Selection of aptamers based on a protein microarray integrated with a microfluidic chip

Abstract

We developed an efficient and fast method based on a protein microarray integrated with a microfluidic chip for the process of SELEX (systematic evolution of ligands by exponential enrichment). Lactoferrin from bovine milk was used as a target protein, while bovine serum albumin (BSA), α-lactalbumin, β-lactoglobulin and casein were used as negative proteins. They were separately dotted and immobilized to prepare the protein microarray and the resulting microarray was further integrated into a microfluidic chip for the SELEX (PMM-SELEX) process. The interaction between aptamer candidates and targets could be monitored using a fluorescence microarray scanner and the whole PMM-SELEX process was performed through seven-round selection. As a result, five aptamers (Lac-14, Lac-6a, Lac-9, Lac-5, Lac-3a) with high specificity and affinity can be repeatedly obtained during three times of independent repeated selection. Surface plasmon resonance (SPR) was used to calculate the dissociation constants (K). The aptamer Lac-6a was then used for detection of lactoferrin by fluorescence polarization. A linear response was observed for lactoferrin concentrations in the range of 0.78-50 μg mL and the detection limit was 0.39 μg mL. Thus, the innovative PMM-SELEX presented shows stability, accuracy and high efficiency for aptamer screening.

Rapid selection of aptamers based on protein microarray

“…Information about them is shown in Table S1. † Especially, the sequences from YFL-1 to YFL-14 were the same as the sequences in PMM-SELEX, 33 which showed the reliability of Microarray-SELEX.…”

“…SA-MPs and NaOH were used to turn PCR products to ssDNA according to our previous reports. 33 Only the sixth-round PCR product of the rst cycle selection was cloned by Sangon Bio-Technology.…”

“…Prior to this, our research group established two screening platforms, which were named microuidic-SELEX 33 and SPR-Imaging-SELEX. 34 First, microuidic-SELEX screened aptamers for lactoferrin by combining microuidics with a protein chip.…”

A systematic evolution of ligands by exponential enrichment workflow with consolidated counterselection to efficiently isolate high‐affinity aptamers

“…In this work, we describe a selection approach that builds off previous SELEX advancements, as well as incorporates a consolidated counterselection strategy, thereby generating high‐affinity and moderate specificity aptamers . We recently reported the design of a custom‐built multichannel peristaltic pump that could be used for this purpose, continuing on the theme of bringing novel ideas and low‐cost resources to the aptamer community .…”

Discovery of tumoricidal DNA oligonucleotides by response-directed in vitro evolution

“…Additional efficient modalities for aptamer selection include capillary electrophoresis [37][38][39] and magnetic sorting 40 . Electrophoresis and magnetic beads have been combined with microfluidics to create integrated systems for aptamer selection [41][42][43] . All these methods have demonstrated that aptamer selection could be significantly sped up and multiplexed.…”