Screening of DNA Aptamers against Myoglobin Using a Positive and Negative Selection Units Integrated Microfluidic Chip and Its Biosensing Application

Abstract

An aptamer screening method using a positive and negative selection units integrated microfluidic chip was introduced. Here, myoglobin (Myo), one of the early markers to increase after acute myocardial infarction, was used as the model. After 7-round selection, the aptamers, which exhibited dissociation constants (K(d)) in the nanomolar range (from 4.93 to 6.38 nM), were successfully obtained using a positive and negative selection units integrated microfluidic chip. The aptamer with the highest affinity (K(d) = 4.93 nM) was then used for the fabrication of a label-free supersandwich electrochemical biosensor for Myo detection based on target-induced aptamer displacement. The detection limit of this aptamer-based electrochemical biosensor was 10 pM, which was significantly lower than that of those previous antibody-based biosensors for Myo detection. This work may not only develop a strategy for screening aptamer but also offer promising alternatives to the traditional analytical and immunological methods for Myo detection.

Visual detection of myoglobin via G-quadruplex DNAzyme functionalized gold nanoparticles-based colorimetric biosensor

“…Hemin and ABTS were purchased from Sigma-Aldrich (USA). DNA1 probe (5 -CCC TCC TTT CCT TCG ACG TAG ATC TGC TGC GTT GTT CCG ATT TTT AGA TGG CTA TGC AA-3 ) [27] and DNA2 probe (5 -T 17 GGG TAG GGC GGG TTG GGT 10 TTG CAT AGC CAT CT-3 ) were synthesized by Sangon Biotech. (Shanghai) Co., Ltd.…”

Rapid selection of aptamers based on protein microarray

“…All oligonucleotides used in this study were synthesized according to Yang's group 35 by Sangon Biotech Co. Ltd. (Shanghai, China). Each single-stranded DNA (ssDNA) library member consisted of 40 nucleotide (nt) randomized sequences and 20 nt primer sites (5 0 -TAMRA-GACAGGCAGGACACCGTAAC-N40-CTGCTACCTCCCTCCTCTTC-3 0 ).…”

Devices and approaches for generating specific high-affinity nucleic acid aptamers

“…A more conventional chromatographic device was recently demonstrated that uses pairs of on-chip microcolumns which overcome these sampling limitations and allow for multiplexing and simultaneous negative selections. 124 …”