Name: phthalic acid ester-binding dna aptamer selection, characterization, and application to an electrochemical aptasensor
Uploaded: 2018-03-21T12:30:00
Description: Watch this Scientific Journal Video about Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor at JoVE.com

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.

1. Library and Primer Design and Synthesis
<ol>
	<li>Design the initial library and primers. 
	Library (Pool0): 5&#39;-TCCCACGCATTCTCCACATC-N40-CCTTTCTGTCCTTCCGTCAC-3&#39; 
	Forward primer (FP): 5&#39;-TCCCACGCATTCTCCACATC-3&#39; 
	Phosphorylated reverse primer (PO4-RP): 5&#39;-PO4-GTGACGGAAGGACAGAAAGG-3&#39;</li>
	<li>Synthesize Pool0, FP, and PO4-RP using standard phosphoramidite chemistry18,<sup ...

<ol>
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Acta. 686 (1-2), 9-18 (2011).</li><li>Mehta, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+and+characterization+of+PCB-binding+DNA+aptamers.">Selection and characterization of PCB-binding DNA aptamers.</a> Anal Chem. 84 (3), 1669-1676 (2012).</li><li>Matsumoto, M., Hirata-Koizumi, M., Ema, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+adverse+effects+of+phthalic+acid+esters+on+human+health:+A+review+of+recent+studies+on+reproduction.">Potential adverse effects of phthalic acid esters on human health: A review of recent studies on reproduction.</a> Regul Toxicol Pharm. 50 (1), 37-49 (2008).</li><li>Goodchild, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugates+of+oligonucleotides+and+modified+oligonucleotides:+a+review+of+their+synthesis+and+properties.">Conjugates of oligonucleotides and modified oligonucleotides: a review of their synthesis and properties.</a> Bioconjug Chem. 1 (3), 165-187 (1990).</li><li>Brown, D. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligo-and+poly-nucleotides:+50+years+of+chemical+synthesis.">Oligo-and poly-nucleotides: 50 years of chemical synthesis.</a> Org Biomol Chem. 3 (21), 3851-3868 (2005).</li><li>Sproat, B., Colonna, F., Mullah, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+the+isolation+and+purification+of+oligoribonucleotides.">An efficient method for the isolation and purification of oligoribonucleotides.</a> Nucleos Nucleot Nucl. 14 (1-2), 255-273 (1995).</li><li>Han, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+group-specific+phthalic+acid+esters+binding+DNA+aptamers+via+rationally+designed+target+immobilization+and+applications+for+ultrasensitive+and+highly+selective+detection+of+phthalic+acid+esters.">Selection of group-specific phthalic acid esters binding DNA aptamers via rationally designed target immobilization and applications for ultrasensitive and highly selective detection of phthalic acid esters.</a> Anal Chem. 89 (10), 5270-5277 (2017).</li><li>Bartlett, J. M. S., Stirling, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+short+history+of+the+polymerase+chain+reaction.">A short history of the polymerase chain reaction.</a> PCR protocols. , 3-6 (2003).</li><li>Saiki, R. K., Scharf, S., Faloona, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+amplification+of+beta-globin+genomic+sequences+and+restriction+site+analysis+for+diagnosis+of+sickle+cell+anemia.">Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.</a> Science. 230, 1350-1354 (1985).</li><li>Albright, L. M., Slatko, B. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+measuring+aptamer-protein+equilibria:+a+review.">Methods for measuring aptamer-protein equilibria: a review.</a> Anal Chim Acta. 686 (1), 9-18 (2011).</li><li>Sharma, T. K., Bruno, J. G., Dhiman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABCs+of+DNA+aptamer+and+related+assay+development.">ABCs of DNA aptamer and related assay development.</a> Biotechnol Adv. 35 (2), 275-301 (2017).</li><li>Liu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling-probe+displacement+electrochemical+aptamer-based+sensor+(SD-EAB)+for+detection+of+nanomolar+kanamycin+A.">Signaling-probe displacement electrochemical aptamer-based sensor (SD-EAB) for detection of nanomolar kanamycin A.</a> Electrochim Acta. 182, 516-523 (2015).</li><li>Mendonsa, S. D., Bowser, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+evolution+of+functional+DNA+using+capillary+electrophoresis.">In vitro evolution of functional DNA using capillary electrophoresis.</a> J Am Chem Soc. 126, 20-21 (2004).</li><li>Lou, X. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromagnetic+selection+of+aptamers+in+microfluidic+channels.">Micromagnetic selection of aptamers in microfluidic channels.</a> Proc Natl Acad Sci USA. 106 (9), 2989-2994 (2009).</li><li>Cho, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+selection+of+DNA+aptamers+through+microfluidic+selection+and+high-throughput+sequencing.">Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 15373-15378 (2010).</li><li>Cho, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+selection+and+parallel+characterization+of+aptamers.">Quantitative selection and parallel characterization of aptamers.</a> Proc Nat Acad Sci USA. 110 (46), 18460-18465 (2013).</li><li>Song, K. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanoparticle-based+colorimetric+detection+of+kanamycin+using+a+DNA+aptamer.">Gold nanoparticle-based colorimetric detection of kanamycin using a DNA aptamer.</a> Anal Biochem. 415 (2), 175-181 (2011).</li><li>Yang, Z., Ding, X., Guo, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+generation+of+signaling-probe+displacement+electrochemical+aptasensor+for+detection+of+picomolar+ampicillin+and+sulfadimethoxine.">Second generation of signaling-probe displacement electrochemical aptasensor for detection of picomolar ampicillin and sulfadimethoxine.</a> Sens Actuators B. 253 (2017), 1129-1136 (2017).</li><li>Lou, X., Zhao, T., Liu, R., Ma, J., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembled+DNA+monolayer+buffered+dynamic+ranges+of+mercuric+electrochemical+sensor.">Self-assembled DNA monolayer buffered dynamic ranges of mercuric electrochemical sensor.</a> Anal Chem. 85 (15), 7574-7580 (2013).</li><li>Zhao, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoprobe-enhanced,+split+aptamer-based+electrochemical+sandwich+assay+for+ultrasensitive+detection+of+small+molecules.">Nanoprobe-enhanced, split aptamer-based electrochemical sandwich assay for ultrasensitive detection of small molecules.</a> Anal Chem. 87 (15), 7712-7719 (2015).</li><li>Lou, X., He, L. 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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 &#215; reaction buffer is provided along with &#955; exonuclease by the provider.</td>
		</tr>
		<tr>
			<td>Dr. GenTLE 
			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&#39;-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 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...

Watch this Scientific Journal Video about Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor at JoVE.com

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

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 ...

The overall goal of this procedure is to select group-specific DNA aptamers to highly hydrophobic, small molecules and to use the selected aptamer to develop a sensitive electrochemical biosensor. This method can help to answer key questions in the aptamer selection field, about how to select and characterize group-specific aptamers when targets are highly hydrophobic molecules. While the most useful part of this method is the development of electrochemical biosensors, for the detection of titles.These biosensors should also work for our aptamer affinity measurements. To begin the reaction, add 100 microliters of RNase-Free water to the purified aptamer PCR product. Vortex the mixture until the precipitate has dissolved.Lambda exonuclease reaction is sensitive to the salt concentration and is a product should be proliferated by ethanol precipitation but another isopropanol. Place into each of five micro-centrifuge tubes, five microliters of the aptamer solution, 11 microliters of Rnase free water and two microliters of 10x reaction buffer. Add two microliters of Rnase free water and solutions of two, five, eight, and 10 units of lambda exonuclease in Rnase free water to one tube each.Mix well with gentle pipetting. Incubate the tubes at 37 degrees Celsius for 35 minutes and at 80 degree Celsius for 15 minutes. Then hold the tubes at four degree Celsius and prepare a 12%native page gel.Add to each tube one microliter of loading dye and four microliters of Rnase free water. Run the mixtures in the native page gel at 150 volts for 45 minutes. Identify the lowest amount of lambda exonuclease needed to completely generate single stranded DNA.Perform a large scale reaction to generate single-stranded DNA. For each binding target to be tested, incubate 10 microliters of mean functionalized DBP coded magnetic beads with 500 microliters of one micromolar solution of DBP-1 for one hour at room temperature while rotating. Then discard the supernatant.Wash the beads four times with 200 microliters portions of PAE binding buffer and resuspend the beads in 10 microliters of PAE binding buffer. Obtain 10 micromolar binding target test dispersions in PAE binding buffer. It's great go to dispersion of the hydrophobic targets in PAE binding buffer.To ensure that, there is enrichment of the library, were selected under affinity tests. Add 10 microliters of DBP-1 coded beads to 110 microliters of each target test solution. Incubate the mixtures for one hour and collect the supernatants by magnetic separation.Dilute the supernatants 100 fold and use three microliters for each quantitative PCR. Calculate the relative affinities by dividing the number of DBP-1 released in the presence of the test sample by the number of DBP-1 released in PAE binding buffer alone. Prior to performing the measurements synthesize and purify the DBP-1 based thiolated core sequence probe and the signaling probe.Reconstitute the thiolated probe and the signaling probe and as 100 micromolar solutions in nuclease free water. Next polish a two millimeter diameter gold electrode to a mirror like surface with one, 0.3, and 0.5 micron alumina powder and a microfiber cloth for five minutes each. Sonicate the electrode in ultra pure water for five minutes after each polishing.Immerse the polished electrode in a 0.5 molar solution of sulfuric acid. Clean the electrode with 35 successive cyclic voltammetry scans from minus 0.4 to positive 1.2 volts versus mercury calomel at 100 millivolts per second. Next prepare in a thin walled centrifuge tube a mixture of 0.5 micromolar thiolated probe DBP-1.And 0.5 micromolar FC modified DBP-1 in 100 microliters of PBS. Heat the mixture in a water bath set to 95 degrees Celsius for 10 minutes. Then allow the mixture to cool to room temperature in the water bath.Add one microliter of a 10 millimolar TCEP stock solution to the cooled mixture and maintain at room temperature for one hour. Then immerse the clean gold electrode in the mixture for 12 hours at room temperature. Rinse the electrode with PBS and then immerse the electrode in a one millimolar solution of thiolated PEG in PBS for one hour.Thoroughly rinse the electrode with target free PAE binding buffer and immerse the electrode in the buffer. Next sonicate electrolytic cells of platinum counter electrode and a saturated calomel reference electrode for two minutes each in ultra pure water and PAE binding buffer in sequence. Open the square wave of voltammetry instrument software and enter the experiment parameters.Fill a clean electrolytic cell with target free PAE binding buffer. Connect the three clean electrodes to a potentiostat and immerse the electrodes in the buffer. Acquire a background SWV scan.Then immerse the gold working electrode in a 10 picamolar DEHP solution in PAE binding buffer for 30 minutes at room temperature. Thoroughly rinse the electrode with PAE binding buffer. Immerse all three electrodes in fresh PAE binding buffer and collect another SWV curve using the same parameters as before.Repeat this process with increasing concentrations of DEHP to obtain a titration curve. The minimum amount of lambda exonuclease necessary to obtain only single strand DNA was found to be two units based on small scale reactions. The aptamer candidate DBP-1 was identified by SELEX followed by high throughput sequencing.DBP-1 showed good group specificity to PAE congeners. An electrochemical aptasensor using DBP-1 selectively responded to DEHP over other common environmental pollutants. The aptasensor was highly sensitive to DEHP with the response at concentrations as low as 10 picamolar.Laser particle where selection and characterization of aptamers for hazard hydrophobic small molecules. Characterization and volatilization of aptamers of hydrophobic small molecules like PAE is generally quite challenging, due to the extremely limited water solubility and the low molecule weight of the PAEs. After watching this video, you should have a good understanding of how to make an electrochemical aptasensors and how to use it to detect the titles.

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