5 ng/ml and a dynamic range of 1.0-100 ng/ml were found for the immunoassay of Tal 1 protein with the developed immunoassay system.     

Sensitive sandwich immunoassay based on single particle mode inductively coupled plasma mass spectrometry detection

A sensitive sandwich type immunoassay has been proposed with the detection by inductively coupled plasma mass spectrometry (ICP-MS) in a single particle mode (time resolved analysis). The signal induced by the flash of ions (¹⁹⁷Au⁺) due to the ionization of single Au-nanoparticle (Au-NP) label in the plasma torch can be measured by the mass spectrometer. The frequency of the transient signals is proportional to the concentration of Au-NPs labels. Characteristics of the signals obtained from Au-NPs of 20, 45 and 80 nm in diameters were discussed. The analytical figures for the determination of Au-labeled IgG using ICP-MS in conventional integral mode and single particle mode were compared in detail. Rabbit-anti-human IgG was used as a model analyte in the sandwich immunoassay. A detection limit (3σ) of 0.1 ng mL⁻¹ was obtained for rabbit-anti-human IgG after immunoreactions, with a linear range of 0.3-10 ng mL⁻¹ and a RSD of 8.1% (2.0 ng mL⁻¹). Finally, the proposed method was successfully applied to spiked rabbit-anti-human IgG samples and rabbit-anti-human serum samples. The method resulted to be a highly sensitive ICP-MS based sandwich type immunoassay.     

Development of an immunomagnetic bead-based time-resolved fluorescence immunoassay for rapid determination of levels of carcinoembryonic antigen in human serum

A novel immunoassay for the determination of tumor markers in human serum was established by combining a time-resolved fluoroimmunoassay (TRFIA) and immunomagnetic separation. Based on a sandwich-type immunoassay format, analytes in samples were captured by magnetic beads coated with one monoclonal antibody and “sandwiched” by another monoclonal antibody labeled with europium chelates. The immunocomplex was separated and washed by exposure to a magnetic field and treatment with enhancement solution; fluorescence was then measured according to the number of europium ions dissociated. Levels of the model analyte, carcinoembryonic antigen (CEA), were determined in a linear range (1–1000 ng mL⁻¹) with a limit of detection of 0.5 ng mL⁻¹ under optimal conditions. The reproducibility, recovery, and specificity of the immunoassay were demonstrated to be acceptable. To evaluate this novel assay for clinical applications, 239 serum samples were evaluated. Compared with the conventional TRFIA and chemiluminescence immunoassay (CLIA), the correlation coefficients of the developed immunoassay were 0.985 and 0.975, respectively. These results showed good correlation and confirmed that our method is feasible and could be used for the clinical determination of CEA (or other tumor antigens) in human serum.     

Lateral-flow colloidal gold-based immunoassay for the rapid detection of deoxynivalenol with two indicator ranges

A lateral-flow immunoassay using a colloidal gold-labelled monoclonal antibody was developed for the rapid detection of deoxynivalenol (DON). Different parameters, such as the amount of immunoreagents, type of the materials, composition of the blocking solution and of the detector reagent mixture, were investigated to provide the optimum assay performance. The experimental results demonstrated that such a visual test had an indicator range rather than a cut-off value. Thus, tests for DON determination with two different indicator ranges of 250-500 and 1000-2000 μg kg⁻¹ were designed. The method allowed detection of DON at low and high concentration levels, which could be useful for research and practical purposes. The assay applied to spiked wheat and pig feed samples demonstrated accurate and reproducible results. The applicability of the developed lateral-flow test was also confirmed under real field conditions. The test strips prepared in Belgium were sent to Mexico, where they were used for the screening of DON contamination in different bread wheat entries from Fusarium Head Blight inoculated plots. The results were compared with those obtained by ELISA and LC-MS/MS. A poor correlation between ELISA and LC-MS/MS was observed. Visual results of the dipstick tests were in a good agreement with the results of the LC-MS/MS method. Coupled with a simple and fast sample preparation, this qualitative one-step test based on the visual evaluation of results did not require any equipment. Results could be obtained within 10 min. The described assay format can be used as a simple, rapid, cost-effective and robust on-site screening tool for mycotoxin contamination in different agricultural commodities.     

Phenylboronic acid immunoaffinity reactor coupled with flow injection chemiluminescence for determination of α-fetoprotein

A reusable and sensitive immunoassay based on phenylboronic acid immunoaffinity reactor in combination with flow injection chemiluminescence (CL) for determination of glycoprotein was described. The reactor was fabricated by immobilizing 3-aminophenylboronic acid (APBA) on glass microbeads with γ-glycidoxypropyltrimethoxysilane (GPMS) as linkage. The α-fetoprotein (AFP) could be easily immobilized on the APBA coated beads through sugar-boronic interaction. After an off-line incubation, the mixture of the analyte AFP with horseradish peroxidase-labeled AFP antibody (HRP-anti-AFP) was injected into the reactor. This led the trapping of free HRP-anti-AFP by the surface coated AFP on glass beads. The trapped HRP-anti-AFP was detected by chemiluminescence due to its sensitizing effect on the reaction of luminol and hydrogen peroxide. Under optimal conditions, the chemiluminescent signal was proportional to AFP concentration in the range of 10-100 ng mL⁻¹. The whole assay process including regeneration of the reactor could be completed within 31 min. The proposed system showed acceptable detection and fabrication reproducibility, and the results obtained with the present method were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. The described method enabled a low-cost, time saving and was potential to detect the serum AFP level in clinical diagnosis.