Elsevier

Analytica Chimica Acta

Volume 580, Issue 2, 24 November 2006, Pages 128-135
Analytica Chimica Acta

Flow-injection immuno-bioassay for interleukin-6 in humans based on gold nanoparticles modified screen-printed graphite electrodes

https://doi.org/10.1016/j.aca.2006.07.068Get rights and content

Abstract

A flow-injection electrochemical immunoassay system based on a disposable immunosensor for the determination of interleukin-6 (IL-6) was proposed. The immunosensor was prepared by entrapping horseradish peroxidase (HRP)-labeled IL-6 antibody into gold nanoparticles-modified composite membrane at a screen-printed graphite electrode. With a non-competitive immunoassay format, the immunosensor was inserted in the flow system with an injection of sample, and the injected sample containing IL-6 antigen was produced transparent immunoaffinity reaction with the immobilized HRP-labeled IL-6 antibody. The formed antigen–antibody complex inhibited partly the active center of HRP, and decreased the immobilized HRP to H2O2 reduction. The performance and factors influencing the performance of the immunosensor were investigated. Under optimal conditions, the current change obtained from the labeled HRP relative to thionine–H2O2 system was proportional to the IL-6 concentration in the range of 5–100 ng L−1 with a detection limit of 1.0 ng L−1 (at 3δ). The flow-injection immunoassay system could automatically control the incubation, washing and measurement steps with acceptable reproducibility and good stability. Moreover, the proposed immunosensors were used to analyze IL-6 in human serum specimens. Analytical results of clinical samples show the developed immunoassay has a promising alternative approach for detecting IL-6 in the clinical diagnosis.

Introduction

Interleukin-6 (IL-6), a pleiotropic cytokine that has a critical role in the inflammatory response, has been implicated in the pathogenesis of a number of inflammatory conditions, such as psoriasis, rheumatoid arthritis, cardiovascular disease, and inflammatory bowel disease [1], [2], [3]. IL-6 elicits B cells to undergo proliferation and differentiation into antibody-forming cells; assists in IL-4 dependant IgE synthesis and T cell activation, growth and differentiation. IL-6 also acts in conjunction with IL-3 to induce the proliferation of pluripotent haematopoietic progenitors [4]. As an important member of the cytokine network, IL-6 mediates acute phase response in the liver, stimulates the production of C-reactive protein (CRP) and fibrinogen. A variety of infectious diseases can cause an increase in serum IL-6 level, and increased IL-6 is in turn closely associated with mortality [5], [6]. Thus, the determination of IL-6 level is very useful to clinical diagnoses.

Recently, human research to IL-6 has profited from the development of models that are increasingly biologically relevant, potentially clinical significance, and also permit the functional assessment of immunity [7], [8]. However, much of this research relies upon recall responses to previously encountered infectious agents or is confounded by variable degrees of prior immunity to the immunogen [9]. This study utilized a standardized method of functional assessment of immunity based on the immunization of subjects with the novel antigen [10]. This model may be considered a biochemical challenge that broadly models the events occurring upon host exposure to a previously unencountered pathogen. Such a response necessitates processing and presentation of the novel antigens, followed by expansion of the effector arm of the response and generation of immunological memory [11]. These researching techniques, unfortunately, require highly qualified personnel, tedious assay time, or sophisticated instrumentation. Therefore, searching for a novel analytical biochemical method in combination with biological knowledge and chemical technique is of considerable interest.

Immunoassays, the measurement of antibody or antigen concentrations based on biospecific recognition interactions, have been considered a major analytical tool in the fields of clinical diagnostics and environmental analysis [12]. Despite the predominance of high sensitivity, the conventional immunoassay methods such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunoaffinity column assay (ICA) and immunoaffinity fluorimetric biosensor (APL sensor) have some limitations such as the short shelf life of 125I-labeled antibody, the radiation hazards, the complicated wash procedure, and the requirements for a long analysis time, expensive and cumbersome instruments and/or skillful operators [13], [14]. The electrochemical method has many advantages including simple instrumentation that enables device miniaturization and easy signal quantification [15]. In electrochemical immunosensor development, an important issue resides in the design and fabrication of immunosensing interfaces. To detect target biomolecules, the target-specific ligands must be effectively immobilized on the sensor surface. The analytes must be able to access their immobilized recognition couple under satisfactory frequency without severe steric hindrance and non-specific binding. Thus, the design and implementation of a unique sensing surface for facile ligand functionalization and biospecific interaction are crucial [12]. Screen-printing technique seems to be one of the most promising approaches allowing simple, rapid and inexpensive biosensors production [16]. The biosensors based on screen-printed electrodes have been extensively used for detections of biomolecules, pesticides, antigens, and anions [17]. Different configurations for the design of screen-printed electrodes and for the protein immobilization using various techniques have been reviewed [17].

The purpose of this paper is to describe the design, fabrication and application of the disposable immunosensor, and develop a flow-injection immunoassay system based on immobilization HRP-labeled IL-6 antibody onto colloidal gold-modified screen-printed graphite electrodes. The system was capable of continuously carrying out all steps in less than 50 min for one sample, including incubation of 45 min, washing, enzymatic reaction, and determination procedures. The quantitative detection of IL-6 showed good analytical performance and good consistency with the ELISA method. Additionally, the performance and factors influencing the performance of the resulting immunosensors have been proposed.

Section snippets

Chemicals

IL-6-ELISA kits, including 96 assay tubes pre-coated with mouse polyclonal anti-IL-6, were purchased from Sigma (USA). The ELISA kits consisted of a series of IL-6 standard solutions with various concentrations from 0 to 200 ng L−1. Glutaraldehyde (GA), bovine serum albumin (BSA), horseradish peroxidase (HRP) (EC. 1.11.1.7) and gold nanoparticles (24 nm in diameter) were purchased from Sigma (USA). Thionine (TH) and hydrogen peroxide (30%, w/w) were obtained from the Shanghai Biochem. Reag.

HRP enzymatic activity detection

As mentioned above, the activities of the immobilized antibodies can be largely influenced by the surface properties of the transducer in clinical immunoassays [18]. In this study, we tried to fabricate an improved interface using gold nanoparticles and BSA for immobilization of antibodies. Use of gold colloids was expected to favor a particle-enhanced immobilization of antibodies due to the unique physical and chemical features of nanoparticles [18]. BSA, an inert protein commonly used as

Conclusions

This work describes a new, simple enzyme immunoassay for IL-6 based on a disposable immunosensor integrated into a flow-injection electrochemical system. To construct such an immunoassay system, HRP-labeled IL-6 antibodies were immobilized onto an SPGE surface with gold nanoparticles and BSA in a saturated glutaraldehyde vapour. The present of gold nanoparticles could improve the reversibility of the system and enhance the sensitivity of the immunosensor to IL-6 detection. The proposed

Acknowledgments

The authors gratefully acknowledge financial support of the Natural Science Foundation of Chongqing City and the Education Committee of Chongqing City, China.

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