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A paper-based microfluidic immunodevice has recently attracted considerable interest for point-of-care testing (POCT) and a washing procedure was used as a standard procedure in immunoassay to eliminate the nonspecific binding protein from a paper surface. However, the traditional washing method cannot get rid of the nonspecific binding protein more completely to get a lower background. In this work, a novel washing strategy with a ring-oven technique integrated on a paper-based immunodevice was presented, which can effectively wash a nonspecific binding protein and enable a low background for sensitive detection of the carcinoembryonic antigen (CEA). By immobilizing the antibody on the detection area and incorporating the temperature-controlled ring-oven under the paper-based device, the continuous washing solution can carry the nonspecific binding protein to the waste area freely by capillary force and then the waste area dried quickly by heating. The paper device, which is matched to the size of the ring-oven, is composed of eight microfluidic channels by the simple and rapid paper-cutting fabrication method. With the HRP-catalyzed 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 colorimetric detection method, a lower detection limit of 0.03 ng/mL CEA can be obtained by enzyme-linked immunosorbent assay (ELISA). The washing efficiency for the nonspecific binding protein was improved a lot compared to the traditional washing methods, and the established paper-based device can be used in the determination of CEA in human serum with high sensitivity. The paper-based device provides a new washing strategy for sensitive immunoassay and point-of-care diagnostics.

Enzyme-linked immunosorbent assay (ELISA), horseradish peroxidase (HRP)-catalyzed fluorescent reaction, and oxalate chemiluminescence analysis have been combined to develop a highly sensitive, simple, and rapid method for analysis of bovine serum albumin (BSA) based on a pair of specific monoclonal antibodies in vaccines. A typical "sandwich type" immunoassay was used. Reaction of 3-(4-hydroxyphenyl propionate) (PHPPA) with hydrogen peroxide-urea, catalyzed by HRP, produced fluorescence of 3-(4-hydroxyphenyl propionate) dimer, which was detected by chemiluminescence analysis with the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H(2)O(2)-glyoxaline-PHPPA dimer chemiluminescent system. This method exhibited high performance with a linear correlation between response and amount of bovine serum albumin (BSA) in the range 0.1 to 100.0 ng mL(-1) (r = 0.9988), and the detection limit was 0.03 ng mL(-1) (S/N = 3). Intra- and interassay coefficient variations were all lower than 9.0% at three concentrations (1.0, 20.0, and 80.0 ng mL(-1)). The proposed method has been used for successful analysis of the amount of residual BSA in vaccines. The results obtained compared well with those obtained by conventional colorimetric ELISA and luminol chemiluminescent ELISA.

Electrochemical immunosensors have attracted great interest in the search for a selective, simple and reliable system for molecular recognition. Presently, electrochemical immunosensors have been widely studied for biomedical molecular's detection, but the regeneration of these immunosensors has restricted their wide application. To prepare a regeneration-free immunosensor, which may be more suitable for clinical determination, a repeatable immunoassay system was developed based on an electrochemical immunosensor with magnetic nanoparticles, biotin-avidin system (BAS) and Fab antibodies for the heart failure markers aminoterminal pro-brain natriuretic peptides (NT-proBNP). At the same time, a microfluidic system was combined into the proposed system, which enabled continuous determination. Using NT-proBNP as a model system, the proposed immunosensor exhibited rapid and sensitive amperometric response to NT-proBNP with good selectivity, stability, and a wide linear range (0.005-1.67 ng/mL and 1.67-4 ng/mL with a detection limit of 0.003 ng/mL under optimal conditions). Importantly, the proposed immunosensor was also suitable for the detection of other proteins and provided new opportunities for disease diagnosis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Staphylococcal enterotoxin B (SEB) is a potent gastrointestinal toxin and is heat resistant. SEB is also a potential bioterrorism agent. The ability to measure accurately very low amounts of staphylococcal enterotoxin B in food and other samples is very important. A highly sensitive and stable sandwich fluorescence immunoassay based on a pair of monoclonal antibodies against SEB which were produced by us was developed. Classical sandwich immunoassay was adopted and the glass slides were used as the base of the immunologic reaction. The functionalized fluorescent core-shell silica nanoparticles were used as labels. The fluorescence issued from the labels was detected by a laser-induced fluorescence millimeter sensor array detection platform. The fluorescence intensity has a linear relationship with the amount of SEB in the range of 50 pg/mL-5 ng/mL, and the detection limit of SEB was 20 pg/mL (the absolute detection limit was 0.02 pg). The relative standard deviation (RSD) for 5 parallel measurements of SEB (1 ng/mL) was 9.2%., (Copyright © 2011 Elsevier B.V. All rights reserved.)

N-Terminal pro-brain natriuretic peptide (NT-proBNP) is an important marker for heart failure that reflects ventricular volume expansion, ventricular overload and the degree of cardiac injury. To establish a sensitive and simple detection method for it in serum, a regeneration-free immunosensor with novel Fab fragment monoclonal antibodies was prepared. The sensor detected NT-proBNP from 0.04 to 2.5 ng/ml with a limit of 0.03 ng/ml. The immunosensor is therefore a simple, cost-effective method to detect NT-proBNP and the proposed immunoassay system would enable other proteins to be detected and open new opportunities for protein diagnostics.

Macrophage migration inhibitory factor (MIF) has a multitude of biological activity and is associated with a number of inflammatory and immune diseases, including rheumatoid arthritis (RA). The increased serum levels of MIF in patients not only suggest this protein as a marker for disease progression, but also as a potential therapeutic target. The aim of this study is to develop a novel electrochemical method to more precisely and conveniently measure MIF in patient sera. An IgM murine monoclonal antibody (mAb) against human MIF was prepared and used in the electrochemical immunosensor, with modified gold electrode coated with compounds of gold nanoparticles, titanium dioxide nanoparticles and thionine (NGP-NTiP-Thi) followed by adsorption of anti-MIF antibodies with IgM or IgG1 isotype. The IgM immunosensor recognized MIF in a linear relationship in the range of 0.03 and 230 ng/mL with the lower limit (S/N=3) of 0.02 ng/mL. The measurement showed considerable levels of sensitivity, selectivity, stability and long-term maintenance of bioactivity, as shown by testing with serum MIF in RA patients as compared to healthy donors. The performance of the IgM immunosensor was also superior to IgG1 sensor. Thus, we have developed a novel measurement approach for serum MIF, which may have great potential in the clinic for monitoring the course of diseases associated with increased MIF.

A non-competitive immunoassay based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of luteinizing hormone (LH) in human serum. The work involved the development of separation and CL conditions, allowing for routine analysis of serum samples. In this study, horseradish peroxidase (HRP)-labelled monoclonal anti-LH can catalyse the luminol-hydrogen peroxide reaction. The determined LH can react with excessive amount of HRP-labelled anti-LH. Within 14 min, free enzyme conjugate and immune complex could be separated in alkaline borate buffer by means of a high voltage (15 kV). To improve sensitivity, a series of measures were adopted, including the choice of para-iodophenol as a CL enhancer, unique design in detect window. Under the optimal conditions, the calibration curve for LH was established in the concentration range 1-200 mIU/mL and the detection limit was 0.08 mIU/mL. Compared with ELISA, this method decreased the detection limit by about 12 times, and it has been successfully employed in the determination of LH in human serum., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Enzyme-linked immunosorbent assay (ELISA), horseradish peroxidase (HRP)-catalyzed fluorescent reaction, and oxalate chemiluminescence imaging analysis have been combined to develop a sensitive, simple, and rapid method for analysis of interferon alpha (alpha-IFN) in human serum samples. A typical "sandwich type" immunoassay was used. Reaction of o-phenylenediamine (OPD) with hydrogen peroxide (H(2)O(2)), catalyzed by HRP, produced 2,3-diaminophenazine (PDA), which was detected by chemiluminescence imaging analysis with the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H(2)O(2)-glyoxaline-PDA chemiluminescent system. The TCPO chemiluminescent imaging system is more sensitive and the chemiluminescence quantum yield is at least five times higher than for the luminol-H(2)O(2)-HRP-PIP (p-iodophenol) chemiluminescent imaging system. The results showed there was a very good linear correlation between response and amount of alpha-IFN in the range 1.3-156.0 pg mL(-1) (R = 0.9991) and the detection limit was 0.8 pg mL(-1) (S/N=3). The relative standard deviation (n = 9) was 4.7%. The proposed method has been used for successful analysis of the amount of alpha-IFN in human serum. The results obtained compared well with those obtained by conventional colorimetric ELISA and luminol chemiluminescent ELISA.

Abstract: A novel chemiluminescence immunoassay was developed, in which two monoclonal antibodies recognised different epitopes of staphylococcal enterotoxin B (SEB) using HRP-functionalised mesoporous silica nanoparticle (MSN) as label. The enzyme-functionalised MSN was fabricated by co-immobilizing HRP and anti-SEB onto the surface of MSN simultaneously using glutaraldehyde as the linkage. Because the large surface area of MSN carriers increased the amount of HRP and the proportion between the enzyme and antibody molecules on the surface of MSN were optimised, the conjugates provided a much higher signal. This is an improvement over the traditional sandwich immunoassay which often has one or two enzyme molecules per antibody. The analysis showed a linear response within the range of 0.01–1ngmL−1 (r =0.9967), and the detection limit (DL) was 4pgmL−1 (3σ). This approach was successfully demonstrated as a simple, cost-effective, sensitive method to detect SEB in milk, water and serum samples. [Copyright &y& Elsevier]

Abstract: A highly sensitive fluoroimmunoassay for the determination of staphylococcal enterotoxin C1 (SEC1) is proposed. It is based on the functionalized fluorescent core-shell nanoparticles as the labels coated with anti-SEC1 monoclonal antibodies in “sandwich” fluoroimmunoassay. With the simple, inverse microemulsion polymerisation method, the functionalized fluorescent core-shell nanoparticles were prepared easily. The preparation process produces a silica shell on the surface of the Ru(bpy)3Cl2 (Rubpy) dye with one step cohydrolysis of tetraethylorthosilicate (TEOS), and the coupling agent (3-aminopropy1)triethoxysilane (APS) provided the amine groups that can be used for biological conjugation. The nanoparticles were then labeled with the anti-SEC1 monoclonal antibodies and the antibody-labeled nanoparticles were successfully used for the determination of SEC1. The calibration graph for SEC1 was linear over the range 1.0–75.0ngml−1 with a detection limit of 0.3ngml−1. The regression equation of the working curve was I F =24.583+0.6426[SEC1] (ngml−1) (r =0.9991). The relative standard deviation (RSD) for five parallel measurements of 25.0ngml−1 SEC1 was 2.5%. Furthermore, the application of fluorescence microscopy imaging in the study of the antibody labeling and sandwich fluoroimmunoassay with the functionalized fluorescent core-shell silica nanoparticles was also explored. The results demonstrate that the method offers potential advantages of easily labeling to the antibody, sensitivity, simplicity and reproducibility for the determination of SEC1 and is applicable to the determination of SEC1 in real samples and enables fluorescence microscopy imaging for the determination of SEC1. [Copyright &y& Elsevier]

Abstract: A sensitive and homogeneous immunoassay (IA) based on capillary electrophoresis (CE) with enhanced chemiluminescence (CL) detection has been developed for the determination of hepatitis B surface antigen (HBsAg) and antibody (HBsAb) in human serum. The conditions for the CL reaction and electrophoresis were investigated in detail using horseradish peroxidase (HRP) labeled HBsAg (HBsAg*) as a marker because of its catalytic effects on the luminol–hydrogen peroxide reaction. The CL reaction was enhanced by para-iodophenol and the CL detector was designed uniquely without any dead volume or diluents effect. The present method has been used for assaying HBsAg and HBsAb in human serum using a competitive format and a non-competitive format, respectively. Under the optimal conditions, the linear ranges were from 1 to 400pmol/L (R =0.9988) for HBsAg and 2 to 200mIU/mL (R =0.9981) for HBsAb. The detection limits were 0.4pmol/L and 1mIU/mL for HBsAg and HBsAb, respectively. The relative standard deviations of peak area were 4.2% and the errors of it were from −0.03% to +0.05% for 80pmol/L HBsAg* (n =7). In this study, the free HBsAg* and the bound HBsAg* (HBsAg*–HBsAb) were separated in the separation capillary within 6min using a borate run buffer. To verify the experimental reliability, the result was comparable with that of enzyme linked immunosorbent assay (ELISA) and demonstrated the feasibility of the CE–CL immunoassay method for clinical diagnosis. [Copyright &y& Elsevier]

Abstract: A novel fluoroimmunoassay (FIA) method was developed for the determination of tumor necrosis factor-α (TNF-α) in this study. The proposed method has the advantage of showing the specificity of immunoassays and sensitivity of fluorescent nanoparticles label technology. With the well-established inverse microemulsion polymerisation process, the tris(2′,2-bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy)-doped fluorescent silica nanoparticles (RuDFSNs) were prepared. Then a RuDFSNs-labeled anti-TNF-α monoclonal antibody was prepared and used for FIA of TNF-α in human serum samples with a sandwich FIA by using the low fluorescent 96-well transparent microtiter plates. The assay response was linear from 1.0 to about 250.0pg/mL with a detection limit of 0.1pg/mL for TNF-α. The intra- and inter-assay precision are 4.9%, 4.4%, 4.6%; 6.1%, 5.9%, 5.3% for five parallel measurements of 2.0, 20.0, 200.0pg/mL TNF-α respectively, and the recoveries are in the range of 96–104% for human serum sample measurements by standard-addition method. We also explored the application of fluorescence microscopy imaging in the study of the FIA for TNF-α with the fluorescent nanoparticle labels. The results demonstrate that the method offers potential advantages of sensitivity, simplicity and good reproducibility for the determination of TNF-α, and is applicable to the determination of TNF-α in serum samples and being capable of fluorescence microscopy imaging for the determination of TNF-α. [Copyright &y& Elsevier]

Abstract: The paper presented a novel chemiluminescence (CL) immunoassay method, which combines the advantages of traditional enzyme-linked immunosorbent assays (ELISA) and bis (2,4,6-trichlorophenyl) oxalate (TCPO)–hydrogen peroxide CL detection system. A fluorescent product 2,3-diaminophenazine (DAPN) was produced by reaction between o-phenylenediamine (OPDA, 1,2-diaminobenzene) and H2O2 catalyzed by horseradish peroxidase (HRP). DAPN was excited by the reactive intermediate of TCPO–H2O2 chemiluminescent reaction, and led to CL. The dependence of the CL intensity on the concentrations of antigen was studied. As analytical application, the proposed method was used for determination of recombinant human interleukin 6 (rHu IL-6) and β-human chorionic gonadotropin (β-HCG). Under the selected experimental conditions, a linear relationship was obtained between the CL intensity and the concentration of rHu IL-6 in the range of 4.0–625.0pg/ml, and β-HCG in the range of 12.5–400.0mIU/ml. The detection limits were 0.5pg/ml for rHu IL-6 and 3mIU/ml for β-HCG with relative standard deviation of 2.3 for 78.0pg/ml rHu IL-6, and 3.9 for 50.0mIU/ml β-HCG. This method has been applied to the determination of rHu IL-6 in human serum and β-HCG in urine with satisfactory results. [Copyright &y& Elsevier]

Abstract: A highly sensitive and stable sandwich fluorescence immunoassay for the quantitative detection of residual BSA in vaccines based on the labels of the functionalized fluorescent core-shell silica nanoparticles and laser-induced fluorescence millimeter sensor array detection platform has been developed. On a glass slide with low fluorescence background, capture antibody against BSA was immobilized, after BSA was captured, another identify antibody against BSA which was labeled with the new fluorescent silica nanoparticles was used to recognize the BSA. The fluorescence issued from the fluorescent silica nanoparticles was successfully detected by the laser induced fluorescence millimeter sensor assay detection platform which was made by us. This method exhibited high performance with a linear correlation between response and amount of BSA in the range 1.0–100ng/mL and the detection limit was 0.3ng/mL (3σ). The relative standard deviation (R.S.D.) was 6.7% at the concentration of 20ng/mL for 5 parallel measurements of BSA. [Copyright &y& Elsevier]