Development of a Rapid Single‐Drop Analysis Biosensor for Screening of Phenanthrene in Water Samples

Detection techniques for biosensors often require bulky instruments or cells that are not feasible for in‐field analysis. Our single‐drop cell design, optimized in this work, comprised a screen‐printed three‐electrode (SPE), strip in horizontal position onto which a volume of 100 μL of sample or substrate solution was placed to ensure electrical contact (complete circuit). Together with optimized linear sweep voltammetry (LSV), parameters for the detection of the enzyme alkaline phosphatase (AP), the system was applied to a biosensor for the analysis of polycyclic aromatic hydrocarbons (PAHs), in environmental samples. A limit of detection (LOD), of 0.15 ppb was achieved for a model system with an IC₅₀ value of 0.885 ppb and a linear range (LR), of 0.2–10 ppb. Application of the single drop analysis (SDA), format to a PAH biosensor gave a LOD of 1.4 ppb for detection of phenanthrene with an IC₅₀ value of 29.3 ppb and linear range of 2–100 ppb. Proof of concept is shown with spiked sample analysis of phenanthrene in matrices such as sea, river and tap water.     

基于pH敏感相分离技术的新型荧光免疫测定体系

In this paper, it was discovered that a novel pH-sensitive copolymer of N-isopropylacrylamide (NIP) and N-(3-dimethylaminopropyl)methacrylamide (DMAPM) could be gotten by polymerization. The phase transition pH (pHtr) of P(NIP-DMAPM) polymer was found to be 7.4 at 37℃. The polymer was precipitated out of water above a critical pH=7.4 and re-dissolved below pH----7.4. The characteristic of this polymer made it possible to carry out the immunochemical steps of an immunoassay in a true solution and then to quickly separate the resulting product from the reaction mixture. In a competitive fluorescence immunoassay, the standard rabbit IgG and rabbit IgG immobilized on P(NIP-DMAPM) first competitively reacted with the fluorescein isothiocyanate (FITC) labeled antibody, then the pH of solution was adjusted above the pHtr of polymer to precipitate the polymer-immune complex,and the polymer-immune complex precipitate was separated and re-dissolved by the adjustment of pH, finally the FITC-labeled antibody in the immune complex was quantified by fluorescence measurement. The calibration graph for rabbit IgG was linear over the range of 100-1000 ng/mL with a detection limit of 11 ng/mL. The method is rapid, sensitive and simple. Owing to neutral pHtr of P(NIP-DMAPM), the damage to antigen-antibody immune complex was greatly decreased in the course of separation. In addition, a sandwich enzyme-linked fluorescence immunoassay method for the determination of human IgG was also developed, showing that the pH-sensitive phase separating immunoassay could be performed in the competitive method as well as the sandwich method.     

Development of a tetramethylrhodamine-labeled probe for a capillary electrophoresis-based competitive immunoassay of staphylococcal enterotoxin B

Staphylococcal enterotoxin B (SEB) was labeled with tetramethylrhodamine isothiocyanate (TRITC) and used as a probe for a competitive immunoassay. Labeling conditions such as solution pH and time were varied to observe the effect on the fluorescent product. It was found that solution pH of the labeling reaction had little effect on the fluorescence signal of the resulting products. However, labeling at pH 7.0 produced a probe that had a higher affinity for the antibody used in this study than the probes produced at pH 8.0 and 9.0. The fluorescent probes were used to perform a competitive assay for SEB in model skim milk samples. Detection limit was approximately 300 fg of SEB. Quantitation was achieved by curve fitting of fluorescent signals for bound/free probe versus log[SEB] with logarithmic functions. Accuracy in the model skim milk samples was acceptable for 3 and 5 nM SEB, but decreased considerably for a concentration of less than 1 nM SEB. The error was attributed to deviation in linearity in the standard curve at lower concentrations. Reproducibility for the analysis of both standard solutions used for the calibration curves and the model skim milk samples was excellent, with standard deviations of approximately 10% from data collected over a 3-week period. No cross-reactivity was found when the assay was tested with a 700 nM sample of staphylococcal enterotoxin A. Although competitive immunoassays are usually used for small molecules, such as therapeutic drugs, the results demonstrate that relatively large molecules (SEB, 27 kDa) can also be assayed with the technique.     

IgA肾病免疫吸附剂的研究(Ⅰ)

本文采用碳酰二咪唑（N，N'-Carbonyldiimidazole,CDI)为活化剂^[1]，活化琼脂糖载体，并以人免疫球蛋白G（IgG)，为配基制成的免疫吸附剂首次探讨了对高IgA肾病病人血清进行体外清除IgA的实验，吸附率为30－35％，具有一定的清除效果。     

The electrogenerated chemiluminescent behavior of hemin and its catalytic activity for the electrogenerated chemiluminescence of lucigenin

The electrogenerated chemiluminescent (ECL) behavior of hemin at a platinum electrode in the alkaline solution has been investigated in detail. Under the optimum conditions the linear response range of hemin is 1.0×10⁻⁵–1.0×10⁻⁸ g ml⁻¹, the detection limit was 1.0×10⁻⁸ g ml⁻¹, and the relative standard derivation for 1×10⁻⁷ g ml⁻¹ hemin was 2.8%. It has been also found that hemin would catalyze the ECL of lucigenin at a platinum electrode in a neutral solution in the presence of hydrogen peroxide, the catalytic ECL intensity was linear with the concentration of hemin in the range of 1.0×10⁻¹⁴–1.0×10⁻¹⁰ g ml⁻¹. IgG labeled with hemin was used to examine the ECL catalytic activity of hemin after conjugating to protein, and the results showed that hemin retained ECL catalytic activity when conjugated to protein.     

芯片式流通池用于顺序注射可更新表面荧光免疫法测定人血清中的IgG

采用芯片式流通池作为非均相免疫反应和原位固相荧光检测的场所,用双岔光纤将芯片式流通池与荧光光度计耦联,以双抗夹心式非均相免疫反应的模式,研究建立了测定人血清中IgG的顺序注射可更新表面非均相免疫分析新方法.     

以pH敏感相分离高分子为载体的酶联荧光免疫分析法测定人IgG

p H敏感高分子因其独特的 p H敏感性质而在药物的控制释放 [1,2 ] 、分子分离 [3 ] 以及生物传感器 [4 ]等方面得到应用 .应用于免疫分析时 ,要求 p H敏感高分子在溶液 p H 7.4左右波动时做出响应 ,过多偏离生理 p H会对免疫反应生成的抗原 -抗体复合物造成不同程度的破坏 .目前 ,p H敏感高分子并未在免疫分析中广泛应用 ,这主要是由于高分子的相转变 p H大多在 4或 1 0左右[5,6] .我们 [7] 曾合成了3 7℃下相转变 p H在 5 .6左右的 p H敏感高分子 ,并将其作为免疫反应载体 ,建立了乙肝表面抗原的分析系统 ,虽然其相转变 p H比以往的高…     

New Enzyme Immunoassay with Visual Detection Based on Membrane Photoimmobilized Antibodies

Abstract

New method for visual enzyme immunoassay of some model antigens in solution by using covalently photoimmobilized antibodies has been developed. This approach is based on the quantitative photoimmobilization of antibodies on the surface of porous matrixes. It is easy to control the dimensions and shape of the activated zones and the quantity of the active groups on it by this technique. A strip of the membrane impregnated with p-azidobenzaldehyde was illuminated by the light. A s a result, .the quantity of aldehyde groups developed on the surface of membrane is proportional to the time of illumination. After the covalent immobilization of antibodies, the membrane has separate zones with an exact surface concentration of antibodies. The antigens of different types were assayed: human IgG, human chorionic gonadotropin, Shigella Sonnei. The lowest detection limit was 1 μg/ml, 20 U/L 1×10₄ cells/ml. The method allows measure of thyroxire concentrations in the range 50 to 200 nM, the precision of replicate measurements has the coefficient of variation 7%. The reasons for the background signal appearance were accurately analyzed. The choice of support was substantiated, the optimal conditions of it.s pretreatment being defined. This method makes possible the visualization of the results based on comparison the color intensity of zones with the control.     

A Novel Fluorescence Immunoassay Based on Two-time Amplified Fluorescence Signal by Hemin and Magnetic Nanoparticles for the Detection of Antigen

Abstract

A novel, selective, and sensitive magnetic-mimetic enzyme fluorescence immunoassay method for antigen detection has been developed by taking advantage of a magnetic separation process and the amplification feature of the hemin label. This method is based on a twice amplified fluorescence signal. The signal is first amplified due to the ultrasmall size and the high surface-to-volume ratio of the silica-coated magnetite nanoparticles, which enable the nanoparticles to carry much more antibodies. Second, the mimetic enzyme (hemin) as a labeling reagent catalyzes the reaction of p-hydroxyphenyl acetic acid and H₂O₂ can further amplify the fluorescence signal. This protocol was also evaluated for a sandwich-type immunoassay of human IgG, and the calibration graph for human IgG was linear over the range of 0–100 ng mL^?1 with a detection limit of 9.8 ng mL^?1. This method can easily separate magnetic nanoparticles from the solution, which simplified the process and played a promising role for various applications in immunoassay.     

Depletion of high-abundance proteins from human plasma using a combination of an affinity and pseudo-affinity column

Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These high-abundance proteins prevent the detection of low-abundance proteins which are potential markers for various diseases. The depletion of HSA and IgG is therefore essential for further proteome analysis. In this paper we describe the optimization of conditions for selective depletion of HSA and IgG using affinity and pseudo-affinity chromatography. A BIA Separations CIM (convective interaction media) Protein G disk was applied for the removal of IgG and the Mimetic Blue SA A6XL stationary phase for the removal of HSA. The binding and the elution buffer for CIM Protein G disk were chosen on the basis of the peak shape. The dynamic binding capacity was determined. It was shown to be dependent on the buffer system used and independent of the flow rate and of the concentration of IgG. Beside the binding capacity for the IgG standard, the binding capacity was also determined for IgG in human plasma. The Mimetic Blue SA A6XL column was characterized using human plasma. The selectivity of the depletion was dependent on the amount of human plasma that was loaded on the column. After the conditions on both supports had been optimized, the Mimetic Blue SA A6XL stationary phase was combined with the CIM Protein G disk in order to simultaneously deplete samples of human plasma. A centrifuge spin column that enables the removal of IgG and HSA from 20 μL of human plasma was designed. The results of the depletion were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis.