Toward sensitive, quantitative point-of-care testing (POCT) of protein markers: miniaturization of a homogeneous time-resolved fluoroimmunoassay for prostate-specific antigen detection

Abstract

Point-of-care testing (POCT) systems which allow for a sensitive, quantitative detection of protein markers are extremely useful for the early detection and therapy progress monitoring of cancer. However, currently commercially available POCT devices are mainly limited to the qualitative detection of protein markers. In this study we demonstrate the successive miniaturization of a sensitive and fast assay for the quantitative detection of prostate-specific antigen (PSA) using a well established and clinically approved homogeneous time-resolved fluoroimmunoassay technology (TRACE®) on a commercial plate-reader system (KRYPTOR®). Regarding the initial requirements for the development of POCT devices we applied a 30-fold assay volume reduction (150 µL to 5 µL) to achieve a reasonable lab-on-a-chip volume and a 24-fold and 120-fold excitation pulse energy reduction to achieve reasonable pulse energies for low-cost miniature excitation sources. Due to highly efficient optimization of key POCT parameters our miniaturized PSA assay achieved a 30% increased sensitivity and a 2-fold improved limit of detection compared to the standard plate-reader method. Our results demonstrate the successful implementation of key parameters for a significant miniaturization and for cost reduction in the clinically approved KRYPTOR® platform for protein detection. The technological alterations required are easy-to-implement and can be immediately adapted for more than 30 diagnostic protein markers already available for the KRYPTOR® platform. These features strongly recommend our assay format to be utilized in innovative, sensitive, quantitative POCT of protein markers.