Portable chemiluminescence multiplex biosensor for quantitative detection of three B19 DNA genotypes
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TECHNICAL NOTE
Portable chemiluminescence multiplex biosensor for quantitative detection of three B19 DNA genotypes Mara Mirasoli & Francesca Bonvicini & Luisa Stella Dolci & Martina Zangheri & Giorgio Gallinella & Aldo Roda
Received: 19 October 2012 / Revised: 8 November 2012 / Accepted: 12 November 2012 / Published online: 28 November 2012 # Springer-Verlag Berlin Heidelberg 2012
Abstract A miniaturized multiplex biosensor exploiting a microfluidic oligonucleotide array and chemiluminescence (CL) lensless imaging detection has been developed for parvovirus B19 genotyping. The portable device consists of a reaction chip, comprising a glass slide arrayed with three B19 genotype-specific probes and coupled with a polydimethylsiloxane microfluidic layer, and a charge-coupled device camera modified for lensless CL imaging. Immobilized probes were used in DNA hybridization reactions with biotin-labeled targets, and then hybrids were measured by means of an avidin-horseradish peroxidase (HRP) conjugate and CL detection. All hybridization assay procedures have been optimized to be performed at room temperature through the microfluidic elements of the reaction chip, with sample and reagents delivery via capillary force exploiting adsorbent pads to drive fluids along the microchannels. The biosensor enabled multiplex detection of all B19 genotypes, with detectability down to
Published in the special issue Analytical Science in Italy with guest editor Aldo Roda. Electronic supplementary material The online version of this article (doi:10.1007/s00216-012-6573-7) contains supplementary material, which is available to authorized users. M. Mirasoli (*) : L. S. Dolci : M. Zangheri : A. Roda Department of Chemistry “G. Ciamician”, University of Bologna, Via Selmi 2, 40126 Bologna, Italy e-mail: [email protected] M. Mirasoli : A. Roda National Institute of Biostructure and Biosystems, N.I.B.B, Interuniversity Consortium, Viale medaglie d’Oro 305, 00136 Rome, Italy F. Bonvicini : G. Gallinella Department of Pharmacy and BioTechnology, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy
80 pmolL−1 for all B19 genotype oligonucleotides and 650 pmolL−1 for the amplified product of B19 genotype 1, which is comparable with that obtained in traditional PCRELISA formats and with notably shorter assay time (30 min vs. 2 h). The specificity of the assay has been evaluated by performing DNA–DNA hybridization reactions among sequences with different degrees of homology, and no cross hybridizations among B19 genotypes have been observed. The clinical applicability has been demonstrated by assaying amplified products obtained from B19 reference serum samples, with results completely consistent with the reference PCRELISA method. The next crucial step will be integration in the biosensor of a miniaturized PCR system for DNA amplification and for heat treatment of amplified products. Keywords Biosensor . Chemiluminescence lensless imaging . Point-of-care testing . Gene probe hybridization . Multiplex assay . Parvovirus B19 genot
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