Genosensor on gold films with enzymatic electrochemical detection of a SARS virus sequence
Identifieur interne : 004E21 ( Main/Exploration ); précédent : 004E20; suivant : 004E22Genosensor on gold films with enzymatic electrochemical detection of a SARS virus sequence
Auteurs : Patricia Abad-Valle [Espagne] ; M. Teresa Fernandez-Abedul [Espagne] ; Agustin Costa-Garcia [Espagne]Source :
- Biosensors & bioelectronics [ 0956-5663 ] ; 2005.
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Abstract
A hybridisation-based genosensor was designed on a 100 nm sputtered gold film. This material worked as an immobilisation and transduction surface. A 30-mer sequence that encodes a short lysine-rich region, unique to SARS (severe acute respiratory syndrome) virus, was chosen as target. A complementary strand (probe), labelled with a thiol group at the 3'-end, was immobilised on the film. After blocking the surface, hybridisation with the biotin-conjugated SARS strand (at the 3'-end) took place. Interaction with alkaline phosphatase-labelled streptavidin permits amplified indirect electrochemical detection. The analytical signal is constituted by an electrochemical process of indigo carmine, the soluble product of the enzymatic hydrolysis of 3-indoxyl phosphate. The use of a sensitive electrochemical technique such as square wave voltammetry allowed a detection limit of 6 pM to be obtained for this DNA sequence, lower than any other found in the bibliography. The parameters affecting the methodology were studied, with special attention being placed on selectivity. Specificity was clearly enhanced when interaction time and stringency (in the form of formamide percentage) were increased. With 1 h of strand interaction and employing 50% of formamide in the hybridisation buffer, a 3-base mismatch strand was perfectly distinguished from the complementary.
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Teresa Fernandez-Abedul</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Departamento de Quimica Física y Analítica, Universidad de Oviedo</s1><s2>Asturias, 33006 Oviedo</s2><s3>ESP</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ></inist:fA14><country>Espagne</country><placeName><region nuts="2" type="communauté">Principauté des Asturies</region></placeName></affiliation></author><author><name sortKey="Costa Garcia, Agustin" sort="Costa Garcia, Agustin" uniqKey="Costa Garcia A" first="Agustin" last="Costa-Garcia">Agustin Costa-Garcia</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Departamento de Quimica Física y Analítica, Universidad de Oviedo</s1><s2>Asturias, 33006 Oviedo</s2><s3>ESP</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ></inist:fA14><country>Espagne</country><placeName><region nuts="2" type="communauté">Principauté des Asturies</region></placeName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">INIST</idno><idno type="inist">05-0394326</idno><date when="2005">2005</date><idno type="stanalyst">PASCAL 05-0394326 INIST</idno><idno type="RBID">Pascal:05-0394326</idno><idno type="wicri:Area/PascalFrancis/Corpus">000618</idno><idno type="wicri:Area/PascalFrancis/Curation">000372</idno><idno type="wicri:Area/PascalFrancis/Checkpoint">000642</idno><idno type="wicri:explorRef" wicri:stream="PascalFrancis" wicri:step="Checkpoint">000642</idno><idno type="wicri:doubleKey">0956-5663:2005:Abad Valle P:genosensor:on:gold</idno><idno type="wicri:Area/Main/Merge">005162</idno><idno type="wicri:Area/Main/Curation">004E21</idno><idno type="wicri:Area/Main/Exploration">004E21</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a">Genosensor on gold films with enzymatic electrochemical detection of a SARS virus sequence</title><author><name sortKey="Abad Valle, Patricia" sort="Abad Valle, Patricia" uniqKey="Abad Valle P" first="Patricia" last="Abad-Valle">Patricia Abad-Valle</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Departamento de Quimica Física y Analítica, Universidad de Oviedo</s1><s2>Asturias, 33006 Oviedo</s2><s3>ESP</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ></inist:fA14><country>Espagne</country><placeName><region nuts="2" type="communauté">Principauté des Asturies</region></placeName></affiliation></author><author><name sortKey="Fernandez Abedul, M Teresa" sort="Fernandez Abedul, M Teresa" uniqKey="Fernandez Abedul M" first="M. Teresa" last="Fernandez-Abedul">M. Teresa Fernandez-Abedul</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Departamento de Quimica Física y Analítica, Universidad de Oviedo</s1><s2>Asturias, 33006 Oviedo</s2><s3>ESP</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ></inist:fA14><country>Espagne</country><placeName><region nuts="2" type="communauté">Principauté des Asturies</region></placeName></affiliation></author><author><name sortKey="Costa Garcia, Agustin" sort="Costa Garcia, Agustin" uniqKey="Costa Garcia A" first="Agustin" last="Costa-Garcia">Agustin Costa-Garcia</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Departamento de Quimica Física y Analítica, Universidad de Oviedo</s1><s2>Asturias, 33006 Oviedo</s2><s3>ESP</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ></inist:fA14><country>Espagne</country><placeName><region nuts="2" type="communauté">Principauté des Asturies</region></placeName></affiliation></author></analytic><series><title level="j" type="main">Biosensors & bioelectronics</title><title level="j" type="abbreviated">Biosens. bioelectron.</title><idno type="ISSN">0956-5663</idno><imprint><date when="2005">2005</date></imprint></series></biblStruct></sourceDesc><seriesStmt><title level="j" type="main">Biosensors & bioelectronics</title><title level="j" type="abbreviated">Biosens. bioelectron.</title><idno type="ISSN">0956-5663</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Biosensor</term><term>Detection</term><term>Electrochemistry</term><term>Gold</term><term>Immobilization</term><term>Molecular hybridization</term><term>Molecular probe</term><term>Severe acute respiratory syndrome</term><term>Virus</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Sonde moléculaire</term><term>Electrochimie</term><term>Détection</term><term>Syndrome respiratoire aigu sévère</term><term>Virus</term><term>Biodétecteur</term><term>Immobilisation</term><term>Or</term><term>Hybridation moléculaire</term></keywords><keywords scheme="Wicri" type="topic" xml:lang="fr"><term>Or</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">A hybridisation-based genosensor was designed on a 100 nm sputtered gold film. This material worked as an immobilisation and transduction surface. A 30-mer sequence that encodes a short lysine-rich region, unique to SARS (severe acute respiratory syndrome) virus, was chosen as target. A complementary strand (probe), labelled with a thiol group at the 3'-end, was immobilised on the film. After blocking the surface, hybridisation with the biotin-conjugated SARS strand (at the 3'-end) took place. Interaction with alkaline phosphatase-labelled streptavidin permits amplified indirect electrochemical detection. The analytical signal is constituted by an electrochemical process of indigo carmine, the soluble product of the enzymatic hydrolysis of 3-indoxyl phosphate. The use of a sensitive electrochemical technique such as square wave voltammetry allowed a detection limit of 6 pM to be obtained for this DNA sequence, lower than any other found in the bibliography. The parameters affecting the methodology were studied, with special attention being placed on selectivity. Specificity was clearly enhanced when interaction time and stringency (in the form of formamide percentage) were increased. With 1 h of strand interaction and employing 50% of formamide in the hybridisation buffer, a 3-base mismatch strand was perfectly distinguished from the complementary.</div></front></TEI><affiliations><list><country><li>Espagne</li></country><region><li>Principauté des Asturies</li></region></list><tree><country name="Espagne"><region name="Principauté des Asturies"><name sortKey="Abad Valle, Patricia" sort="Abad Valle, Patricia" uniqKey="Abad Valle P" first="Patricia" last="Abad-Valle">Patricia Abad-Valle</name></region><name sortKey="Costa Garcia, Agustin" sort="Costa Garcia, Agustin" uniqKey="Costa Garcia A" first="Agustin" last="Costa-Garcia">Agustin Costa-Garcia</name><name sortKey="Fernandez Abedul, M Teresa" sort="Fernandez Abedul, M Teresa" uniqKey="Fernandez Abedul M" first="M. Teresa" last="Fernandez-Abedul">M. Teresa Fernandez-Abedul</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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