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.

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

Research

JoVE Journal

Chemistry

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in understanding contaminant mass transport and the ability to decontaminate materials. If a material is contaminated, over time, the transport of highly toxic chemicals (such as chemical warfare agent species) out of the material can result in vapor exposure or transfer to the skin, which can result in percutaneous exposure to personnel who interact with the material. Due to the high toxicity of chemical warfare agents, the release of trace chemical quantities is of significant concern. Mapping subsurface concentration distribution and transport characteristics of absorbed agents enables exposure hazards to be assessed in untested conditions. Furthermore, these tools can be used to characterize subsurface reaction dynamics to ultimately design improved decontaminants or decontamination procedures. To achieve this goal, an inverse analysis mass transport modeling approach was developed that utilizes time-resolved mass spectroscopy measurements of vapor emission from contaminated paint coatings as the input parameter for calculation of subsurface concentration profiles. Details are provided on sample preparation, including contaminant and material handling, the application of mass spectrometry for the measurement of emitted contaminant vapor, and the implementation of inverse analysis using a physics-based diffusion model to determine transport properties of live chemical warfare agents including distilled mustard (HD) and the nerve agent VX.

In this paper, a procedure for quantifying the mass transport parameters of chemicals in various materials is presented. This process involves employing an inverse-analysis based diffusion model to vapor emission profiles recorded by real-time, mass spectrometry in high vacuum.

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in ...

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

An Aptamer-based Sensor for Unchelated Gadolinium(III)

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples of gadolinium-based contrast agents as a result of incomplete reactions between the ligand and the ion, or as a dissociation product. Since the ion is toxic, its detection is of critical importance. Herein, the design and usage of an aptamer-based sensor (Gd-sensor) for Gd3+ are described. The sensor produces a fluorescence change in response to increasing concentrations of the ion, and has a limit of detection in the nanomolar range (~100 nM with a signal-to-noise ratio of 3). The assay may be run in an aqueous buffer at ambient pH (~7 - 7.4) in a 384-well microplate. The sensor is relatively unreactive toward other physiologically relevant metal ions such as sodium, potassium, and calcium ions, although it is not specific for Gd3+ over other trivalent lanthanides such as europium(III) and terbium(III). Nevertheless, the lanthanides are not commonly found in contrast agents or the biological systems, and the sensor may therefore be used to selectively determine unchelated Gd3+ in aqueous conditions.

An Aptamer-based Sensor for Unchelated GadoliniumIII

The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor.

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples ...

Electrochemical Impedance Spectroscopy as a Tool for Electrochemical Rate Constant Estimation

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). In the case of fast reversible electrochemical processes, current is predominantly affected by the rate of diffusion, which is the slowest and limiting stage. EIS is a powerful technique that allows separate analysis of stages of charge transfer that have different AC frequency response. The capability of the method was used to extract the value of charge transfer resistance, which characterizes the rate of charge exchange on the electrode-solution interface. The application of this technique is broad, from biochemistry up to organic electronics. In this work, we are presenting the method of analysis of organic compounds for optoelectronic applications.

Electrochemical impedance spectroscopy (EIS) of species that undergo reversible oxidation or reduction in solution was used for determination of rate constants of oxidation or reduction.

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). ...

An Electrochemical Cholesteric Liquid Crystalline Device for Quick and Low-Voltage Color Modulation

We demonstrate a method for fabricating a prototype reflective display device that contains cholesteric liquid crystal (LC) as an active component. The cholesteric LC is composed of a nematic LC 4&#39;-pentyloxy-4-cyanobiphenyl (5OCB), redox-responsive chiral dopant (FcD), and a supporting electrolyte 1-ethyl-3-methylimidazolium trifluoromethanesulfonate (EMIm-OTf). The most important component is FcD. This molecule changes its helical twisting power (HTP) value in response to redox reactions. Therefore, in situ electrochemical redox reactions in the LC mixture allow for the device to change its reflection color in response to electrical stimuli. The LC mixture was introduced, by a capillary action, into a sandwich-type ITO glass cell comprising two glass slides with patterned indium tin oxide (ITO) electrodes, one of which was coated with poly(3,4-ethylenedioxythiophene)-co-poly(ethylene glycol) doped with perchlorate (PEDOT+). Upon application of +1.5 V, the reflection color of the device changed from blue (467 nm) to green (485 nm) in 0.4 s. Subsequent application of 0 V made the device recover the original blue color in 2.7 s. This device is characterized by its fastest electrical response and lowest operating voltage among any previously reported cholesteric LC device. This device could pave the way for the development of next generation reflective displays with low energy consumption rates.

A protocol for the fabrication of a reflective cholesteric liquid crystalline display device containing a redox-responsive chiral dopant allowing quick and low-voltage operation is presented.

We demonstrate a method for fabricating a prototype reflective display device that contains cholesteric liquid crystal (LC) as an active component. The ...

Electrochemical Roughening of Thin-Film Platinum Macro and Microelectrodes

This protocol demonstrates a method for electrochemical roughening of thin-film platinum electrodes without preferential dissolution at grain boundaries of the metal. Using this method, a crack free, thin-film macroelectrode surface with up to 40 times increase in active surface area was obtained. The roughening is easy to do in a standard electrochemical characterization laboratory and incudes the application of voltage pulses followed by extended application of a reductive voltage in a perchloric acid solution. The protocol includes the chemical and electrochemical preparation of both a macroscale (1.2 mm diameter) and microscale (20 &#181;m diameter) platinum disc electrode surface, roughening the electrode surface and characterizing the effects of surface roughening on electrode active surface area. This electrochemical characterization includes cyclic voltammetry and impedance spectroscopy and is demonstrated for both the macroelectrodes and the microelectrodes. Roughening increases electrode active surface area, decreases electrode impedance, increases platinum charge injection limits to those of titanium nitride electrodes of same geometry and improves substrates for adhesion of electrochemically deposited films.

This protocol demonstrates a method for electrochemical roughening of thin-film platinum electrodes without preferential dissolution at grain boundaries. The electrochemical techniques of cyclic voltammetry and impedance spectroscopy are demonstrated to characterize these electrode surfaces.

This protocol demonstrates a method for electrochemical roughening of thin-film platinum electrodes without preferential dissolution at grain boundaries ...

Asymmetric Thermoelectrochemical Cell for Harvesting Low-grade Heat under Isothermal Operation

Low-grade heat is abundantly available in the environment as waste heat. The efficient conversion of low-grade heat into electricity is very difficult. We developed an asymmetric thermoelectrochemical cell (aTEC) for heat-to-electricity conversion under isothermal operation in the charging and discharging processes without exploiting the thermal gradient or the thermal cycle. The aTEC is composed of a graphene oxide (GO) cathode, a polyaniline (PANI) anode, and 1M KCl as the electrolyte. The cell generates a voltage due to the pseudocapacitive reaction of GO when heating from room temperature (RT) to a high temperature (TH, ~40-90 &#176;C), and then current is successively produced by oxidizing PANI when an external electrical load is connected. The aTEC demonstrates a remarkable temperature coefficient of 4.1 mV/K and a high heat-to-electricity conversion efficiency of 3.32%, working at a TH = 70 &#176;C with a Carnot efficiency of 25.3%, unveiling a new promising thermoelectrochemical technology for low-grade heat recovery.

Low-grade heat is abundant, but its efficient recovery is still a great challenge. We report an asymmetric thermoelectrochemical cell using graphene oxide as a cathode and polyaniline as an anode with KCl as the electrolyte. This cell works under isothermal heating, exhibiting a high heat-to-electricity conversion efficiency in low-temperature regions.

Low-grade heat is abundantly available in the environment as waste heat. The efficient conversion of low-grade heat into electricity is very difficult. We ...

Probing Surface Electrochemical Activity of Nanomaterials using a Hybrid Atomic Force Microscope-Scanning Electrochemical Microscope (AFM-SECM)

Scanning electrochemical microscopy (SECM) is used to measure the local electrochemical behavior of liquid/solid, liquid/gas and liquid/liquid interfaces. Atomic force microscopy (AFM) is a versatile tool to characterize micro- and nanostructure in terms of topography and mechanical properties. However, conventional SECM or AFM provides limited laterally resolved information on electrical or electrochemical properties at nanoscale. For instance, the activity of a nanomaterial surface at crystal facet levels is difficult to resolve by conventional electrochemistry methods. This paper reports the application of a combination of AFM and SECM, namely, AFM-SECM, to probe nanoscale surface electrochemical activity while acquiring high-resolution topographical data. Such measurements are critical to understanding the relationship between nanostructure and reaction activity, which is relevant to a wide range of applications in material science, life science and chemical processes. The versatility of the combined AFM-SECM is demonstrated by mapping topographical and electrochemical properties of faceted nanoparticles (NPs) and nanobubbles (NBs), respectively. Compared to previously reported SECM imaging of nanostructures, this AFM-SECM enables quantitative assessment of local surface activity or reactivity with higher resolution of surface mapping.

Probing Surface Electrochemical Activity of Nanomaterials using a Hybrid Atomic Force Microscope-Scanning Electrochemical Microscope AFM-SECM

Atomic force microscopy (AFM) combined with scanning electrochemical microscopy (SECM), namely, AFM-SECM, can be used to simultaneously acquire high-resolution topographical and electrochemical information on material surfaces at nanoscale. Such information is critical to understanding heterogeneous properties (e.g., reactivity, defects, and reaction sites) on local surfaces of nanomaterials, electrodes and biomaterials.

Scanning electrochemical microscopy (SECM) is used to measure the local electrochemical behavior of liquid/solid, liquid/gas and liquid/liquid interfaces. ...