Development of fluorescence change-based, reagent-less optic immunosensor

Abstract

A reagent-less, regenerable and portable optic immunosensor was developed. A model sample, immunoglobulin G (IgG), was detected with this system based on changes in fluorescent intensity of fluorescent labeled protein A with specific reactivity to IgG depending on a reaction between the proteins.

A glass plate immobilized with Qdot™-labeled protein A was placed on the top of optic fibers designed for both excitation and fluorescence emission. The optic fibers with the Qdot™-labeled protein A-immobilized glass plate were inserted into a solution of pH 7.4 phosphate buffered saline. After stabilization of the fluorescence intensity, IgG was added and the time-course of the fluorescence intensity was measured on a fluorometer connected with the optic fibers. Furthermore, the fluorescence response of a transient state was evaluated with the same system.

When the Qdot-labeled protein A bound to IgG, fluorescence intensity decreased because of the inhibition by IgG. The degree of fluorescence decrease depends on the IgG concentration at a steady state and also in a transient state.

Introduction

Immunoassay is one of the most important assay systems because of the potential variability of antibodies produced coupled with the ability of antibodies to bind to antigenic determinants with high affinity and specificity. Antibodies are able to recognize and bind to a defined epitopic site on an antigen, which is able to induce immediate environmental changes around a probe labeled to antibody. Non-separation immunoassays, such as fluorescence-enhancement and quenching, are based on the environmental changes caused by an antigen–antibody reaction (Hemmila, 1991, Price and Newman, 1997). Though the non-separation immunoassays are, basically, demonstrated in homogeneous systems, they can be reagent-less immunoassays when labeled-antibodies are immobilized. There are, however, few reports (Bright et al., 1990, Aoyagi et al., 2003, Fan et al., 2003) on a non-separation immunoassay such as fluorescence-enhancement immunoassay in a heterogeneous system. A new reagent-less and regenerable immunosensor based on fluorescence-enhancement immunoassay, one of the non-separation immunoassays, with a fluorescent reagent immobilized on a glass plate, has been developed (Aoyagi et al., 2003), and it measures the immunoglobulin G (IgG) concentration co-existing with other proteins. A previous reagent-less immunosensor system was a batch system and is not suitable for in situ monitoring, and its detecting principle was based on the enhancement of fluorescein isothiocyanate (FITC) fluorescence caused by reactions between proteins. Protein A participates in bio-affinity reactions, such antigen–antibody reactions, with human IgG sub-classes 1, 3 and 4 accounting for more than 95% of total IgG (Duhamel et al., 1979). IgG bonding to protein A is separated from protein A in a low pH solution, approximately pH 2–4 (Lindmark et al., 1983). The previous system was demonstrated in a batch cell fluorometer.

The objective of the present study was to develop an in situ measurement system with optic fibers. The signs of fluorescence intensity change, such as fluorescence-enhancement and quenching are, however, less than the signal of conventional separation fluorescence immunoassays. Therefore, it is difficult to detect the signs of fluorescence intensity change caused by sample protein binding with fluorescent labeled-reagent by means of an optic-fiber system, because of the loss of light detection through optic fibers. Quantum dot fluorescent particles (Peng et al., 1997, Murray et al., 1993) were then employed to the fluorescence probe to obtain a higher and more stable response reducing the influence of the excitation light. The quantum dots are extremely small devices that contain free electrons, are fabricated in semiconductor materials, and have typical dimensions ranging in size between a few nanometers to a few microns. The size and shape of these structures and the number of electrons they contain can be precisely controlled so as to be produced as materials having the requisite functions. The material properties can vary dramatically because quantum effects arise not only from the confinement of electrons but also holes in the material. Size changes crucial material properties such as the electrical and nonlinear optical properties of a given material, making these properties very different from those of the bulk form the materials. When a quantum dot is excited, the smaller the quantum dot, the higher the energy and intensity of its emission light. The color of the emission light depends on the size of the quantum dots: the larger the dot, the longer the wavelength of emission light. As the quantum dots shrink in size, the emission light becomes shorter in wavelength.

Quantum dots have been applied to obtain imaging techniques such as fluorescent labels and also to evaluate the interaction of macromolecules labeled with organic fluorescence probes. Recently, quantum dots have been applied to biosensors, making use of fluorescent properties such as a wide range of excitation light, high quantum yield, high stability and sensibility to environmental change which causes fluorescence resonance energy transfer (Wang et al., 2002, Lin et al., 2004, Medintz et al., 2003). Fluorescence resonance energy transfer occurs, originally, based on distance-dependent transfer of energy from a donor fluorescent probe to an acceptor of fluorescence probe (Slvin, 2000). In the present study, an optic-fiber system with a quantum dot-labeled protein A-immobilized substrate for detecting IgG was developed, and its capability was evaluated.