Design of biosensor probes based on biological templates for sensitive in vitro and in vivo diagnostics

Abstract

Early detection of many diseases including cancers, have recently been considered as an emerging field with an aid of disease markers that have been well characterized by previous studies. The reason being that the technology provides a high probability of effective treatment, and has successful results in improving the survival rates and quality of a patients’ life. Especially, therapeutic efficiency of cardiovascular diseases and cancers are tightly related with the time of detection. One of them, such as heart disease can cause the whole body to dysfunction within a very short amount of time and thus early and quick diagnosis is essential for prevention of related death. Based on these reasons, development of highly sensitive diagnostic devices has received a lot of attention in the clinical fields. Typically, point-of-care testing (POCT) is a rapid and simple in vitro diagnostic method. Also, medical imaging plays an important role in early diagnosis and disease prevention by directly visualizing and identifying abnormalities. For highly sensitive in vitro and in vivo diagnosis, we designed novel diagnostic systems by combining sensing probes with biological materials. In the first part, we designed the Protein G-mediated antibody immobilization method for the construction of spatially oriented antibody-conjugated magnetic beads, and adopted the technique to lateral flow assay system for enhanced detection sensitivity of an important cardiac marker, cardiac troponin I (cTnI). Our new assay system remarkably showed an improvement in detection sensitivity for cTnI to 0.01 ng/ml in comparison with that of conventional random immobilization method. In the second part, we designed an advanced conjugation strategy on the basis of the biologically specific interactions between double-stranded DNA (dsDNA) and zinc-finger protein (ZnF), to synthesize linear magnetic nanoparticle (MNP) clusters with different chain lengths in a controlled manner. We effectively constructed precisely controlled MNP clusters by using different length of designed dsDNAs, which consists of a tandem array of ZnF binding sites. The novel MNP clusters give rise to enhanced results on both magnetic resonance imaging (MRI) contrast effect which is 3-fold than Feridex and specific targeting of folate-receptor-positive cancer cells. Based on the results, we demonstrated that the synthesis strategy was straightforward and effective for the construction of the dsDNA-templated nanoclusters. This can be generally used in nano and biotechnology fields for development of highly ordered and controllable nanocluster systems. In the last part, we constructed nucleotide-based drug delivery system by using repebody-ZnF fusion proteins. The resulting construct exhibited effective receptor-mediated delivery of intercalated doxorubicin into EGFR-expressed cancer cells. Our results suggest that the DNA-templated nanoclusters can be developed as a novel medical material for various theranostic applications.