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

Vi er taknemmelige for finansiel støtte fra National Natural Science Foundation (21675112), Key projekt af videnskab og teknologi plan af Beijing uddannelse Kommissionen (KZ201710028027) og Yanjing Young Scholars Program af kapital Normal University.

1. bibliotek og Primer Design og syntese
<ol><li>
Designe det første bibliotek og primere. Bibliotek (Pool0): 5'-TCCCACGCATTCTCCACATC-N40-CCTTTCTGTCCTTCCGTCAC-3' Videresende primer (FP): 5'-TCCCACGCATTCTCCACATC-3' Fosforyleret omvendt primer (PO4- RP): 5'-PO4- GTGACGGAAGGACAGAAAGG-3'</li>
	<li>Syntetisere Pool0, FP og PO4- RP ved hjælp af standard phosphoramidite kemi18,19,<...

<ol>
	<li>Vorkamp, K., Riget, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+new+and+current-use+contaminants+in+the+Arctic+environment:+Evidence+of+long-range+transport+and+indications+of+bioaccumulation.">A review of new and current-use contaminants in the Arctic environment: Evidence of long-range transport and indications of bioaccumulation.</a> Chemosphere. 111, 379-395 (2014).</li><li>Net, S., Sempere, R., Delmont, A., Paluselli, A., Ouddane, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence,+fate,+behavior+and+ecotoxicological+state+of+phthalates+in+different+environmental+matrices.">Occurrence, fate, behavior and ecotoxicological state of phthalates in different environmental matrices.</a> Environ Sci Technol. 49 (7), 4019-4035 (2015).</li><li>Xie, Q. L., Liu, S. H., Fan, Y. Y., Sun, J. Z., Zhang, X. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+phthalate+esters+in+edible+oils+by+use+of+QuEChERS+coupled+with+ionic-liquid-based+dispersive+liquid-liquid+microextraction+before+high-performance+liquid+chromatography.">Determination of phthalate esters in edible oils by use of QuEChERS coupled with ionic-liquid-based dispersive liquid-liquid microextraction before high-performance liquid chromatography.</a> Anal BioanalChem. 406 (18), 4563-4569 (2014).</li><li>Ierapetritis, I., Lioupis, A., Lampi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+phthalates+into+vegetable+oils+by+isotopic+dilution+gas+chromatography+mass+spectrometry.">Determination of phthalates into vegetable oils by isotopic dilution gas chromatography mass spectrometry.</a> Food Anal Methods. 7 (7), 1451-1457 (2014).</li><li>Sun, J. Z., He, H., Liu, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+phthalic+acid+esters+in+Chinese+white+spirit+using+dispersive+liquid-liquid+microextraction+coupled+with+sweeping+beta-cyclodextrin-modified+micellar+electrokinetic+chromatography.">Determination of phthalic acid esters in Chinese white spirit using dispersive liquid-liquid microextraction coupled with sweeping beta-cyclodextrin-modified micellar electrokinetic chromatography.</a> J Sep Sci. 37 (13), 1679-1686 (2014).</li><li>Yilmaz, P. K., Ertas, A., Kolak, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+determination+of+seven+phthalic+acid+esters+in+beverages+using+ultrasound+and+vortex-assisted+dispersive+liquid-liquid+microextraction+followed+by+high-performance+liquid+chromatography.">Simultaneous determination of seven phthalic acid esters in beverages using ultrasound and vortex-assisted dispersive liquid-liquid microextraction followed by high-performance liquid chromatography.</a> J Sep Sci. 37 (16), 2111-2117 (2014).</li><li>Sun, R., Zhuang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultrasensitive+gold+nanoparticles+improved+real-time+immuno-PCR+assay+for+detecting+diethyl+phthalate+in+foodstuff+samples.">An ultrasensitive gold nanoparticles improved real-time immuno-PCR assay for detecting diethyl phthalate in foodstuff samples.</a> Anal Biochem. 480, 49-57 (2015).</li><li>Sun, R. Y., Zhuang, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+heterogeneous+biotin-streptavidin+enzyme-linked+immunosorbent+assay+for+the+determination+of+di-(2-ethylhexyl)phthalate+(DEHP)+in+beverages+using+a+specific+polyclonal+antibody.">A sensitive heterogeneous biotin-streptavidin enzyme-linked immunosorbent assay for the determination of di-(2-ethylhexyl)phthalate (DEHP) in beverages using a specific polyclonal antibody.</a> Anal Methods. 6 (24), 9807-9815 (2014).</li><li>Zhou, L., Lei, Y., Zhang, D., Ahmed, S., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultra-sensitive+monoclonal+antibody-based+enzyme-linked+immunosobent+assay+for+dibutyl+phthalate+in+human+urinary.">An ultra-sensitive monoclonal antibody-based enzyme-linked immunosobent assay for dibutyl phthalate in human urinary.</a> Sci Total Environ. 541, 570-578 (2016).</li><li>Shen, J., Li, Y., Gu, H., Xia, F., Zuo, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+development+of+sandwich+assay+based+on+the+nanobiotechnologies+for+proteins,+nucleic+Acids,+small+Molecules,+and+ions.">Recent development of sandwich assay based on the nanobiotechnologies for proteins, nucleic Acids, small Molecules, and ions.</a> Chem Rev. 114 (15), 7631-7677 (2014).</li><li>Yin, X. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+nucleic+acids+for+electrochemical+and+electrochemiluminescent+sensing+applications.">Functional nucleic acids for electrochemical and electrochemiluminescent sensing applications.</a> TrAC, Trends Anal Chem. 33, 81-94 (2012).</li><li>Nguyen, V. -. T., Kwon, Y. S., Gu, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamer-based+environmental+biosensors+for+small+molecule+contaminants.">Aptamer-based environmental biosensors for small molecule contaminants.</a> Curr Opin Biotechnol. 45, 15-23 (2017).</li><li>Groher, F., Suess, B. <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+selection+of+antibiotic-binding+aptamers.">In vitro selection of antibiotic-binding aptamers.</a> Methods. 106, 42-50 (2016).</li><li>Yang, K. -. A., Pei, R., Stojanovic, M. N. <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+selection+and+amplification+protocols+for+isolation+of+aptameric+sensors+for+small+molecules.">In vitro selection and amplification protocols for isolation of aptameric sensors for small molecules.</a> Methods. 106, 58-65 (2016).</li><li>Jing, M., 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=Methods+for+measuring+aptamer-protein+equilibria:+a+review.">Methods for measuring aptamer-protein equilibria: a review.</a> Anal. Chim. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+history+of+oligonucleotide+synthesis.">A brief history of oligonucleotide synthesis.</a> Protocols for Oligonucleotides and Analogs: Synthesis and Properties. , 1-17 (1993).</li><li>Reese, C. 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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denaturing+polyacrylamide+gel+electrophoresis.">Denaturing polyacrylamide gel electrophoresis.</a> Current Protocols in Nucleic Acid Chemistry. , A. 3B 1-A. 3B 5 (2001).</li><li>Summer, H., Gr&#228;mer, R., Dr&#246;ge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denaturing+urea+polyacrylamide+gel+electrophoresis+(Urea+PAGE).">Denaturing urea polyacrylamide gel electrophoresis (Urea PAGE).</a> JoVE. (32), e1485 (2009).</li><li>Jing, M., 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=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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+passivation+using+oligo(ethylene+glycol)+in+ATRP-assisted+DNA+detection.">Surface passivation using oligo(ethylene glycol) in ATRP-assisted DNA detection.</a> Sens Actuators,B. 129 (1), 225-230 (2008).</li></ol>

En enestående fordel ved aptamers er, at de er identificeret gennem in vitro- metoden SELEX, mens antistoffer er genereret via i vivo immunoreactions. Derfor kan aptamers vælges med ønskede mål specificitet under veldesignet forsøgsbetingelser, antistoffer er begrænset til fysiologiske tilstande.
For at lette adskillelsen af bundne sekvenser fra gratis sekvenser, flere modificerede SELEX er for nylig blevet rapporteret, i hvilke kapillær elektroforese...

Sammen med hurtig økonomisk udvikling er acceleration af industrialisering og bybygning, miljøforurening mere alvorlige end nogensinde. Typiske miljøforurenende omfatter heavy metal ioner, toksiner, antibiotika, pesticider, endokrine disruptorer og persistente organiske miljøgifte (pop). Udover metal-ioner og toksiner, andre forurenende stoffer er små molekyler der ganske ofte består af en række forskellige kongenere. For eksempel, de mest giftige POP'er omfatter polychlorerede biphenyler (PCB), polycykliske aromatiske kulbrinter (PAH), polybromerede diphenyl ethere (PBDE), polychlorerede dibenzo-p-dioxiner (PCDD), polychlorerede dibenzofuraner (PCDF), og phthalic Acid estere (PAEs)1,2, som alle består af mange kongenere. Lille molekyle opdagelse er blevet primært udført af kromatografi/masse massespektrometri-baseret teknikker på grund af deres mangfoldighed af programmer3,4,5,6. For on-site opdagelser, er antistof-baserede metoder for nylig blevet udviklet7,8,9. Men da disse metoder er meget specifikke for en bestemt kongeners, flere prøver skal udføres. Hvad er mere alvorligt er at de roman kongenere vokser så hurtigt at deres antistoffer ikke kan oprettes i tid. Derfor kan udviklingen af gruppespecifikke biosensorer til at overvåge de samlede niveauer af alle kongenere i én test udgør en uvurderlig metrikværdi for at vurdere miljøforurening status.
For nylig, nukleinsyre aptamers er blevet bredt anvendt som anerkendelse elementer i forskellige biosensing platforme på grund af deres evne til erkendelse af en bred vifte af mål, fra ioner og små molekyler til proteiner og celler10,11 ,12. Aptamers er identificeret gennem en in vitro- metode, der kaldes systematisk udvikling af ligander af eksponentielle berigelse (SELEX)13,14. SELEX begynder med tilfældige syntetiske enkelt streng oligonukleotid bibliotek, som indeholder ca 1014-1015 sekvenser. Størrelsen af den tilfældige bibliotek sikrer mangfoldighed af RNA eller DNA kandidat strukturerne. Den typiske SELEX proces består af flere runder af berigelse indtil biblioteket er beriget i sekvenser med høj affinitet og specificitet til målet. Den endelige beriget pool er derefter sekventeret, og dissociation konstanter (Kd) og selektivitet mod potentielle interfererende stoffer bestemmes ved hjælp af forskellige teknikker såsom filter bindende affinitet kromatografi, overflade Plasmon resonans (SPR), osv. 15
På grund af ekstremt fattige vandopløselighed og manglen på funktionelle grupper for overflade immobilisering er aptamer Udvalget af dukker teoretisk vanskeligt. Betydelige fremskridt for SELEX har fremskyndet opdagelsen af aptamers. Dog har udvalg af gruppe-specifikke aptamers for pop ikke endnu blevet rapporteret. Hidtil har kun PCB-bindende DNA aptamers med høj specificitet for en bestemt kongeners blevet identificeret16. PAEs anvendes hovedsageligt i polyvinylchlorid materialer, skiftende polyvinylchlorid fra en hård plast til en elastisk plast, dermed fungere som en blødgører. Nogle PAEs er blevet identificeret som hormonforstyrrende stoffer, kan forårsage alvorlige skader til lever- og nyrefunktion, reducere motilitet af mandlige sædceller, og kan resultere i abnorm sperm morfologi og testikelkræft17. Hverken stof- eller gruppe-specifikke PAE-bindende aptamers er blevet rapporteret.
Målet med dette arbejde er at give en repræsentativ protokol for at vælge gruppe-specifikke DNA aptamers til yderst hydrofobe små molekyler såsom PAEs, en repræsentativ gruppe af dukker. Vi viser også anvendelsen af de valgte aptamer til registrering af miljøforurening. Denne protokol giver vejledning og indsigt for aptamer opdagelsen af andre hydrofobe små molekyler.

<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

Phthalater acid estere (PAEs) areone i de store grupper af persistente organiske miljøgifte. Gruppe-specifik påvisning af PAEs er meget eftertragtede på grund af den hurtige voksende kongeneres. DNA aptamers er blevet i stigende grad anvendt som anerkendelse elementer på biosensor platforme, men at vælge aptamers mod stærkt hydrofobe lille molekyle mål, såsom PAEs, er sjældent rapporteret. Dette arbejde beskriver en perle-baserede metode designet til Vælg gruppe-specifikke DNA aptamers til PAEs. Aminogruppen functionalized dibutylphthalat (DBP-NH2) som anker mål blev syntetiseret og immobiliseret på epoxy-aktiveret Agarosen perler, tillader visning af gruppen phthalic ester på overfladen af matrixen immobilisering og Derfor valget af gruppespecifikke bindemidler. Vi bestemt dissociation konstanter aptamer kandidater af kvantitative polymerisering kædereaktion kombineret med magnetisk separation. Den relative tilhørsforhold og selektivitet af aptamers til andre PAEs blev bestemt af de konkurrencedygtige assays, hvor aptamer kandidater var pre afgrænset til DBP-NH2 knyttet magnetiske perler og frigivet til supernatanten ved inkubering med de testede PAEs eller andre potentielle interfererende stoffer. Konkurrencedygtige analysen blev anvendt, fordi den gav en letkøbt affinitet sammenligning blandt PAEs, der havde ingen funktionelle grupper for overflade immobilisering. Endelig, vi viste fabrikation af en elektrokemisk aptasensor og brugte den til ultrasensitive og selektiv påvisning af bis(2-ethylhexyl) phthalat. Denne protokol giver indsigt aptamer opdagelsen af andre hydrofobe små molekyler.

Vi designet og syntetiseret aminogruppen functionalized dibutylphthalat (DBP-NH2) som anker mål (figur 1F). Vi har derefter gennemført DNA aptamer udvalg af PAEs ved hjælp af DBP-NH2 som anker mål og efter klassisk mål immobilisering-baseret metode (figur 2). I hver runde, blev en pilot PCR udført ved hjælp af denatureret side for at optimere cyklus antallet af PCR (figur 3</stron...

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

En protokol til in vitro- udvælgelse og karakterisering af gruppespecifikke phthalater syre ester-bindende DNA aptamers præsenteres. Anvendelsen af den valgte aptamer i en elektrokemisk aptasensor er også inkluderet.

Phthalater syre Ester-bindende DNA Aptamer udvalg, karakterisering og ansøgning til en elektrokemisk 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 ...

Research

JoVE Journal

Chemistry

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

Foldning og karakterisering af en Bio-responsiv Robot fra 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 ...

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

Nucleosidtriphosphater - Fra Syntese til Biokemisk karakterisering

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

En omvendt analyse tilgang til karakterisering af kemiske Transport i Maling

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

Dynamisk Elektrokemisk Måling af chloridioner

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)

An Aptamer-based Sensor for Unchelated GadoliniumIII

En aptamer-baserede sensor til ucheleret 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 ...

Electrochemical Impedance Spectroscopy as a Tool for Electrochemical Rate Constant Estimation

Elektrokemiske impedans spektroskopi som et redskab for elektrokemisk sats konstant estimering

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

En elektrokemisk Cholesteric flydende krystallinske enhed for hurtig og simpel-spænding farve graduering

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

Elektrokemisk Skruening af tynde film platin makro og Mikroelektroder

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

Asymmetrisk termoelektrokemisk celle til høst af varme af lav kvalitet 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 ...

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

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

Sondering Overflade elektrokemisk aktivitet af nanomaterialer ved hjælp af en Hybrid Atomic Force Mikroskop-Scanning elektrokemiske mikroskop (AFM-SECM)

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