Name: Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor (Video) | JoVE Journal
Uploaded: 2018-03-21T12:30:00.000+00:00
Duration: 9 min 33 s
Description: Learn to select and characterize group-specific DNA aptamers for hydrophobic small molecules using SELEX and electrochemical biosensor development. This protocol enables sensitive detection of challenging targets like phthalic acid esters.

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We are grateful for financial support from the National Natural Science Foundation (21675112), Key project of science and technology plan of Beijing Education Commission (KZ201710028027) and Yanjing Young Scholar Program of Capital Normal University.

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</ol>

The authors declare no competing financial interest.

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...

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 acid esters (PAEs)1,2, which all consist of many congeners. Small molecule detection has been mainly performed by chromatography/mass spectrometry-based techniques due to their diversity of applications3,4,5,6. For on-site detections, antibody-based methods have recently been developed7,8,9. However, since these methods are highly specific for a certain congener, multiple tests must be performed. What is more serious is that the novel congeners grow so fast that their antibodies can&#39;t be generated in time. Therefore, the development of group-specific biosensors to monitor the total levels of all congeners in one test may provide an invaluable metric for assessing environmental pollution status.
Recently, nucleic acid aptamers have been widely applied as recognition elements in various biosensing platforms due to their capability of recognizing a wide variety of targets, from ions and small molecules to proteins and cells10,11,12. Aptamers are identified through an in vitro method called systematic evolution of ligands by exponential enrichment (SELEX)13,14. SELEX begins with the random synthetic single strand oligonucleotide library, which contains approximately 1014-1015 sequences. The size of the random library ensures the diversity of the RNA or DNA candidate structures. The typical SELEX process consists of multiple rounds of enrichment until the library is enriched in sequences with high affinity and specificity to the target. The final enriched pool is then sequenced, and the dissociation constants (Kd) and selectivity against potential interfering substances are determined by different techniques such as filter binding, affinity chromatography, surface plasmon resonance (SPR), etc.15
Due to the extremely poor water solubility and lack of functional groups for surface immobilization, the aptamer selection of POPs is theoretically difficult. Significant advances for SELEX have speeded up the discovery of aptamers. However, the selection of group-specific aptamers for POPs has not yet been reported. So far, only PCB-binding DNA aptamers with high specificity for a certain congener have been identified16. PAEs are mainly used in polyvinyl chloride materials, changing polyvinyl chloride from a hard plastic to an elastic plastic, thus acting as a plasticizer. Some PAEs have been identified as endocrine disruptors, can cause serious damage to liver and kidney function, reduce the motility of male sperm, and may result in abnormal sperm morphology and testicular cancer17. Neither the compound- nor group-specific PAE-binding aptamers have been reported.
The goal of this work is to provide a representative protocol for selecting group-specific DNA aptamers to highly hydrophobic small molecules such as PAEs, a representative group of POPs. We also demonstrate the application of the selected aptamer for environmental pollution detection. This protocol provides guidance and insights for the aptamer discovery of other hydrophobic small molecules.

<table><tbody><tr><td>UV-2550</td><td>Shimadzu,Japan</td><td></td><td>protocol, section 3.8.2</td></tr><tr><td>DNA Engnine Thermal cycler,PTC0200</td><td>BIO-RAD</td><td></td><td>section 3.5.1.2 and 3.5.2</td></tr><tr><td>C1000 Touch</td><td>BIO-RAD</td><td></td><td>section 5.3.6 and 6.3</td></tr><tr><td>VMP3 multichannel potentiostat</td><td>Bio-Logic Science, Claix, France</td><td></td><td>section 7.4,7.8 and 7.11</td></tr><tr><td>Epoxy-activated Sepharose 6B</td><td>GE Healthcare (Piscataway, NJ, USA)</td><td>10220020</td><td>argarose beads, section 2.3 and 3.3</td></tr><tr><td>Dynabeads M-270 carboxylic acid magnetic beads</td><td>Invitrogen, USA</td><td>420420</td><td>magnetic beads,section 5.2. and 5.3</td></tr><tr><td>Premix Taq Hot Start Version</td><td>Takara,Dalian,China</td><td>R028A</td><td>polymerase, section 3.5.1.1</td></tr><tr><td>PARAFILM Sealing Membrane</td><td>Bemis, USA</td><td>PM-996</td><td>section 3.6.5</td></tr><tr><td>Lambda Exonuclease</td><td>Invitrogen, USA</td><td>EN0561</td><td>section3.7.1.2.The 10 &amp;times; reaction buffer is provided along with &amp;lambda; exonuclease by the provider.</td></tr><tr><td>Dr. GenTLE&lt;br/&gt; Precipitation Carrier</td><td>Takara,Dalian,China</td><td>9094</td><td>section 3.6.2 and 3.8.1</td></tr><tr><td>UNIQ-10 PAGE DNA recovery kit</td><td>Sangon Biotech (Shanghai)</td><td>B511135</td><td>section 4.2</td></tr><tr><td>SYBR Gold nucleic acid gel stain</td><td>Invitrogen, USA</td><td>1811838</td><td>nucelic acid stain dye, section 3.5.1.5</td></tr><tr><td>SYBR Premix Ex Taq II</td><td>Takara,Dalian,China</td><td>RR820A</td><td>polymerase mix contaning polymerase and dNTPs, section 5.3.5</td></tr><tr><td>2-(N-Morpholino)ethanesulfonic acid (MES)</td><td>Sigma-Aldrich</td><td>CAS: 1132-61-2</td><td>section 5.2.1</td></tr><tr><td>1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)</td><td>Invitrogen, USA</td><td>CAS: 25952-53-8</td><td>section 5.2.2</td></tr><tr><td>N-hydroxysuccinimide (NHS)</td><td>Sigma-Aldrich</td><td>6066-82-6</td><td>section 5.2.3</td></tr><tr><td>mercaptohexanol (MCH)</td><td>Sigma-Aldrich</td><td>CAS: 1633-78-9</td><td>section 7.7</td></tr><tr><td>Gold electrode</td><td>Shanghai Chenhua</td><td>CHI101</td><td>section 7.4. - 7.11</td></tr><tr><td>tris(2-carboxyethyl) phosphine hydrochloride (TCEP)</td><td>Sigma-Aldrich</td><td>CAS: 51805-45-9</td><td>section 7.5</td></tr><tr><td>O-(2-Mercaptoethyl)-O&#039;-methyl-hexa-(ethylene glycol)</td><td>Sigma-Aldrich</td><td>CAS: 651042-82-9</td><td>section 7.7</td></tr><tr><td>diethylhexyl phthalate (DEHP)</td><td>National Institute of Metrology, China</td><td>CAS: 117-81-7</td><td>section 7.11</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>CAS: 9005-64-5</td><td>polyoxyethy-lene(20) sorbaitan monolaurate</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>CAS: 9002-93-1</td><td>non-ionic surface active agent</td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>P5368</td><td>10 mM phosphate buffer containing 1 M NaCl, pH 7.4</td></tr></tbody></table>

phthalic acid ester-binding dna aptamer selection, characterization, and application to an electrochemical aptasensor

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

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.

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...

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.

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 ...

phthalic-acid-ester-binding-dna-aptamer-selection-characterization

Research

JoVE Journal

Chemistry

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions.  These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

Primer-Free Aptamer Selection Using A Random DNA Library

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes.  Here we present a general procedure for the fabrication and use of such sensors.

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental...

Detection of Bacteria Using Fluorogenic DNAzymes

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

The present protocol describes the use of Isothermal Titration Calorimetry (ITC) to analyze the association and dissociation kinetics of the binding between a DNA aptamer and tetracycline, including sample preparation, running standards and samples, and interpreting the resulting data.

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry

A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular...

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

The present protocol demonstrates the use of a fast, 3-stage, aptamer-based exponential amplification assay to detect targets. Sample preparation, signal amplification, and color development are covered to implement this system to recognize the presence of theophylline over that of caffeine.

Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction

This protocol outlines the design of split hybridization probes utilizing fluorescent light-up aptamer DAP-10-42 as a signal reporter and their application for nucleic acid detection and differentiation between targets with single-nucleotide substitutions.

Split Hybridization Probe Utilizing a DNA Fluorescent Light-up Aptamer as a Signal Reporter for Sequence-Specific Nucleic Acid Analysis

Learn to select and characterize group-specific DNA aptamers for hydrophobic small molecules using SELEX and electrochemical biosensor development. This protocol enables sensitive detection of challenging targets like phthalic acid esters.

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

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