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Quantitative detection of multiple chicken cytokines is a good evaluation of cell-mediated immunity in chickens after disease infection or vaccination. However, current assay methods for chicken cytokines cannot meet the needs of clinical diagnosis due to unsatisfactory sensitivity and low assay throughput. Herein, a sensitive chemiluminescence (CL) imaging immunosensor array has been developed for high-throughput detection of multiple chicken cytokines. The chicken cytokines immunosensor array was prepared by assembling different cytokine capture antibodies onto a disposable silanized glass chip, where horseradish peroxidase and antibody-conjugated gold nanoparticles were used as multienzymatic amplification probe for CL imaging signal amplification. By using a sandwich assay mode, the amplified CL signals from each sensing array cell were collected for quantitation. Using chicken interleukin-4 and chicken interferon-γ as model cytokines, this novel multiplexed and amplified method demonstrated simultaneous measurement of the two chicken cytokines in the linear ranges of 0.008-0.12 ng/mL and 0.005-0.20 ng/mL, respectively, which yields limits of detection down to 2 pg/mL and 3 pg/mL. The CL imaging array method reported here also demonstrated high specificity, good repeatability, and high stability and accuracy, providing a novel multiplex immunoassay strategy for highly sensitive and high-throughput detection of chicken cytokines and further disease diagnosis in poultry., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Microcantilever-based immunosensor is a next-generation electromechanical technique with broad application in biological detection. In this paper, we reported a microcantilever-based immunosensor that quantitatively detect the carbofuran, by using monoclonal antibodies to carbofuran as the receptor molecules. The surface of gold-coated microcantilever was chemically modified by the crosslinking of l-cysteine (l-cys)/glutaraldehyde (GA). The monoclonal antibodies to carbofuran were then immobilized on the side of the microcantilever to fabricate the immunosensor, the mechanical bending induced by antigen-antibody specific binding under an experimental environment. Under the optimized conditions, immunosensor detected carbofuran showed a good linear relationship over the range from 1.0×10 -7 to 1.0×10 -3 g/L (R=0.998), with a detection limit of 0.1ng/mL. Moreover, the proposed immunosensor exhibited high sensitivity, specificity and good stability and can be successfully applied in the carbofuran determination in soil and vegetable samples with satisfactory results., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Chicken interleukin-4 (ChIL-4), which is released by activated type 2 helper (Th2) cells following their stimulation in vitro, is an important indicator for the study of cell-mediated immunity in chickens after infection or vaccination. In this work, the first ChIL-4 chemiluminescent (CL) immunosensor was developed via the immobilization of monoclonal ChIL-4 antibodies on a nitrogen-doped graphene (NG)-chitosan nanocomposite matrix. NG nanosheets were used for the first time in the CL immunoassay to provide a biocompatible microenvironment for the immobilized capture antibody. The ChIL-4 immunosensor was characterized systematically. The proposed immunosensor displayed a wide linear range from 0.05 to 70ngmL -1 and a low detection limit of 0.02ngmL -1 at a signal-to-noise ratio of 3. Compared to traditional assay methods, this system was more flexible, simple, rapid, and sensitive. Moreover, this CL immunoassay system had an excellent detection and fabrication reproducibility, a high specificity, an acceptable accuracy, and a high stability. This work enables the specific detection of ChIL-4 and the further study of its role in the immune responses of poultry., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this work, an efficient and universal streptavidin-functionalized nitrogen-doped graphene (NG) was for the first time proposed and used to develop a highly sensitive electrochemical immunosensor for the detection of tumor markers. Transmission electron microscopy, electrochemical impedance spectrum, static water contact measurement, and cyclic voltammetry were used to characterize the streptavidin-functionalized NG platform and immunosensor. The biofunctionalized NG showed excellent hydrophilicity, larger specific surface area, and high electrochemical activity. These properties of the platform enhanced the loading capacity of proteins, and retained the bioactivity of the immobilized proteins, and thus remarkably improved the sensitivity of the immunosensor. Using carcinoembryonic antigen (CEA) as model analyte, the proposed immunosensor demonstrated a wide linear range of 0.02-12ngmL -1 with a low detection limit of 0.01ngmL -1 . The CEA immunosensor could be applied to detect human serum samples with satisfactory results. The streptavidin-functionalized NG material provided an universal and promising platform for the electrochemical immunosensing applications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A new label-free chemiluminescence (CL) immunoassay method is proposed for the cheap, convenient and sensitive detection of proteins through the co-immobilization of a capture antibody and horseradish peroxidase on a sensing interface. The specific immunoreaction on the interface effectively inhibits the enzymatic CL reaction, thus causing a decrease in CL signals.

Bovine interferon gamma (BoIFN-γ) released by T cells plays very important roles in early diagnosis of Mycobacterium tuberculosis (MTB) infections and control of bovine tuberculosis. In this work, a label-free electrochemical impedance immunosensor is for the first time developed for highly sensitive determination of BoIFN-γ. Cylinder-shaped TiO2 nanorods are synthesized by a facile hydrothermal method, and show high surface area and good hydrophicility. The immunosensor is fabricated by the immobilization of BoIFN-γ monoclonal antibody on the TiO2 nanorods modified glassy carbon electrode. The prepared TiO2 and immunosensor are characterized using transmission electron microscopy, scanning electron microscopy, X-ray diffraction, contact angle measurement, cyclic voltammetry, and electrochemical impedance spectra. The BoIFN-γ concentration is measured through the relative increase of impedance values in corresponding specific binding of BoIFN-γ antigen and BoIFN-γ antibody. The relative increased impedance values are proportional to the logarithmic value of BoIFN-γ concentrations in a wide range of 0.0001 to 0.1 ng/mL with a low detection limit of 0.1 pg/mL. The developed BoIFN-γ immunosensor shows a 249-fold decrease in detection limit in comparison with current enzyme-linked immunosorbent assay. This study provides a new, simple, and highly sensitive approach for very potential application in early diagnosis of MTB infections and control of bovine tuberculosis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this work, a novel streptavidin functionalized graphene oxide/Au nanoparticles (streptavidin/GO/AuNPs) composite is prepared and for the first time used to construct sensitive chemiluminescent immunosensor for the detection of tumor marker. The streptavidin/GO/AuNPs composite and the immunosensor are characterized using scanning electron microscopy, static water contact angle measurement and electrochemical impedance spectroscopy. The biofunctionalized composite has large reactive surface area and excellent biocompatibility, thus the capture antibody can be efficiently immobilized on its surface based on the highly selective recognition of streptavidin to biotinylated antibody. Using α-fetoprotein (AFP) as a model, the proposed chemiluminescent immunosensor shows a wide linear range from 0.001 to 0.1 ng mL(-1) with an extremely low detection limit down to 0.61 pg mL(-1). The resulting AFP immunosensor shows high detection sensitivity, fast assay speed, acceptable detection and fabrication reproducibility, good specificity and stability. The assay results of serum samples with the proposed method are in an acceptable agreement with the reference values. This work provides a promising biofunctionalized nanostructure for sensitive biosensing applications., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The quantitative detection of chicken interferon-γ (ChIFN-γ) released by T cells after in vitro stimulation is a good evaluation of cell-mediated immunity in chickens after infection or vaccination. This work reports a new flow-through the chemiluminescent (CL) immunoassay method for rapid and specific determination of ChIFN-γ at a level of sub-picogram per milliliter. The biocompatible graphene oxide nanosheet is introduced into CL immunoassay for highly efficient immobilization of capture antibody. The detection limit of the proposed method at a signal to noise ratio of 3 is 0.36 pg ml(-1), which is 138-fold lower than the current lowest value of 50 pg ml(-1) for ChIFN-γ. Coupling with a flow-through system, the whole immunoassay process can be completed within 25 min. The resulting ChIFN-γ immunosensor shows excellent detection and fabrication reproducibility, good specificity and stability. The assay results of nature ChIFN-γ samples with the proposed method are in an acceptable agreement with the reference values. Compared to the present assay methods, this method is more flexible, simple, rapid and sensitive. The aim of this work is to demonstrate that the ultrasensitive, specific and rapid CL immunoassay format can become a very potential application for quantifying ChIFN-γ and further studying its role in immune response in poultry., (© 2013 Elsevier B.V. All rights reserved.)

In this experiment, fenvalerate antibodies were immobilised on the electrode by the crosslinking with glutaraldehyde modified on the glassy carbon electrode (GCE) via chitosan. Fenvalerate was measured by the increase of electron transfer resistance when the immune reaction occurred with Fe(CN)6(3-)/Fe(CN)6(4-) as the probe. Under optimal conditions, the change of resistance is in a linear relationship with the logarithm of the concentration in the range of 1.0×10(-3)∼1.0×10(1)mg/L (R=0.998) with a detection limit of 0.80 μg/L. This method bears such merits as simplicity of operation, high sensitivity, wide linear range, specificity, reproducibility and good stability. The immunosensor was applied in the detection of real samples of tea, achieving satisfactory results, and it could be regenerated after being placed alternately in 0.5 mol/L HCl and 0.5 mol/L NaOH solutions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

An ultrasensitive chemiluminescent (CL) immunoassay system was developed for the detection of tumor marker. This sandwich CL assay method was for the first time designed based on a highly efficient streptavidin-functionalized multi-walled carbon nanotubes (MWCNTs) platform. The glass slide was firstly silylanized with 3-gycidoxypropyltrimethoxysilane (GPTMS) to generate surface epoxy group functionality. Subsequently, the MWCNTs/chitosan solution was mixed with streptavidin solution, and a certain amount of the resulting suspension was dropped on the surface of the epoxy-activated glass substrate to form a firm streptavidin-functionalized MWCNTs platform. The biofunctionalized-MWCNTs platform shows large reactive surface area and excellent biocompatibility. The capture antibody can be efficiently immobilized on the biosensing platform surface based on the highly selective recognition of streptavidin to biotinylated antibody. Using α-fetoprotein (AFP) as model analyte, the proposed method exhibits wide linear range of 0.001-0.1 ng mL(-1) with a low detection limit down to 0.52 pg mL(-1). The CL immunoassay system displays 7.9-fold increase in the detection sensitivity compared to the immunosensor without using MWCNTs. Moreover, the resulting immunosensor demonstrates excellent specificity, good reproducibility, and acceptable stability. This streptavidin-functionalized MWCNTs platform opens a novel and promising avenue for fabricating ultrasensitive CL immunoassay system., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We describe an automation-friendly chemiluminescent immunoassay method for sensitive and rapid determination of chicken interferon-γ at the sub-picogram per millilitre level based on the monoclonal antibody immobilized paramagnetic microspheres and flow-through system.

A novel chemiluminescent immunosensor for carbohydrate antigen 19-9 (CA19-9) based on the immobilization of CA19-9 on the cross-linked chitosan membrane was developed. The different membranes were characterized by atomic force microscopy (AFM) and infrared spectrum, respectively. Based on a noncompetitive immunoassay format, this proposed chemiluminescent immunosensor enabled a low-cost, flexible and rapid determination for CA19-9 in combination with flow injection analysis (FIA). After an off-line incubation of the analyte CA19-9 with horseradish peroxidase (HRP)-labeled anti-CA19-9, the mixture was injected into the immunosensor, which led to the trapping of free HRP-labeled anti-CA19-9 by the immobilized antigen in the immunosensor. The trapped HRP-labeled antibody was detected by chemiluminescence due to its catalytic activity following the reaction of luminol and H2O2. Under optimal conditions, the decreased chemiluminescent signal of the immunosensor was proportional to the CA19-9 concentration in the range of 2.0-25 U/ml with a detection limit of 1.0 U/ml. The immunosensor showed an acceptable accuracy and good reproducibility. The results of 20 human serum samples detected by this method were in acceptable agreement with those obtained by immunoradiometric assay. The proposed immunosensor provided a new promising tool for practical clinical detection of the serum CA19-9 level.

A novel immunosensor for rapid separation-free determination of carcinoembryonic antigen (CEA) in human serum is proposed. The immunosensor is prepared by co-immobilizing thionine and horseradish peroxidase (HRP)-labeled CEA antibody on a glassy carbon electrode (GCE) through covalently binding them to GCE with a glutaraldehyde (GA) linkage. The electrochemical behavior of the immobilized thionine displays a surface-controlled electrode process with an average electron transfer rate constant of 4.74+/-2.99 s(-1). It can be used as an electron transfer mediator for enzymatic activity detection of the HRP-labeled antibody to CEA. After the immunosensor is incubated with CEA solution at 23 degrees C for 40 min, the access of activity center of the HRP to thionine is partly inhibited, which leads to a linear decrease in the catalytic efficiency of the HRP to the oxidation of immobilized thionine by H(2)O(2) at -300 mV over two CEA concentration ranges from 0.5 to 3.0 and 3.0 to 167 ng/ml. Under optimal conditions, the detection limit for the CEA immunoassay is 0.1 ng/ml at three times background noise. The immunosensor shows good accuracy and acceptable storage stability, precision and reproducibility with intra-assay CVs of 6.1% and 5.8% at 2.5 and 50 ng/ml CEA, respectively, and an inter-assay CV of 6.3% at 50 ng/ml. This method is economical and shortens the analytical time, making it potentially attractive for clinical immunoassays.

Dual-functional cupric oxide nanorods (CuONRs) as peroxidase mimics are proposed for the development of a flow-through, label-free chemiluminescent (CL) immunosensor. Forming the basis of this cost-efficient, label-free immunoassay, CuONRs, synthesized using a simple hydrothermal method, were deposited onto epoxy-activated standard glass slides, followed by immobilization of biotinylated capture antibodies through a streptavidin bridge. The CuONRs possess excellent catalytic activity, along with high stability as a solid support. Antigens could then be introduced to the sensing system, forming large immunocomplexes that prevent CL substrate access to the surface, thereby reducing the CL signal in a concentration dependent fashion. Using carcinoembryonic antigen (CEA) as a model analyte, the proposed label-free immunosensor was able to rapidly determine CEA with a wide linear range of 0.1-60ngmL-1 and a low detection limit of 0.05ngmL-1. This nanozyme-based immunosensor is simple, sensitive, cost-efficient, and has the potential to be a very promising platform for fast and efficient biosensing applications.

Metal–organic frameworks (MOFs) show excellent catalytic activity and have been widely applied in diagnosis of diseases and tumors. However, current assay methods usually involve cumbersome configurations and complicated procedures, which inhibit their practical applications. Hence, a Cu–Ni MOF/carbon printed electrode (CPE)-based integrated electrochemical immunosensor was constructed for highly sensitive and efficient determination of carcinoembryonic antigen (CEA). First, highly conductive carbon ink was screen-printed onto a polyethylene terephthalate substrate to manufacture a CPE. Afterward, an aminated Cu-Ni MOF was prepared by a typical solvothermal strategy and modified on the CPE. Owing to its excellent peroxidase activity, the Cu–Ni MOF can catalytically oxidize hydroquinone using hydrogen peroxide, which greatly amplifies the peak current signal. Then the formation of an immune complex inhibited the catalytic activity of the MOF, thus enabling the quantitative determination of CEA content with a wide linear range of 0.5 pg mL−1–500 ng mL−1 and a low detection limit of 0.16 pg mL−1. Furthermore, the Cu–Ni MOF/CPE-based integrated portable electrochemical immunosensor also showed satisfactory performance in the detection of CEA in clinical serum samples with excellent accuracy, showing great potential for application in point-of-care disease diagnosis. [ABSTRACT FROM AUTHOR]

Herein, we report the synthesis of novel nanosized reduced graphene oxide@polystyrene nanospheres (rGO@PS NSs) through an electrostatic interaction method, and further their exploitation for highly sensitive label-free electrochemical immunoassay applications. These rGO@PS NSs provide a universal and promising carrier platform to develop excellent biosensors for clinical screening and diagnostic applications. [ABSTRACT FROM AUTHOR]

We report on label-free immunosensors for the highly sensitive detection of avian influenza virus. The method makes use of the microcantilevers of an atomic force microscope onto which monoclonal antibodies against avian influenza virus were covalently immobilized. The factors influencing the performance of the resulting immunosensors were optimized by measuring the deflections of the cantilever via optical reflection, and this resulted in low detection limits and a wide analytical range. The differential deflection signals revealed specific antigen binding and their intensity is proportional to the logarithm of the concentrations of the virus in solution. Under optimal conditions, the immunosensors exhibit a linear response in the 7.6 ng mL to 76 μg mL concentration range of avian influenza virus, and the detection limit is 1.9 ng mL. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Rapid and highly sensitive detection of tumor marker (TM) is critical for the early diagnosis and treatment of cancers. Herein, utilizing highly bright and water-stable CsPbBr 3 perovskite nanocrystals (NCs) capped with amphiphilic polymer ligand of octylamine-modified polyacrylic acid (OPA) and gold nanoparticles (AuNPs) as reporters, a lateral flow immunoassay (LFIA) strip is developed for fluorescence and colorimetric dual-mode detection of carcinoembryonic antigen (CEA). The prepared CsPbBr 3 NCs capped by an amphiphilic polymeric of OPA ligand showed high stability and bright fluorescence. Moreover, the AuNPs immunoprobes were captured with CEA antigen and quench the green fluorescence of CsPbBr 3 /OPA NCs on the T line due to the inner filter effect (IFE). Therefore, CEA could be quantitative analyzed by the dual-readout of fluorescence and colorimetric signal. The detection limits of CEA can reach as low as 0.023 ng/mL and 0.027 ng/mL for the fluorescence and colorimetric mode, respectively. Good specificity and reproducibility were also demonstrated for this method. Finally, the CsPbBr3/OPA NCs-based LFIA showed good accuracy in detection of CEA level from clinical serum samples. This work firstly enables the application of CsPbBr 3 perovskite NCs in a LFIA, displaying great potential in point-of-care clinical diagnosis. [Display omitted] • A dual read-out LFIA strip based on CsPbBr 3 /OPA NCs for rapid detection of CEA. • The work firstly enables the application of CsPbBr 3 perovskite NCs in a LFIA. • The CsPbBr 3 /OPA NCs showed high photoluminescence and good water stability. • With the aid of AuNPs, dual read-out competitive immunoassay analysis of CEA realized. • The LFIA strips showed high performance for CEA detection. [ABSTRACT FROM AUTHOR]

Abstract: Time-resolved fluorescence has been developed for immunoassay to obtain higher sensitivity than usual immunoassays. In this paper, a simple, sensitive and specific method was developed for immunoassay of serum carcinoembryonic antigen (CEA) by combining time-resolved fluoroimmunoassay (TRFIA) and flow injection analysis. Based on a sandwich immunoassay format, a monoclonal antibody immobilized immunoaffinity column inserted in a flow system was used for immunoreactions. The cleaved solution was collected after the reaction between the immunocomplex in the immunoaffinity column and the enhancement solution that was used to cleave the Eu-labels from the immunocomplex, and then detected by time-resolved fluorescence. Serum CEA could be detected in the linear range from 2.5 to 100 ng/ml with a correlation coefficient of 0.997 and the detection limit of 1.0 ng/ml. Twenty human serum samples detected by this method were in good agreement with the results obtained by the electrochemiluminescence immunoassay. This method could be further developed for fast practical clinical detection of serum CEA levels. [Copyright &y& Elsevier]