Digitized Molecular Detection on Off-the-shelf Blu-ray Discs: Upgraded Resolution and Enhanced Sensitivity

Beyond the intrinsic capability of storing and archiving high-definition movies and games, off-the-shelf Blu-ray discs have been adopted for the preparation of molecular binding assays, which are subsequently read and quantitated with a standard computer drive in conjunction with the disc-quality check program. The performance of this digitized molecular detection system has been examined first with an artificial “ink assay” (an array of microsize ink dots) to define the quantitation capability and the lateral resolution; the conventional biotin-streptavidin binding assay was then tested with the Blu-ray detection platform, and the results compared with that obtained on a DVD. The upgraded lateral resolution (<100 μm) and enhanced assay performance (linear response up to 0.4 μg/mL and LOD estimated to be >< 0.1 μg/mL for the trial biotin-streptavidin system augments its potential to be adapted as a cost-effective and quantitative diagnostic tool for on-site analysis and point-of-care medical diagnosis at trace amounts

Isothermal DNA amplification strategies for duplex microorganism detection

[EN] A valid solution for micro-analytical systems is the selection of a compatible amplification reaction with a simple, highly-integrated efficient design that allows the detection of multiple genomic targets. Two approaches under isothermal conditions are presented: recombinase polymerase amplification (RPA) and multiple displacement amplification (MDA). Both methods were applied to a duplex assay specific for Salmonella spp. and Cronobacter spp., with excellent amplification yields (0.2 8.6 108 fold). The proposed approaches were successfully compared to conventional PCR and tested for the milk sample analysis as a microarray format on a compact disc (support and driver). Satisfactory results were obtained in terms of resistance to inhibition, selectivity, sensitivity (101 102 CFU/mL) and reproducibility (below 12.5%). The methods studied are efficient and cost-effective, with a high potential to automate microorganisms detection by integrated analytical systems working at a constant low temperature.Funding projects MINECO CTQ2013-45875-R and GV PrometeoII/2014/040. MECD provided S.S.F with a PhD grant.Santiago Felipe, S.; Tortajada-Genaro, LA.; Morais, S.; Puchades, R.; Maquieira Catala, Á. (2015). Isothermal DNA amplification strategies for duplex microorganism detection. Food Chemistry. 174:509-515. https://doi.org/10.1016/j.foodchem.2014.11.080S50951517

One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method

[EN] An integrated sensor comprising isothermal DNA amplification and in situ detection is presented. The method principle is based on recombinase polymerase amplification (RPA) and detection in the microarray format by compact disc technology as a high-throughput sensing platform. Primers were immobilised on the polycarbonate surface of digital versatile discs (DVD) and, after hemi-nested amplification, multiplexing identification of each tethered product was achieved by optical scanning with a 650 nm-laser of the DVD drive. The efficiency of one-pot hybridisation/elongation/detection depended strongly on probedensity and other factors such as the concentration of the unbound primers present in solution. The optimised conditions provided equivalent amplification factors (7.3 x 10(8) -8.9 x 10(8) fold) to those obtained by conventional reactions performed in vials. The proposed method was applied to Salmonella detection (generic by hns and oriC genes, and specific for subspecies I by STM4507 gene). A triplex assay was satisfactorily compared to the non-integrated protocols. Food and vaccine samples were analysed in a shorter time with less handling. The results indicate that the multiplex DVD assay is a simple, competitive, isothermal, portable system that is particularly useful for microbiological routine analysis. (C) 2014 Elsevier B.V. All rights reserved.This research has been funded through Projects GVA-PROMETEO/2010/008 (Generalitat Valenciana) and CTQ/2013/ 45875-R (MINECO). The Spanish Ministry of Education and Science provided S.S.F. with a grant for her PhD studies.Santiago Felipe, S.; Tortajada-Genaro, LA.; Morais, S.; Puchades, R.; Maquieira Catala, Á. (2014). One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method. Sensors and Actuators B: Chemical. 204:273-281. https://doi.org/10.1016/j.snb.2014.07.073S27328120

Detection of food-borne pathogens with DNA arrays on disk

A DNA oligonucleotide array for duplex pathogen detection on a DVD platform is developed. The assay involves hybridization of PCR products and optical detection using compact disc technology. Different DNA array constructions for attachment of synthetic oligonucleotides on to DVD surface are evaluated, finding that streptavidin-biotin coupling method yielded the highest sensitivity in combination with enzymatic signal amplification. Issues of importance for the DNA array construction such immobilized probes design, PCR product labeling strategy and composition of the hybridization buffer were addressed. The methodology was proved scoring single nucleotide polymorphisms with high selectivity. The assay capability was also demonstrated by the identification of two pathogenic microorganisms in powder milk samples. In fifty minutes, the DVD-array system identifies Salmonella spp. and Cronobacter spp. (previously named Enterobacter sakazakii) precise and simultaneously with a sensitivity of 100 and 102 cfu/mL, respectively, in infant milk. Results were in good agreement with those obtained by quantitative real-time PCR. © 2012 Elsevier B.V. All rights reserved.This work was funded by the projects PATSENS, PSE-010000-2008-6 (Spanish Government and EU FEDER funds), FEDER CTQ2010-15943 (CICYT, Spain), PROMETEO 2010/008 and ACOMP/2012/158 (Generalitat Valenciana). The Spanish MEC provided T.A-C with a grant for her PhD studies.Arnandis Chover, T.; Morais, S.; Tortajada-Genaro, LA.; Puchades, R.; Maquieira Catala, Á.; Berganza, J.; Olabarria, G. (2012). Detection of food-borne pathogens with DNA arrays on disk. Talanta. 101:405-412. https://doi.org/10.1016/j.talanta.2012.09.049S40541210

Disc-based microarrays: principles and analytical applications

[EN] The idea of using disk drives to monitor molecular biorecognition events on regular optical discs has received considerable attention during the last decade. CDs, DVDs, Blu-ray discs and other new optical discs are universal and versatile supports with the potential for development of protein and DNA microarrays. Besides, standard disk drives incorporated in personal computers can be used as compact and affordable optical reading devices. Consequently, a CD technology, resulting from the audio-video industry, has been used to develop analytical applications in health care, environmental monitoring, food safety and quality assurance. The review presents and critically evaluates the current state of the art of disc-based microarrays with illustrative examples, including past, current and future developments. Special mention is made of the analytical developments that use either chemically activated or raw standard CDs where proteins, oligonucleotides, peptides, haptens or other biological probes are immobilized. The discs are also used to perform the assays and must maintain their readability with standard optical drives. The concept and principle of evolving disc-based microarrays and the evolution of disk drives as optical detectors are also described. The review concludes with the most relevant uses ordered chronologically to provide an overview of the progress of CD technology applications in the life sciences. Also, it provides a selection of important references to the current literature.This work was supported by the Spanish Ministry of Economy and Competitiveness (project CTQ2013-45875-R) and Generalitat Valenciana (PROMETEO II 2014/040 and ACOMP 2012/158). All authors were partially sponsored by the European Regional Development Fund.Morais, S.; Puchades, R.; Maquieira Catala, Á. 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