利用微型生物光学传感器的竞争免疫检测法检测牛奶中氨苄青霉素

利用微型表面等离子体共振的生物传感器测量了残留在牛奶中的氨苄青霉素的浓度.采用竞争抑制试验的方法,即先将定量的单克隆抗氨苄青霉素抗体（3H295）和含氨苄青霉素的牛奶样品混合,样品中氨苄青霉素即与抗体结合,然后将该混合样品通入传感器表面,传感器的表面共价固定了氨苄青霉素分子,通过生物特异相互作用分析,检测样品中剩余的抗体,从而得到氨苄青霉素的浓度.样品的测量时间为10min,最低检测限为2.5 ng/mL,低于欧盟标准4 ng/mL.该检测方法的测量时间短、重复性好,批间测量的变异系数为5.4%,表明该方法能满足实际测量要求.     

Immunoassay techniques for detection of the herbicide simazine based on use of oppositely charged water-soluble polyelectrolytes.

Linear water-soluble polyelectrolytes, i.e., poly(methacrylate) polyanion and poly(N-ethyl-4-vinylpyridinium) polycation, were used as carriers for the reactants in immunoassay. The strength of ionic forces through distance and the cooperative binding of oppositely charged chains, the carriers interact with each other at an extremely high rate and affinity. These properties of the polyelectrolytes made it possible to carry out the immunochemical steps of the assay in true solution and then to quickly separate the resulting products from the reaction mixtures. The above approach was applied to an assay for the herbicide simazine. Both enzyme-linked immunosorbent assay (ELISA) and dot blot formats of the immunoassay were evaluated. In the ELISA format, the polycation was adsorbed on the surface of a microtiter plate. A tracer antigen (simazine) was allowed to interact in solution with components of the reaction mixture containing simazine-peroxidase conjugate, specific antibodies, and staphylococcal protein A conjugated with the polyanion, and then the mixture was added to the immobilized polycation. Quick separation of the immunoreactants was achieved due to formation of interpolyelectrolyte complexes between polycation and polyanion molecules. After washing, the microplate wells were filled with a solution of substrate, and the optical density of the reaction products was measured. In the second format, a solution of the same reaction mixture (after incubation) was filtered through a porous membrane, with the polycation adsorbed. The subsequent addition of substrate led to the development of colored spots. Sensitivity of the dot blot format was close to that of the traditional ELISA format using the same reactants, i.e., 0.5 ng/mL. However, the assay was much faster (assay time decreased from 100-120 to 45 min). Sensitivities of the dot immunoassay were 1 ng/mL for densitometric detection and 10 ng/mL for visual detection with a duration of 20 min. The techniques developed here were used for simazine determination in water, milk, and juices.     

Label-free detection of leptin antibody-antigen interaction by using LSPR-based optical biosensor

In the recent research, the development of optical biosensing devices has been focused on finding new method and technologies to exploit the optical properties of noble metal nanostructure, especially localized surface plasmon resonance (LSPR). In this study, we fabricated a LSPR-based label-free optical biosensor with the multi-spot gold-capped nanoparticle array (MG-NPA) biochip based on the deposition of a thin gold (Au) film on the silica nanoparticles layer with the simple process. The MG-NPA biochip used the silica nanoparticles as the core and a thin Au film as a shell on the surface. This structure can excite the LSPR signal easily with the high reproducibility. The anti-leptin antibody was immobilized on the surface of MG-NPA biochip, which could recognize only leptin antigen. The leptin antibody-antigen interaction was performed by the introduction of different concentration (1 pg/mL-100 microg/mL) of leptin antigen solutions for 1 h. The detection limit was found to be 100 pg/mL by using the anti-leptin antibody immobilized MG-NPA biochip. This LSPR-based label-free optical biosensor employing the MG-NPA biochip brings several advantages such as low cost, easy to fabricate, using a simple optical system and can be applied in a wide immunoassay with the similar antibody-antigen model.     

配方奶粉中核苷酸含量高效液相色谱测定

研究建立了定量测定配方奶粉中5种核苷一磷酸(钠盐)的高效液相色谱方法。通过添加适量乙酸、离心浓缩、调节pH等技术方法,快捷有效地去除奶粉中影响核苷酸含量定量测量的蛋白质、脂肪及其他杂质成分,使用核苷酸国家级标准物质,采用外标法对样品中的核苷酸含量进行了定量测量。结果表明:该方法检出限低,准确度在90.8%～99.3%之间,方法的重复性限为1.43,再现性限为2.44。该方法准确度高,重复性好,可行性强,易于掌握,并且快捷、成本低,为奶粉中核苷酸含量的定量测量及相关产品的质量技术监督检验提供了方法学依据。     

Development of magnetosomes‐based biosensor for the detection of Listeria monocytogenes from food sample

Listeriosis through contaminated food is one of the leading causes of premature deaths in pregnant women and new born babies. Here, the authors have developed a magnetosomes‐based biosensor for the rapid, sensitive, specific and cost‐effective detection of Listeria monocytogenes from food sample. Magnetosomes were extracted from Magnetospirillum sp. RJS1 and then directly bound to anti‐Listeriolysin antibody (0.25–1 µg/ml), confirmed in spectroscopy. Listeriolysin (LLO) protein (0.01–7 µg/ml) was optimised in enzyme‐linked immunosorbent assay. Magnetosomes was conjugated with LLO antibody (0.25 µg/ml) in optimum concentration to detect LLO protein (0.01 µg/ml). Magnetosomes–LLO antibody complex was 25% cost effective. The magnetosomes–LLO antibody complex was directly stabilised on screen printed electrode using external magnet. The significant increase in resistance (R _CT value) on the electrode surface with increase in concentration of LLO protein was confirmed in impedance spectroscopy. The L. monocytogenes contaminated milk and water sample were processed and extracted LLO protein was detected in the biosensor. The specificity of the biosensor was confirmed in cross‐reactivity assay with other food pathogens. The detection limit of 10¹ Cfu/ml in both water and milk sample manifests the sensitive nature of the biosensor. The capture efficiency and field emission scanning electron microscopy confirmed positive interaction of Listeria cells with magnetosomes–antibody complex.Inspec keywords: proteins, food safety, enzymes, contamination, biosensors, dairy products, microorganisms, cost reduction, separation, electrodes, magnets, electric resistance, field emission scanning electron microscopyOther keywords: anti‐Listeriolysin antibody, LLO protein, monocytogenes contaminated milk, food pathogens, magnetosomes‐based biosensor, Listeria monocytogenes, contaminated food, sensitive cost‐effective detection, listeriosis, extraction process, Magnetospirillum sp. RJS1, enzyme‐linked immunosorbent assay, LLO antibody complex, screen printed electrode, magnet, electric resistance, RCT value, electrode surface, impedance spectroscopy, field emission scanning electron microscopy     

In situ amplified electrochemical immunoassay for carcinoembryonic antigen using horseradish peroxidase-encapsulated nanogold hollow microspheres as labels

Methods based on sandwich-type electrochemical enzyme immunoassay protocol have been extensively developed for the detection of biomolecules, but most often exhibit low detection signals and low detection sensitivity, and are unsuitable for routine use. In this study, we initially synthesized specially horseradish peroxidase-encapsulated nanogold hollow microspheres (HRP-GHS), and then the prepared HRP-GHS was conjugated to the secondary carcinoembryonic antibody (HRP-GHS- anti-CEA). Carcinoembryonic antigen (CEA), as a model protein, was monitored by using the electrochemical sandwich-type enzyme immunoassay format. Under optimized conditions, the linear range of the immunoassay by using single HRP-labeled anti-CEA (HRP- anti-CEA) as secondary antibodies is 2.5-120 ng/mL with a detection limit of 1.5 ng/mL CEA, while the assay sensitivity by using HRP-GHS- anti-CEA as secondary antibodies is further increased from 0.01 to 200 ng/mL with a lower detection limit of 1.5 pg/mL CEA. The intra- and interassay reproducibility is acceptable. The CEA concentrations of the clinical serum specimens assayed by the developed immunoassay show consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay. This immunoassay system has many desirable merits including sensitivity, accuracy, and little required instrumentation. Significantly, the new protocol may be quite promising, with potentially broad applications for clinical immunoassays.     

Array biosensor for simultaneous identification of bacterial, viral, and protein analytes.

The array biosensor was fabricated to analyze multiple samples simultaneously for multiple analytes. The sensor utilized a standard sandwich immunoassay format: Antigen-specific "capture" antibodies were immobilized in a patterned array on the surface of a planar waveguide and bound analyte was subsequently detected using fluorescent tracer antibodies. This study describes the analysis of 126 blind samples for the presence of three distinct classes of analytes. To address potential complications arising from using a mixture of tracer antibodies in the multianalyte assay, three single-analyte assays were run in parallel with a multianalyte assay. Mixtures of analytes were also assayed to demonstrate the sensor's ability to detect more than a single species at a time. The array sensor was capable of detecting viral, bacterial, and protein analytes using a facile 14-min assay with sensitivity levels approaching those of standard ELISA methods. Limits of detection for Bacillus globigii, MS2 bacteriophage, and staphylococcal enterotoxin B (SEB) were 10(5) cfu/mL, 10(7) pfu/mL, and 10 ng/mL, respectively. The array biosensor also analyzed multiple samples simultaneously and detected mixtures of the different types of analytes in the multianalyte format.     

Detection of antistreptolysin O antibody: application of an initial rate method of latex piezoelectric immunoassay.

Latex plezoelectric immunoassay (LPEIA) is a new latex immunoassay using a plezoelectric quartz crystal (Kurosawa et al. Chem. Pharm. Bull. 1990, 38, 1117). This assay requires no immobilization of antigen or antibody on an electrode surface of a plezoelectric crystal, while the immobilization is indispensable for ordinary immunoassays using a plezoelectric crystal as a microbalance. The present paper improves a previous method (end-point analysis) by introducing the initial rate method using a batch cell; reduction of assay volume (1.2 mL) and shortening of assay time (2-3 min) were achieved. This assay was applied to the detection of antistreptolysin O antibody (ASO) in serum. The frequency change was proportional to the ASO concentration up to 1040 IU mL-1, and the method has good sensitivity for actual clinical application. The volume of serum required for the assay was 0.02 mL. Twenty-four clinical specimens were analyzed with this LPEIA, and the values obtained were compared with those obtained with a turbidimetric latex agglutination method. The correlation coefficient between these values was 0.950 (P < 0.01).     

Ultrasensitive electrochemical immunosensor for clinical immunoassay using thionine-doped magnetic gold nanospheres as labels and horseradish peroxidase as enhancer

A new signal amplification strategy based on thionine (TH)-doped magnetic gold nanospheres as labels and horseradish peroxidase (HRP) as enhancer holds promise to improve the sensitivity and detection limit of the immunoassay for carcinoembryonic antigen (CEA), as a model protein. This immunoassay system was fabricated on a carbon fiber microelectrode (CFME) covered with a well-ordered anti-CEA/protein A/nanogold architecture. The reverse micelle method was initially used for the preparation of TH-doped magnetic gold nanospheres (nanospheres), and the synthesized nanospheres were then labeled on HRP-bound anti-CEA as a secondary antibody (bionanospheres). Sandwich-type protocol was successfully introduced to develop a new high-efficiency electrochemical immunoassay with the labeled bionanospheres toward the reduction of H2O2. Under optimized conditions, the linear range of the proposed immunoassay without HRP as enhancer was 1.2-125 ng/mL CEA, whereas the assay sensitivity by using HRP as enhancer could be further increased to 0.01 ng/mL with the linear range from 0.01 to 160 ng/mL CEA. The developed immunoassay method showed good precision, high sensitivity, acceptable stability and reproducibility, and could be used for the detection of real samples with consistent results in comparison with those obtained by the enzyme-linked immunosorbent assay (ELISA) method.     

Carbon nanotubes with enhanced chemiluminescence immunoassay for CCD-based detection of Staphylococcal enterotoxin B in food

Enhanced chemiluminescence (ECL) detection can significantly enhance the sensitivity of immunoassays but often requires expensive and complex detectors. The need for these detectors limits broader use of ECL in immunoassay applications. To make ECL more practical for immunoassays, we utilize a simple cooled charge-coupled device (CCD) detector combined with carbon nanotubes (CNTs) for primary antibody immobilization to develop a simple and portable point-of-care immunosensor. This combination of ECL, CNT, and CCD detector technologies is used to improve the detection of Staphylococcal enterotoxin B (SEB) in food. Anti-SEB primary antibodies were immobilized onto the CNT surface, and the antibody-nanotube mixture was immobilized onto a polycarbonate surface. SEB was then detected by an ELISA assay on the CNT-polycarbonate surface with an ECL assay. SEB in buffer, soy milk, apple juice, and meat baby food was assayed with a LOD of 0.01 ng/mL using our CCD detector, a level similar to the detection limit obtained with a fluorometric detector when using the CNTs. This level is far more sensitive than the conventional ELISA, which has a LOD of approximately 1 ng/mL. Our simple, versatile, and inexpensive point-of-care immunosensor combined with the CNT-ECL immunoassay method described in this work can also be used to simplify and increase sensitivity for many other types of diagnostics and detection assays.     

Colorimetric determination of melamine by pyridine-3-boronic acid modified gold nanoparticles

A rapid, simple and sensitive colorimetric detection method for melamine was proposed based on pyridine-3-boronic acid (PBA) modified gold nanoparticles (AuNPs). The formation of supramolecular hydrogen-bonded structure between PBA and melamine resulted in the aggregation of PBA modified AuNPs and the color change from red to blue. Melamine could be detected by colorimetric response of AuNPs that could be detected by naked eyes or a UV-vis spectrophotometer. The detection concentration of melamine ranged from 6.0 x 10(-8) to 1.6 x 10(-6) M, and the detection limit (3sigma) was 3.0 x 10(-8) M (i.e., 3.2 ppb). This provided an effective and facile colorimetric sensor for real-time and on-site determination of melamine. Particularly, the proposed method could be used to detect melamine in pretreated liquid milk products with high sensitivity and low interference, and the recoveries were from 95% to 102%.     

高效液相色谱-紫外检测禽蛋及其制品中三聚氰胺

建立了用高效液相色谱-紫外检测法检测禽蛋及其制品中三聚氰胺的方法。采用乙腈作为样品中蛋白质的沉淀剂和三聚氰胺提取剂,流动相为0.05mol/L(pH=3.00)的磷酸二氢钾乙腈缓冲溶液,检测波长为240nm,流速为1.50mL/min,SCX(Luna 5u SCX250×4.6mm)色谱柱进行分离,HPLC-UV检测,外标法定量。结果表明,该方法简便快捷,线性范围0.20～10.00mg/L,回收率在99.8%-109.4%,线性关系良好R2=1.000,可广泛应用于禽蛋及其制品中三聚氰胺的检测。     

Fabrication of a disposable biosensor for Escherichia Coli O157:H7 detection

As the safety in the food supply becomes critical, the demand for a rapid, low-volume, and sensitive microbial detection device has dramatically increased. A biosensor based on an electrochemical sandwich immunoassay using polyaniline has been developed for detecting foodborne pathogens, such as Escherichia coli (E. coli) O157:H7. The biosensor is comprised of two types of proteins: capture protein and reporter protein. The capture protein is immobilized on a pad between two electrodes, while the reporter protein is attached to conductive polymers. After adding the sample, the target protein binds to the reporter protein and forms a sandwich complex with the capture protein. The conductive polymer that is attached to the reporter protein serves as a messenger, reporting the amount of target protein captured in the form of an electrical signal. The architecture of the biosensor utilizes a lateral flow format, which allows the liquid sample to move from one pad to another by capillary action. Experiments to evaluate the best construction materials, the optimal polyaniline and antibody concentrations, and the distance between electrodes are highlighted in this paper. Results show that the biosensor could detect approximately 7.8/spl times/10/sup 1/ colony forming unit per milliliter of E. coli O157:H7 in 10 min.     

Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay

Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). Although conventional antibodies dominate current assay development, recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. We expressed VHHs from an immunized alpaca and developed a VHH-based immunoassay using 3-phenoxybenzoic acid (3-PBA), a major metabolite of pyrethroid insecticides as a model system. A phage VHH library was constructed, and seven VHH clones were selected by competitive binding with 3-PBA. The best immunoassay developed with one of these VHHs showed an IC(50) of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameters were further improved by using the phage borne VHH, IC(50) = 0.1 ng/mL and LOD = 0.01 ng/mL. Both assays showed a similar tolerance to methanol and dimethylsulfoxide up to 50% in assay buffer. The assay was highly specific to 3-PBA and its 4-hydroxylated derivative, 4-hydroxy 3-PBA, (150% cross reactivity) with negligible cross reactivity with other tested structural analogues, and the recovery from spiked urine sample ranged from 80 to 112%. In conclusion, a highly specific and sensitive VHH for 3-PBA was developed using sequences from immunized alpaca and phage display technology for antibody selection.     

Integrating waveguide biosensor

A capillary biosensor is demonstrated which uses the waveguiding properties of the capillary to integrate the signal over an increased surface area without simultaneously increasing the background noise from the detector. This biosensor achieves limits of detection of 30-50 pg/mL in immunoassays using a diode laser for excitation and a PMT for detection. This is approximately 2 orders of magnitude greater sensitivity than was achieved using the same immunoassay reagents in a fiber optic biosensor or a planar array biosensor. Two different approaches to using the capillaries as immunosensors are described, either of which could be adapted for multianalyte sensing.     

Assessment of specific binding proteins suitable for the detection of paralytic shellfish poisons using optical biosensor technology

Paralytic shellfish poisoning (PSP) toxin monitoring in shellfish is currently performed using the internationally accredited AOAC mouse bioassay. Due to ethical and performance-related issues associated with this bioassay, the European Commission has recently published directives extending procedures that may be used for official PSP control. The feasibility of using a surface plasmon resonance optical biosensor to detect PSP toxins in shellfish tissue below regulatory levels was examined. Three different PSP toxin protein binders were investigated: a sodium channel receptor (SCR) preparation derived from rat brains, a monoclonal antibody (GT13-A) raised to gonyautoxin 2/3, and a rabbit polyclonal antibody (R895) raised to saxitoxin (STX). Inhibition assay formats were used throughout. Immobilization of STX to the biosensor chip surface was achieved via amino-coupling. Specific binding and inhibition of binding to this surface was achieved using all proteins tested. For STX calibration curves, 0-1000 ng/mL, IC50 values for each binder were as follows: SCR 8.11 ng/mL; GT13-A 5.77 ng/mL; and R895 1.56 ng/mL. Each binder demonstrated a different cross-reactivity profile against a range of STX analogues. R895 delivered a profile that was most likely to detect the widest range of PSP toxins at or below the internationally adopted regulatory limits.     

Optical resonance-enhanced absorption-based near-field immunochip biosensor for allergen detection

An optical immunochip biosensor has been developed as a rapid method for allergen detection in complex food matrixes, and its application evaluated for the detection of the egg white allergens, ovalbumin and ovomucoid. The optical near-field phenomenon underlying the basic principle of the sensor design is called resonance-enhanced absorption (REA), which utilizes gold nanoparticles (Au NPs) as signal transducers in a highly sensitive interferometric setup. Using this approach, a novel, simple, and rapid colorimetric solid-phase immunoassay on a planar chip substrate was realized in direct and sandwich assay formats, with a detection system that does not require any instrumentation for readout. Semiquantitative immunochemical responses are directly visible to the naked eye of the analyst. The biosensor shows concentration-dependent color development by capturing antibody-functionalized Au NPs on allergen-coated chips and has a detection limit of 1 ng/mL. To establish a rapid method, we took advantage of the physicochemical microenvironment of the Au NP-antibody bioconjugate to be bound directly over an interacting poly(styrene-methyl methacrylate) interlayer by an immobilized antigen. In the direct assay format, a coating time with allergen of only 5 min under "soft" nondenaturing conditions was sufficient for accurate reproducibility and sensitivity. In conclusion, the REA-based immunochip sensor is easy to fabricate, is reproducible and selective in its performance, has minimal technical requirements, and will enable high-throughput screening of affinity binding interactions in technological and medical applications.     

Demonstration of four immunoassay formats using the array biosensor

The ability of a fluorescence-based array biosensor to measure and quantify the binding of an antigen to an immobilized antibody has been demonstrated using the four different immunoassay formats: direct, competitive, displacement, and sandwich. A patterned array of antibodies specific for 2,4,6-trinitrotoluene (TNT) immobilized onto the surface of a planar waveguide and used to measure signals from different antigen concentrations simultaneously. For direct, competitive, and displacement assays, which are one-step assays, measurements were obtained in real time. Dose-response curves were calculated for all four assay formats, demonstrating the array biosensor's ability to quantify the amount of antigen present in solution.     

Microfabricated on-chip-type electrochemical flow immunoassay system for the detection of histamine released in whole blood samples

This paper describes an on-chip-type electrochemical flow immunoassay system with a multichanneled matrix column. The multichanneled matrix column was functionally coated with cation-exchange resin and used for separation of proteins. Antihistamine immunoglobulin G (IgG) antibody conjugated with ferrocenemonocarboxylic acid (Fc) was also prepared and used as a novel analytical reagent. Antibody-antigen complexes were separated from free Fc-conjugated IgG antibody (Fc-IgG) on the basis of differences in isoelectric point (pI) using the multichanneled matrix column coated with cation-exchange resin. The assay yields a good relationship between current and histamine concentration in the range of 200-2000 ng/mL. This simple technique enables the assay of histamine released in whole blood within 2 min. Furthermore, a good correlation was found between the response of the electrochemical immunoassay described in this paper and the conventional RIA (radioimmunoassay). This on-chip-type electrochemical flow immunoassay requires only minute quantities of whole blood samples and generates highly reproducible results.     

Multifunctional gold-silica nanostructures for ultrasensitive electrochemical immunoassay of streptomycin residues

A facile and simple electrochemical immunoassay for ultrasensitive determination of streptomycin residues (STR) in food was designed by using nanogold-assembled mesoporous silica (GMSNs) as bionanolabels on a three-dimensional redox-active organosilica-functionalized sensing interface. To construct such a sensing interface, we initially synthesized organosilica colloids by using wet chemical method, and then utilized the prepared colloidal organosilica nanocomposites for the immobilization of monoclonal anti-STR antibodies on a glassy carbon electrode based on a sol-gel method. The bionanolabels were prepared based on coimmobilization of horseradish peroxidase (HRP) and STR-bovine serum albumin conjugates (STR-BSA) on the GMSNs. With a competitive-type immunoassay format, the assay toward STR analyte was carried out in pH 5.5 acetate acid buffer (ABS) by using redox-active organosilica nanocomposites as electron mediators, biofunctionalized GMSNs as traces, and hydrogen peroxide (H(2)O(2)) as enzyme substrate. Under optimal conditions, the reduction current of the electrochemical immunosensor decreased with the increase in STR level in the sample, and displayed a wide dynamic range of 0.05-50 ng mL(-1) with a low detection limit (LOD) of 5 pg mL(-1) at 3s(B). Intra- and interassay coefficients of variation were less than 8.7 and 9.3% for STR detection, respectively. In addition, the methodology was validated with STR spiked samples including honey, milk, kidney, and muscle, receiving a good correspondence with the results obtained from high-performance liquid chromatography (HPLC).