Fluorescence Immunoassay of Prostate-Specific Antigen Using 3D Paddle Screw-Type Devices and Their Rotating System

Abstract

In this paper, we present a sensitive and highly reproducible fluorescence immunosensor for detecting PSA in human serum. A unique feature of this study is that it uses creatively designed paddle screw-type devices and their custom-made rotating system for PSA immunoassay. The paddle screw devices were designed to maximize the surface-to-volume ratio over which the immunoassay reaction could occur to improve detection sensitivity. This paddle screw-based immunoassay offers an accessible and efficient method with a short analysis time of less than 30 min. Active rotation of the paddle screw plays a crucial role in fast and accurate analysis of PSA. Additionally, a paddle screw-based immunoassay and subsequent fluorescence detection using a custom prototype fluorescence detection system were compared to a typical well plate-based immunoassay system. Results of PSA detection in human serum showed that the detection sensitivity through the paddle screw-based analysis improved about five times compared to that with a well plate-based analysis.

Keywords: 3D paddle screw; detection sensitivity; fluorescence measurement; immunoassay; prostate-specific antigen.

Scheme 1

Schematic illustration of a sandwich immunoassay reaction for PSA occurring on the surface of a paddle screw-type device utilized in this study.

Figure 1

Various shapes of 3D paddle structures. The paddle screw shown in the blue box was selected considering that it was convenient to manufacture it with 3D printing and that it matched well with 1.5 mL microtubes for PSA immunoassay.

Figure 2

A custom-made fluorescence detection system consisting of an LED light source, fluorescence filters, and a photodiode detector.

Figure 3

A custom-made paddle screw rotating system. Microtubes (1.5 mL) for PSA assay located at the bottom of the system consisted of one solution containing complex of PSA and anti-PSA detecting antibody–FITC conjugates, three PBS buffer solutions, and one DTT solution.

Figure 4

Computational fluid dynamics (CFD) simulations for mixing effects of two liquids. The difference in mixing effect between the two liquids due to simple diffusion and active rotation with a single-blade paddle (200 rpm) was approximately 13 times.

Figure 5

(a) Fluorescence spectra of FITC released by DTT treatment after sandwich immunoassay according to PSA concentration, and (b) fluorescence intensity versus PSA concentration.

Figure 6

Changes in fluorescence signal ratio in the paddle screw-based immunoassay according to PSA concentration. The lower plot (solid square marks) shows the results of a well plate sandwich immunoassay. The upper plot (solid circle marks) shows the results of a paddle screw-based sandwich immunoassay. Data in red circles deviated from the logarithmic linear range.

Figure 7

Correlation of the proprietary paddle screw system with the commercial Centaur^®XP immunoassay system in PSA immunoassays for seven blinded samples prepared at different concentrations. A reasonable correlation was observed between the two methods over the entire tested concentration range. The percentage above the solid circle represents the relative error at each concentration.