Abstract
A new sensitive and selective platform, three-dimensional immunosensor, has been developed for a rapid serological diagnosis; detection of a Borrelia infection was considered as a model assay. The immunosensor is based on a 3-dimensional (3D) porous solid surface (sinter body) with dimensions of 2×2.5mm where a recombinant variable lipoprotein surface-exposed protein (VlsE; Borrelia-antigen) is immobilized by different techniques. The sinter body served as a robust and inexpensive carrier, which facilitated a successful hydrophobic adsorption as well as covalent immobilization of the antigen with sufficient amounts of on the surface. Because of sinter body’s porosity, the detection could be performed in an immune affinity flow system based on a little disposable plastic column. The flow of reagents through the column is advantageous in terms of reducing the non-specific interaction and shortening the test time. Furthermore, three labels were tested for a colorimetric detection: i) a horseradish peroxidase (HRP) labeled secondary antibody, ii) nanoparticles based on Sudan IV, and iii) gold nanoparticles modified with protein A. HRP secondary labeled antibody provides the most sensitive test, 1000 fold dilution of serum sample can be clearly detected in only 20min. Gold nanoparticles modified with protein A were used as a direct label or as a catalyst for reduction of silver ions. Direct detection with gold nanoparticles provides short time of analysis (5min) while detection of metallic silver required longer time (12min) but with improved sensitivity. Nanoparticles based on Sudan IV showed high background and were less favorable. The assay is distinctive because of the rapid analysis time with all used labels, longest 20min. Compared to classical serological methods for Borrelia diagnosis, the developed method offers a simple, rapid and reliable tool of analysis with minimal cost and can be easily transferred to other infectious diseases.
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