Fluorometric enzyme immunosensing system based on a renewable immunoreaction platform for the detection of Schistosoma japonicum antibody

A fluoroimmunosensing device which was based on ferulic acid (FA)/horseradish peroxidase system for the detection of Schistosoma japonicum antibody (SjAb) has been developed. To circumvent the difficulty of regeneration of immunocomposite surface, a natural chitosan-epoxy resin matrix was used for the immobilization of SjAg. The surface of the immunocomposite layer reacted was easily regenerated by simple polishing. The renewed surface served as a platform for the competitive immuno-reaction of HRP-SjAb and SjAb with SjAg immobilized at the support body surface and for enzymatic reaction. A novel fluorescent substrate ferulic acid for HRP, which is relatively stable toward H₂O₂, has been adapted in the proposed fluorometric enzyme immunosensing system. FA can been catalyzed to produce a non-fluorescent species. The amount of HRP-SjAb bound to the aforementioned renewable surface layer, which is related to the content of SjAb in samples could be quantitized by measuring the decrease of fluorescence of FA induced by HRP-SjAb. The chitosan incorporated in matrix is favorable for the amplification of this sensing system due to the electrostatic reaction with FA. The proposed method showed a linear response ranging from 45 to 150 ng ml⁻¹, with an improved detection limit of 45 ng ml⁻¹. The method has been employed to determine SjAb in serum samples.     

Silica sol-gel amperometric immunosensor for Schistosoma japonicum antibody assay.

An amperometric immunosensor was constructed by dispersing graphite, schistosoma-japonicum antigen (SjAg) and silica sol-gel at low temperature. The performance characteristics of the prepared immunosensor were examined in the buffered solution of o-aminophenol (o-AP) used as a substrate. It exhibited excellent physical and electrochemical stability with a renewable external surface. A competitive binding assay was employed to determine schistosoma-japonicum antibody (SjAb) with the aid of horseradish peroxidase labeled SjAb (HRP-SjAb). The experimental parameters for SjAb assay were optimized, including the amount of labeled SjAb in incubation solution, incubation time, temperature and the pH of solution. The use of o-AP substrate and amperometric detection at -250 mV (vs. SCE) results in a determination limit of 0.32 microg/ml and a linear range extending up to 0.18 microg/ml. The results of SjAb assay in serum samples demonstrate the feasibility of using the proposed immunosensor for clinical analysis.     

Fluorometric enzyme immunosensing system based on natural product resveratrol for horseradish peroxidase and Ag/SiO<Subscript>2</Subscript> nanoparticles

The properties of resveratrol (3′, 4′, 5-trihydroxystlbene, RST) were for the first time evaluated as a potential substrate for horseradish peroxidase (HRP)-catalyzed fluorogenic reaction. The properties of RST for use as fluorogenic substrates for HRP and its application in immunoassays were compared with commercially available substrates such as p-hydroxyphenylpropionic acid (pHPPA), chavicol and Amplex red by a fluoroimmunosensing method in the use of Schistosomia japonicum antibody (SjAb) as a model analyte. The fluoroimmunosensing device was constructed by dispersing Schistosomia japonicum antigen (SjAg), nano-Ag/SiO₂ particles and sol-gel at low temperature. In pH 5.8 Britton-Robinson buffer (B-R), HRP-SjAb conjugates can catalyze the polymerization reaction of RST by H₂O₂ forming fluorescent dimmers. The increase of the fluorescence intensity of the dimmers product at emission of 462 nm (excitation: 315 nm) is proportional to the concentration of HRP-SjAb binding to the SjAg entrapped in the nano-Ag/SiO₂ particles-sol-gel matrix. A competitive binding assay has been used to determine SjAb in rabbit serum with the aid of SjAb labeled with HRP. Substrate RST showed comparable ability for HRP detection and its enzyme-linked immunosensing reaction system, in a linear detection ranging of 1.5×10⁻⁶–7.3×10⁻⁴ g/L and with a detection limit of 1.5×10⁻⁶ g/L. The immobilized biocomposites surface could be regenerated by simply polishing with an alumina paper, with an excellent reproducibility (RSD = 4.7%). The proposed method has been successfully used for analysis of the rabbit serum sample with satisfactory results. Supported by the Projects of Scientific Research Fund of Hunan Provincial Education Department of China (Grant Nos. 05B020 and 06C098)     

Electrochemical Detection of Schistosoma Japonicum Antibody Using Biocatalytic Deposition

Abstract

An ultrasensitive electrochemical immunoassay based on biocatalytic deposition has been proposed for the detection of Schistosoma japonicum antibody (SjAb) in infected rabbit serum. Schistosoma japonicum antigen (SjAg) was immobilized on the gold electrode surface via glutaraldehyde crosslink and then incubated with infected rabbit serum containing SjAb; finally, the goat anti-rabbit IgG labeled with alkaline phosphatase was sandwiched to form the immunocomplex on the gold electrode surface. The alkaline phosphatase converted nonelectroactive substrate into the reducing agent and the latter, in turn, reduced metal ions to form electroactive metallic product on the electrode surface. Linear sweep voltammetry (LSV) was used to quantify the amount of the deposited silver and give the analytical signal for SjAb. Assay conditions such as the antigen concentration and enzymatic silver deposition time were optimized. The electrochemical immunosensor was able to realize a reliable determination of SjAb in the dilution range from 1:5000 to 1:100 with a detection limit of 1:6457 of dilution ratio. The feasibility of the proposed immunosensor for possible clinical applications was also investigated by analyzing real serum samples.     

An amperometric immunosensor based on a conducting immunocomposite electrode for the determination of Schistosoma japonicum antigen.

A renewable amperometric immunosensor based on a graphite-paraffin-Schistosoma japonicum antibody (SjAb) biocomposite electrode has been prepared for the detection of Schistosoma japonicum antigen (SjAg). Competitive ELISA was employed involving HRP-SjAg as a tracer and 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The product of an enzyme catalytic reaction was detected at +0.1 V (vs. Ag/AgCl reference electrode) for measuring the amount of HRP-labeled SjAg binding to the electrode surface. The assay conditions were optimized, including the amount of SjAb loading in the electrode and HRP-SjAg in the incubation solution, the pH of the measuring solution and the incubation time. The measuring range was 0.5-30 microg/ml under the optimum conditions. Rabbit serum samples of different infection degree were measured, which demonstrated that the immunosensor meets the demands of clinical analysis. It exhibits some advantages, such as simplicity of fabrication, rapidity of measurement, and satisfactory sensitivity and reproducibility.     

Automated chemiluminescent dual-analyte immunoassay based on resolved immunosensing channels

A novel system of series-wound immunosensing channels (SWIC) was proposed for automated chemiluminescent (CL) dual-analyte immunoassay by immobilizing respectively different capture antibodies on the inner walls of series-wound glass channels. This system could use a single enzyme as label to perform multiplex immunoassay in one fluid way. Using α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as model analytes, the mixture including AFP, horseradish peroxidase (HRP)-labeled anti-AFP antibody, CEA and HRP-labeled anti-CEA antibody was introduced into the SWIC for carrying out the on-line incubation. Upon injection of CL substrate the CL signals from the two immunosensing channels were conveniently resolved and near-simultaneously collected with the aid of optical shutter. AFP and CEA could be rapidly assayed in the ranges of 1.0-100 and 1.0-80 ng/ml with detection limits of 0.41 and 0.39 ng/ml, respectively. The assay results of clinical serum samples were in an acceptable agreement with the reference values. This designed flow-through immunosensing system based on SWIC provided an automated, reusable, simple, sensitive and low-cost approach for multianalyte immunoassay.     

A polymer-based microfluidic device for immunosensing biochips

This paper describes the design, fabrication, and test of a PDMS/PMMA-laminated microfluidic device for an immunosensing biochip. A poly(dimethyl siloxane)(PDMS) top substrate molded by polymer casting and a poly(methyl methacrylate)(PMMA) bottom substrate fabricated by hot embossing are bonded with pressure and hermetically sealed. Two inlet ports and an air vent are opened through the PDMS top substrate, while gold electrodes for electrochemical biosensing are patterned onto the PMMA bottom substrate. The analyte sample is loaded from the sample inlet port to the detection chamber by capillary force, without any external intervening forces. For this and to control the time duration of sample fluid in each compartment of the device, including the inlet port, diffusion barrier, reaction chamber, flow-delay neck, and detection chamber, the fluid conduit has been designed with various geometries of channel width, depth, and shape. Especially, the fluid path has been designed so that the sample flow naturally stops after filling the detection chamber to allow sufficient time for biochemical reaction and subsequent washing steps. As model immunosensing tests for the microfluidic device, functionalizations of ferritin and biotin to the sensing surfaces on gold electrodes and their biospecific interactions with antiferritin antiserum and streptavidin have been investigated. An electrochemical detection method for immunosensing by biocatalyzed precipitation has been developed and applied for signal registration. With the biochip, the whole immunosensing processes could be completed within 30 min.     

Bioelectrochemical Magnetic Immunosensing of Trichloropyridinol: A Potential Insecticide Biomarker

A fast, simple and sensitive bioelectrochemical magnetic immunosensing method is developed to monitor a potential insecticide biomarker, trichloropyridinol (TCP), in environmental sample. A magnet/glassy carbon (MGC) working electrode was used to accumulate immunocomplex associated magnetic beads and separate free and unbound reagents after liquid phase competitive immunoreaction among TCP antibody coated magnetic beads, TCP analyte and horseradish peroxidase (HRP) labeled TCP. The activity of HRP tracers was monitored by square‐wave voltammetry (SWV) by scanning electrocactive enzymatic product in the presence of 3,3′,5,5′‐tetramethylbenzidine dihydrochloride and hydrogen peroxide (TMB‐H₂O₂) substrate solution. The electrochemical signal of enzymatic product was greatly enhanced by dual accumulation events: magnetic accumulation of enzyme tracers bound magnetic beads and constant potential accumulation of enzymatic product. The voltammetric characteristics of substrate and enzymatic product were investigated, and the parameters of SWV analysis and immunoassay were optimized. Under the optimal conditions the immunosensor was used to measure as low as 5 ng L^?1 (ppt) TCP, which is 50‐fold lower than the value indicated by the manufacture of the TCP RaPID Assay kit (0.25 μg/L, colorimetric detection). The performance of the developed immunosensing system was successfully evaluated with river water samples spiked with TCP, indicating this convenient and sensitive technique offers great promise for decentralized environmental application. This technique could be readily used for detection of other environmental contaminants by developing specific antibodies against the contaminants and are expected to open new opportunities for environmental monitoring and public health.     

A piezoelectric immunoassay based on self-assembled monolayers of cystamine and polystyrene sulfonate for determination of Schistosoma japonicum antibodies

A piezoelectric immunosensor based on an improved immobilization strategy combining self-assembled monolayers (SAM) of cystamine (Cys) and polystyrene sulfonate (PSS) has been developed for the determination of Schistosoma japonicum antibodies (SjAb) in rabbit serum. Cys SAM were first applied to the gold electrode surface of the crystal, serving as a positively-charged base. Schistosoma japonicum antigen (SjAg) was then electrostatically immobilized on the crystal by means of a negatively-charged PSS layer. When sealed by use of an appropriately selected blocking reagent for BSA and normal rabbit serum (NRS), non-specific adsorption could be substantially reduced.The immunosensor was used to determine SjAb in optimized buffer medium with addition of poly(ethylene glycol) (PEG), which served as an immunoreaction enhancer. It was shown experimentally that SjAg immobilized by the Cys-PSS adsorption procedure had higher immunological activity or binding efficiency than those immobilized by the glutaraldehyde (GLU) binding or direct attachment procedures. The immunosensor developed had satisfactory sensitivity and detection limit, and regeneration of the piezoelectric quartz-crystal was easy. Analytical results obtained with infected rabbit serum samples indicated that the proposed immunosensor is a promising alternative for qualitative and quantitative determination of SjAb in clinical diagnosis of infection with Schistosoma japonicum.     

Disposable multichannel immunosensors for 2,4-dichlorophenoxyacetic acid using acetylcholinesterase as an enzyme label

An improved version of the disposable multichannel immunochemical biosensor for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) based on a screen-printed amperometric transducer and monoclonal antibodies (MAb) against 2,4-D is reported. Entrapment within a thin Nafion film was used for the direct immobilization of MAb at the electrode surface. The amount of the tracer (2,4-D conjugated to acetylcholinesterase) bound in a competitive immunochemical reaction was determined amperometrically using acetylthiocholine iodide as substrate. The measuring procedure (times of incubation with tracer and substrate, pH, tracer concentration) was optimized. The sensor was able to detect less than 0.01 μg/L of free 2,4-D in water. One analysis (8 samples) was completed in 30 min (20 min for immunochemical reaction, 5 min incubation with substrate, 5 min measurement). The performance of the immunosensor (two configurations) was evaluated on real samples (tap water) with added 2,4-D. The determined amounts (mean values 0.097 to 0.105 and 0.89 to 1.13) corresponded well with the added contents of 2,4-D (0.100 and 1.00 μg/L, respectively).     

An optical immunosensor based on surface plasmon resonance for determination of bFGF

An optical immunosensor based on surface plasmon resonance (SPR) has been developed for immunosensing. The sensor is designed on the basis of fixing incident angle of light and measuring the reflected intensities in the wavelength range of 400-800 nm simultaneously. The SPR spectrum was shown in terms of reflected light intensities versus wavelengths of incident light. The intensity of the reflected light reaches the minimum at the resonant wavelength. Molecular self-assembling in solution is used to form the sensing membrane on gold substrate. The kinetic processes of sensing monolayer formation were studied. The basic fibroblast growth factor, a kind of basic polypeptide, was determined in the concentration range of 0.24-9.6 μg/ml. Under optimum experimental conditions, the sensor has a good repeatability, reversibility and selectivity.     

纳米金-蛋白A介导抗体定向固定的压电传感及电化学特性研究

以纳米金为载体标记蛋白A(PA),用于介导抗体在压电石英晶体金电极表面的定向固定化.以补体C_1q抗体为模型,采用压电传感技术实时监察了此敏感界面的免疫反应过程,并考察了与补体C_1q免疫反应的压电响应性能.金标PA固定抗体的方法与传统的直接PA固定化方法相比较,具有传感界面无需活化,固定抗体的免疫活性高等优点,可对相应抗原进行高灵敏的压电免疫检测.分别利用循环伏安和电化学交流阻抗技术对金标PA固定抗体及其免疫反应的动力学过程进行了表征.     

Bacteria-modified amperometric immunosensor for a Brucella melitensis antibody assay.

A novel amperometric immunosensor setup is described which uses horseradish peroxidase (HRP) as a label in conjunction with a current-based Brucella sensor. The Bacteria modified immunosensor was constructed by using a biocomposite formed by dispersing graphite powder into a mixture of Brucella melitensis and silicate polymer gel. The enzyme-labeled antibody can readily diffuse toward the encapsulated antigen (Brucella melitensis), which retains its binding properties, and the association reaction is easily detected at the surface exposed to the solution. The use of an oaminophenol (o-AP) substrate and amperometric detection at -150 mV (vs. SCE) results in a relatively low detection limit of 3.5 ng/ml and a linear detection range of 3.5 ng/ml to 200 ng/ml. Based on an optimized parameter, the prepared sensor was used to detect the Brucella melitensis antibody in serum samples by using a competitive binding assay. The results demonstrate the feasibility of employing the proposed immunosensor for the detection for Brucella melitensis antibody in a clinical analysis.     

Electron-capture gas chromatography of methadone after oxidation to benzophenone.

A procedure for the determination of low levels of methadone (6-dimethylamino-4,4-diphenylheptanone-3) in serum has been developed. Methadone is extracted from serum into n-heptane and re-extracted into an acidic aqueous phase. Methadone is oxidized to benzopheone with barium peroxide in sulphuric acid, during which procedure an n-heptane phase is present into which the oxidation product is continuously extracted. The benzophenone formed is determined by means of electron-capture gas chromatography. The recoveries are 100 +/- 3% and 100 +/- 4.5% at the 120 and 16 ng levels, respectively. The minimum amount that can be determined in 1 ml of serum is 4 ng. Interferences from possible metabolies are probably minor. The main cyclic metabolite is only co-determined to a minor extent if the oxidation time is optimized. Comparison of this oxidation method with a combined gas chromatographic-mass spectrometric determination with selected ion monitoring showed identical serum levels.     

Stereoselective determination of the enantiomers of methadone in plasma using high-performance liquid chromatography.

An enantioselective high-performance liquid chromatographic assay for the quantification of methadone in human and beagle plasma is described. The procedure involves extraction of methadone from alkalized plasma into hexane-isoamyl alcohol (99:1, v/v). Stereoselective separation was achieved with a silica column with covalently bound alpha 1-acid glycoprotein (Chiral-AGP) without any derivatization procedure. The detection wavelength was set at 215 nm. Using an internal standard provided reliable control of the extraction procedure as well as quantification of the enantiomers of methadone. The limit of quantification was found to be 2.5 ng/ml. The method was demonstrated to be sufficiently sensitive for stereoselective pharmacokinetic studies of methadone.     

Studies on Surface Plasmon Resonance Sensors in Optical Immunosensing

A novel optical biochemical sensor based on surface plasmon resonance (SPR) has been developed for immunosensing. The new device is a relatively simple and inexpensive one designed on the basis of fixing angle of incidence and scanning wavelength in the range of 400-800 nm. While the BIAcore SPR sensing method is to fix wavelength and modulate angle of incidence light, which need an expensive and complicated apparatus.     

Non-equilibrium solid-phase microextraction coupled directly to ion-trap mass spectrometry for rapid analysis of biological samples

To determine sub-ppb levels of drugs in biological samples, selective, sensitive and rapid analytical techniques are required. This work shows the possibilities for high-throughput analysis of solid-phase microextraction (SPME) directly coupled to an ion-trap mass spectrometer equipped with an atmospheric pressure chemical ionisation source. As no chromatographic separation is performed, the SPME procedure is the time-limiting step. Direct immersion SPME under non-equilibrium conditions permits the determination of lidocaine in urine within 10 min. After a 5 min sorption time with a 100 microm polydimethylsiloxane-coated fibre, the extraction yield of lidocaine from urine is about 7%. When applying 4 min desorption, using a mixture of ammonium acetate buffer (pH 4.5) and acetonitrile (85 + 15 v/v), about 10% of the analyte is retained on the fibre. An extra cleaning step of the fibre is therefore used to prevent carry-over. By use of tandem MS, no matrix interference is observed. The detection limit for lidocaine is about 0.4 ng ml(-1) and the intraday and interday reproducibility are within 14% over a concentration range of 2-45 ng ml(1).     

Inkjet-printed paperfluidic immuno-chemical sensing device

This paper reports on an inkjet printing method for the fabrication of lateral flow immunochromatographic devices made from a single piece of filter paper by patterning microfluidic channels and dispensing immunosensing inks, requiring only a single printing apparatus. This “paperfluidic” immunosensing device allows for a less time-consuming and more low-cost fabrication compared with the conventional immunochromatographic strips requiring multiple pads, plastic or nylon backing, and a plastic case. A sandwich immunoreaction was performed on the patterned immunosensing paper device, and the sensitivity of the device was optimized with an IgG model analyte. Inkjet-printed antibodies on the test line and the control line were immobilized by physical adsorption, resulting in a very simple fabrication method applicable for pure cellulose surfaces. The color intensity in the test line and the control line was determined both by naked eye and by means of a color scanner in combination with a simple computer program. With the resulting paperfluidic immunosensing device, human IgG concentrations at least down to 10 μg/l could be detected within 20 min. Additionally, in order to demonstrate the feasibility of a total multianalyte sensing system, a combined immuno-chemical sensing device was also fabricated by patterning an additional microfluidic channel for a chemical assay onto the same paper substrate. This low-cost multianalyte paperfluidic sensing device thus demonstrates the feasibility of simple, portable, and disposable tools for pathogen detection in the field of medical, environmental, and food analyses, possibly resulting in useful devices in remote settings and less-industrialized countries.     

Molecular patterning on carbon based surfaces through photobiotin activation

We have demonstrated the site-specific adhesion of photobiotin as a method of producing protein micropatterns. These patterns were created by the selective UV irradiation of a thin film of deposited photobiotin. The UV activated areas of photobiotin were then developed using fluorescently labelled avidin. The size of pattern produced is an order of magnitude smaller than those previously reported by this method. The patterns were characterised, using atomic force microscopy (AFM) to determine their microstructure. It was found that the AFM could discriminate between the areas of protein immobilised to the surface through the activated photobiotin, and the bare substrate surface where the inactivated photobiotin had been removed during the washing process. The potential of these patterns as sensing surfaces is demonstrated through the creation of a spatially patterned immunosensing surface. In this case, a biotinylated antibody was bound to the surface and the pattern developed using a second antibody specific to the immobilised biotinylated antibody. This technique could thus provide a simple and efficient method of producing high density immunoassay systems.     

Improved electrochemical immunosensor for myeloperoxidase in human serum based on nanogold/cerium dioxide-BMIMPF6/L-Cysteine composite film

An electrochemical immunosensing assay for myeloperoxidase (MPO) determination in human serum has been developed. Firstly, L-Cysteine was initially electropolymerized on an Au electrode to form L-Cysteine film. After that cerium dioxide (CeO2) dispersed in 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6) were immobilized on the L-Cysteine film. Then the negatively charged nanogold particles were adsorbed onto the membrane via the positive charge of CeO2, which aimed at assembling more antibody of MPO (anti-MPO). The resulting immunosensor showed a high sensitivity, broad linear response to the MPO concentration comprised between 10 ng/mL and 400 ng/mL with a detection limit of 0.06 ng/mL. Moreover, the surface morphology of the electrode was studied by means of a scanning electron microscope and the electrochemical properties of the fabricated immunosensor were further characterized by cyclic voltammetry. Also, factors influencing the performance of the resulting immunosensors were studied in detail.