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 80 pmol L−1 for all B19 genotype oligonucleotides and 650 pmol L−1 for the amplified product of B19 genotype 1, which is comparable with that obtained in traditional PCR-ELISA 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 PCR-ELISA 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.
A portable multiplex biosensor was developed for detection and genotyping of parvovirus B19 DNA, exploiting lensless CL imaging. The reaction chip is composed of a polydimethylsiloxane microfluidic layer coupled with a glass slide on which oligonucleotide probes specific for three different B19 genotypes are covalently immobilized in a 3 × 3 array. The reaction chip was used in hybridization reactions with biotin-labeled targets and then hybrids were then detected by means of an avidin-HRP conjugate, upon addition of a CL substrate for HRP
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Acknowledgments
This work was supported by grants from the Italian Ministry of Instruction, University and Research (PRIN 2009MB4AYL “Integration of biosensing and nanotechnology for medical diagnostics with high analytical performance”) and from the University of Bologna (“Fundamental Oriented Research”).
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Published in the special issue Analytical Science in Italy with guest editor Aldo Roda.
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Mirasoli, M., Bonvicini, F., Dolci, L.S. et al. Portable chemiluminescence multiplex biosensor for quantitative detection of three B19 DNA genotypes. Anal Bioanal Chem 405, 1139–1143 (2013). https://doi.org/10.1007/s00216-012-6573-7
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DOI: https://doi.org/10.1007/s00216-012-6573-7