Developing a multiplexing platform for the detection of specific biomarkers -

Abstract

The development of simple, sensitive and multiplexed detection system is highly desirable in the field of clinical diagnostics. This is mainly due to the fact that multiplexed systems can reduce labor, time and cost while maintaining a higher accuracy of detection. In view of that, herein we report on the preparation of different sensing platforms for the detection of trace amounts of protein and DNA biomarkers. Using a chip-based electrochemical immunosensor, we were able to detect TBI biomarkers for lab purposes inside purified samples. The assay allowed us to investigate the microscale size regime of gold sensors and achieve sensitivity in the range of pg-mL. On the other hand, we described a fluorescent sandwich assay as an alternative method. The assay was built on gold coated polystyrene microspheres and visualized using flow cytometry with the aid of different labels. Out of three examined fluorescent tags, dye encapsulated liposomes gave the best enhanced sensitivity and allowed the detection of low biomarker levels that are clinically relevant. Building upon our findings, we developed an ultrasensitive universal platform for the detection of oligonucleotide sequences. The basis of this scheme relied on immobilizing separately two DNA strands each complementary to a different portion of target analyte onto the surface of gold coated polystyrene microspheres and dye-encapsulated liposomes. The hybridization event was recognized by an enhancement of the fluorescent signal recorded by flow cytometry. The assay parameters were optimized so that the whole experiment can take less than 2 hours with a detection limit of 2.7 pM. Furthermore, the proposed scheme was shown to perform well in complex matrices such as serum making it promising platform for fast multiplexing diagnosis.