Capture-SELEX: Selection Strategy, Aptamer Identification, and Biosensing Application

Abstract

Small-molecule contaminants, such as antibiotics, pesticides, and plasticizers, have emerged as one of the substances most detrimental to human health and the environment. Therefore, it is crucial to develop low-cost, user-friendly, and portable biosensors capable of rapidly detecting these contaminants. Antibodies have traditionally been used as biorecognition elements. However, aptamers have recently been applied as biorecognition elements in aptamer-based biosensors, also known as aptasensors. The systematic evolution of ligands by exponential enrichment (SELEX) is an in vitro technique used to generate aptamers that bind their targets with high affinity and specificity. Over the past decade, a modified SELEX method known as Capture-SELEX has been widely used to generate DNA or RNA aptamers that bind small molecules. In this review, we summarize the recent strategies used for Capture-SELEX, describe the methods commonly used for detecting and characterizing small-molecule-aptamer interactions, and discuss the development of aptamer-based biosensors for various applications. We also discuss the challenges of the Capture-SELEX platform and biosensor development and the possibilities for their future application.

Keywords: DNA and RNA Capture-SELEX; aptamer; biosensing applications; characterization; nucleic acid aptamer; small molecule contaminant.

Figure 1

The experimental flowchart of DNA Capture-SELEX and RNA Capture-SELEX. (A) DNA Capture-SELEX. Each DNA Capture-SELEX selection round consists of 7 main steps: (1) The designed ssDNA library template is hybridized with biotinylated capture-oligonucleotide. (2) The hybridized product is immobilized on the washed streptavidin-coated magnetic beads. (3–4) Target-bound sequence elution: counter selection is followed by positive selection. (5) PCR amplification was performed on the eluted ssDNA to obtain the amplified dsDNA pool, (6) and regenerate ssDNA for the next DNA Capture-SELEX round (either gel purification or magnetic bead-based method). (7) After DNA Capture-SELEX, aptamer sequences were identified through sequencing. (B) RNA Capture-SELEX. The principles and procedures are similar to (A), except that RNA Capture-SELEX consists of additional transcription (step 1) to convert DNA to RNA and reverse transcription (step 6) to convert RNA to DNA steps.

Figure 2

A schematic illustration of six commonly used methods for detecting interactions between small molecule and aptamer. (A) SYBR green I (SGI) assay. (B) Carbon nanoparticles (CNPs) fluorescence quenching assay. (C) Gold nanoparticles (AuNPs) colorimetric assay. (D) Microscale thermophoresis (MST) assay. (E) Graphene oxide (GO)-based fluorescent assay. (F) Isothermal titration calorimetry (ITC) assay.

Figure 3

The schematic illustration of aptasensors for the detection of small molecules using novel aptamer generated from Capture-SELEX. (A) Biolayer interferometry (BLI)-based aptasensor. (B) A lateral flow aptasensor (LFA) is generally constructed by 4 sections: sample pad, conjugate pad, nitrocellulose membrane with test line and control line, and absorbent pad. The sample flow from left to right laterally (left). When the target is loaded onto the sample pad, positive result (red band for both control and test line) is observed after 15 min (right).