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Electrogenerated chemiluminescence. 77. DNA hybridization detection at high amplification with [Ru(bpy)3]2+-containing microspheres.
Anal Chem. 2004 Sep 15; 76(18):5379-86.AC

Abstract

An ultrasensitive DNA hybridization detection method based on electrogenerated chemiluminescence (ECL) using polystyrene microspheres/beads (PSB) as the carrier of the ECL labels, namely, tris(2,2'-bipyridyl)ruthenium(II) tetrakis(pentafluorophenyl)borate (Ru(bpy)3[B(C6F5)4]2), is reported. Probe single-stranded DNA (p-ssDNA) was attached to the surface of magnetic beads (MB) and hybridized with target-ssDNA (t-ssDNA) with immobilized PSB containing a large number of water insoluble Ru(bpy)3[B(C6F5)4]2 species (approximately 7.5 x 10(9) molecules/bead). With this approach a large amplification factor of Ru(bpy)3[B(C6F5)4]2 molecules for each t-ssDNA can be achieved, when each PSB is attached to a limited number of t-ssDNA. The p-ssDNA-MB <--> t-ssDNA-PSB/Ru(bpy)3(2+) conjugates formed were magnetically separated from the reaction media and dissolved in MeCN containing tri-n-propylamine (TPrA) as an ECL coreactant. ECL was produced with a potential scan from 0 to 3.0 V versus Ag/Ag+, and the integrated ECL intensity was found to be linearly proportional to the t-ssDNA concentration in a range of 1.0 fM to 10 nM under optimized conditions. ECL signals associated with two base pair mismatched ssDNA and noncomplementary ssDNA can be distinguished well from the ECL signal related to the complementary DNA hybridization. A Poisson distribution is followed when a large number of MB reacts with PSB, and the minimum number of 1.0- and 2.8-microm diameter MB required to bind and magnetically separate a single 10-microm diameter PSB from the reaction solution was estimated to be three and one, respectively. The principle described in this paper could be also applied to many other ECL analyses, such as immunoassays.

Authors+Show Affiliations

Miao W
Department of Chemistry and Biochemistry, 1 University Station A5300, University of Texas at Austin, Austin, Texas 78712-0165, USA.
Bard AJ
No affiliation info available

MeSH

2,2'-DipyridylAvidinBase PairingCoordination ComplexesDNADNA ProbesElectrochemistryLuminescenceMicrospheresNucleic Acid HybridizationRuthenium Compounds

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15362895

Citation

Miao, Wujian, and Allen J. Bard. "Electrogenerated Chemiluminescence. 77. DNA Hybridization Detection at High Amplification With [Ru(bpy)3]2+-containing Microspheres." Analytical Chemistry, vol. 76, no. 18, 2004, pp. 5379-86.
Miao W, Bard AJ. Electrogenerated chemiluminescence. 77. DNA hybridization detection at high amplification with [Ru(bpy)3]2+-containing microspheres. Anal Chem. 2004;76(18):5379-86.
Miao, W., & Bard, A. J. (2004). Electrogenerated chemiluminescence. 77. DNA hybridization detection at high amplification with [Ru(bpy)3]2+-containing microspheres. Analytical Chemistry, 76(18), 5379-86.
Miao W, Bard AJ. Electrogenerated Chemiluminescence. 77. DNA Hybridization Detection at High Amplification With [Ru(bpy)3]2+-containing Microspheres. Anal Chem. 2004 Sep 15;76(18):5379-86. PubMed PMID: 15362895.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Electrogenerated chemiluminescence. 77. DNA hybridization detection at high amplification with [Ru(bpy)3]2+-containing microspheres. AU - Miao,Wujian, AU - Bard,Allen J, PY - 2004/9/15/pubmed PY - 2006/5/25/medline PY - 2004/9/15/entrez SP - 5379 EP - 86 JF - Analytical chemistry JO - Anal Chem VL - 76 IS - 18 N2 - An ultrasensitive DNA hybridization detection method based on electrogenerated chemiluminescence (ECL) using polystyrene microspheres/beads (PSB) as the carrier of the ECL labels, namely, tris(2,2'-bipyridyl)ruthenium(II) tetrakis(pentafluorophenyl)borate (Ru(bpy)3[B(C6F5)4]2), is reported. Probe single-stranded DNA (p-ssDNA) was attached to the surface of magnetic beads (MB) and hybridized with target-ssDNA (t-ssDNA) with immobilized PSB containing a large number of water insoluble Ru(bpy)3[B(C6F5)4]2 species (approximately 7.5 x 10(9) molecules/bead). With this approach a large amplification factor of Ru(bpy)3[B(C6F5)4]2 molecules for each t-ssDNA can be achieved, when each PSB is attached to a limited number of t-ssDNA. The p-ssDNA-MB <--> t-ssDNA-PSB/Ru(bpy)3(2+) conjugates formed were magnetically separated from the reaction media and dissolved in MeCN containing tri-n-propylamine (TPrA) as an ECL coreactant. ECL was produced with a potential scan from 0 to 3.0 V versus Ag/Ag+, and the integrated ECL intensity was found to be linearly proportional to the t-ssDNA concentration in a range of 1.0 fM to 10 nM under optimized conditions. ECL signals associated with two base pair mismatched ssDNA and noncomplementary ssDNA can be distinguished well from the ECL signal related to the complementary DNA hybridization. A Poisson distribution is followed when a large number of MB reacts with PSB, and the minimum number of 1.0- and 2.8-microm diameter MB required to bind and magnetically separate a single 10-microm diameter PSB from the reaction solution was estimated to be three and one, respectively. The principle described in this paper could be also applied to many other ECL analyses, such as immunoassays. SN - 0003-2700 UR - https://www.unboundmedicine.com/medline/citation/15362895/Electrogenerated_chemiluminescence__77__DNA_hybridization_detection_at_high_amplification_with_[Ru_bpy_3]2+_containing_microspheres_ DB - PRIME DP - Unbound Medicine ER -