Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

Summary

A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.

Abstract

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH₂) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH₂ attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.

Introduction

Along with rapid economic development, acceleration of industrialization, and urban construction, environmental pollution is more severe than ever. Typical environmental pollutants include heavy metal ions, toxins, antibiotics, pesticides, endocrine disruptors, and persistent organic pollutants (POPs). Besides metal ions and toxins, other pollutants are small molecules that quite often consist of a variety of congeners. For example, the most toxic POPs include polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), polybrominated biphenyl ethers (PBDEs), polychlorinated dibenzo-p-dioxin (PCDDs), polychlorinated dibenzofuran (PCDFs), and phthalic aci....

Protocol

1. Library and Primer Design and Synthesis

1. Design the initial library and primers.
   Library (Pool₀): 5'-TCCCACGCATTCTCCACATC-N40-CCTTTCTGTCCTTCCGTCAC-3'
   Forward primer (FP): 5'-TCCCACGCATTCTCCACATC-3'
   Phosphorylated reverse primer (PO₄-RP): 5'-PO₄-GTGACGGAAGGACAGAAAGG-3'
2. Synthesize Pool₀, FP, and PO₄-RP using standard phosphoramidite chemistry^18,19,20, and purify all the DNAs by standard high-performance liquid chromatography (HPLC)

Results

We designed and synthesized the amino group functionalized dibutyl phthalate (DBP-NH₂) as the anchor target (Figure 1F). We then performed the DNA aptamer selection of PAEs using DBP-NH₂ as the anchor target and following the classical target immobilization-based method (Figure 2). In each round, a pilot PCR was performed using the denatured PAGE to optimize the cycle number of PCR (Figu.......

Discussion

One outstanding benefit of aptamers is that they are identified through the in vitro method SELEX, while antibodies are generated via in vivo immunoreactions. Therefore, aptamers can be selected with desired target specificity under well-designed experimental conditions, whereas antibodies are limited to physiological conditions.

To facilitate the separation of bound sequences from free sequences, several modified SELEX have recently been reported, in which capillary electrop.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
UV-2550	Shimadzu,Japan		protocol, section 3.8.2
DNA Engnine Thermal cycler,PTC0200	BIO-RAD		section 3.5.1.2 and 3.5.2
C1000 Touch	BIO-RAD		section 5.3.6 and 6.3
VMP3 multichannel potentiostat	Bio-Logic Science, Claix, France		section 7.4,7.8 and 7.11
Epoxy-activated Sepharose 6B	GE Healthcare (Piscataway, NJ, USA)	10220020	argarose beads, section 2.3 and 3.3
Dynabeads M-270 carboxylic acid magnetic beads	Invitrogen, USA	420420	magnetic beads,section 5.2. and 5.3
Premix Taq Hot Start Version	Takara,Dalian,China	R028A	polymerase, section 3.5.1.1
PARAFILM Sealing Membrane	Bemis, USA	PM-996	section 3.6.5
Lambda Exonuclease	Invitrogen, USA	EN0561	section3.7.1.2.The 10 × reaction buffer is provided along with λ exonuclease by the provider.
Dr. GenTLE Precipitation Carrier	Takara,Dalian,China	9094	section 3.6.2 and 3.8.1
UNIQ-10 PAGE DNA recovery kit	Sangon Biotech (Shanghai)	B511135	section 4.2
SYBR Gold nucleic acid gel stain	Invitrogen, USA	1811838	nucelic acid stain dye, section 3.5.1.5
SYBR Premix Ex Taq II	Takara,Dalian,China	RR820A	polymerase mix contaning polymerase and dNTPs, section 5.3.5
2-(N-Morpholino)ethanesulfonic acid (MES)	Sigma-Aldrich	CAS: 1132-61-2	section 5.2.1
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)	Invitrogen, USA	CAS: 25952-53-8	section 5.2.2
N-hydroxysuccinimide (NHS)	Sigma-Aldrich	6066-82-6	section 5.2.3
mercaptohexanol (MCH)	Sigma-Aldrich	CAS: 1633-78-9	section 7.7
Gold electrode	Shanghai Chenhua	CHI101	section 7.4. - 7.11
tris(2-carboxyethyl) phosphine hydrochloride (TCEP)	Sigma-Aldrich	CAS: 51805-45-9	section 7.5
O-(2-Mercaptoethyl)-O'-methyl-hexa-(ethylene glycol)	Sigma-Aldrich	CAS: 651042-82-9	section 7.7
diethylhexyl phthalate (DEHP)	National Institute of Metrology, China	CAS: 117-81-7	section 7.11
Tween 20	Sigma-Aldrich	CAS: 9005-64-5	polyoxyethy-lene(20) sorbaitan monolaurate
Triton X-100	Sigma-Aldrich	CAS: 9002-93-1	non-ionic surface active agent
PBS	Sigma-Aldrich	P5368	10 mM phosphate buffer containing 1 M NaCl, pH 7.4