Ultra-sensitive Aptamer-based Lateral Flow Assays for DENV Detection

Abstract

Dengue virus (DENV) is the causative agent of a mosquito-transmitted disease mainly in tropical regions of the earth. Dengue is commonly diagnosed using polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay (ELISA); however, these diagnostic methods both require complicated blood sample preparation, highly trained personnel, and centralized laboratory facilities, all of which are difficult to realize in many clinical settings where resources are limited.

In the current study, a novel ultra-sensitive dendrimer-aptamer-based lateral flow assay (LFA) is designed to detect the presence of the DENV by detecting the envelope protein (E-Protein) of the DENV in phosphate-buffered saline (PBS) buffer and bovine serum albumin (BSA) sample. To achieve this, a “bioink”, a muti-handled streptavidin-dendrimer-aptamer conjugation is used to construct the modified test line in order to enhance the capturing efficiency of the signaling gold nanoparticle complexes on the test line. This work is the first time reported aptamer-based LFA of dengue virus detection. Our results show that the new LFA has a limit of detection of 24 pg/mL when tested using samples in PBS buffer (27 pg/mL in BSA solution), which is more sensitive that of a parallel ELISA test of 32 pg/mL and about ten-fold more sensitive than a conventional aptamer-based LFA. In addition, the new LFA shows that no non-specific binding with other E-protein in the flavivirus family and exhibits a long shelf-time for more than five weeks when stored in ambient conditions under subdued light.

It can be concluded that the use of “bioink” -- a streptavidin-dendrimer-aptamer -- complex on the T-line can significantly enhance the detection sensitivity of the LFA assay. As a result, it is perceivable that the intrinsic portable, rapid, user-friendly, and cost-effective natures of LFAs in combination with the enhanced sensitivity due to the special fishnet-liked design will find broader applications for the LFAs as an effective and sufficiently sensitive diagnostic tool in many resources limited clinical settings.