title: Real-time and label-free ring-resonator monitoring of solid-phase recombinase polymerase amplification.
abstract: In this work we present the use of a silicon-on-insulator (SOI) chip featuring an array of 64 optical ring resonators used as refractive index sensors for real-time and label-free DNA detection. Single ring functionalisation was achieved using a click reaction after precise nanolitre spotting of specific hexynyl-terminated DNA capture probes to link to an azido-silanised chip surface. To demonstrate detectability using the ring resonators and to optimise conditions for solid-phase amplification, hybridisation between short 25-mer single stranded DNA (ssDNA) fragments and a complementary capture probe immobilised on the surface of the ring resonators was carried out and detected through the shift in the resonant wavelength. Using the optimised conditions demonstrated via the solid-phase hybridisation, a 144-bp double stranded DNA (dsDNA) was then detected directly using recombinase and polymerase proteins through on-chip target amplification and solid-phase elongation of immobilised forward primers on specific rings, at a constant temperature of 37°C and in less than 60min, achieving a limit of detection of 7.8·10(-13)M (6·10(5) copies in 50µL). The use of an automatic liquid handler injection instrument connected to an integrated resealable chip interface (RCI) allowed programmable multiple injection protocols. Air plugs between different solutions were introduced to prevent intermixing and a proportional-integral-derivative (PID) temperature controller minimised temperature based drifts.
authors.affiliation: Nanobiotechnology and Bioanalysis Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007 Tarragona, Spain.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Sabaté Del Río
authors.affiliation: IMEC, Smart Systems and Emerging Technologies Unit, Department of Life Science Technologies, Kapeldreef 75, 3001 Leuven, Belgium.
authors.forename: Tim
authors.initials: T
authors.lastname: Steylaerts
authors.affiliation: Nanobiotechnology and Bioanalysis Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007 Tarragona, Spain.
authors.forename: Olivier Y F
authors.initials: OYF
authors.lastname: Henry
authors.affiliation: IMEC-Ghent University, Photonics Research Group, Sint-Pieters-nieuwstraat 41, 9000 Ghent, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Bienstman
authors.affiliation: IMEC, Smart Systems and Emerging Technologies Unit, Department of Life Science Technologies, Kapeldreef 75, 3001 Leuven, Belgium. Electronic address: Tim.Stakenborg@imec.be.
authors.forename: Tim
authors.initials: T
authors.lastname: Stakenborg
authors.affiliation: IMEC, Smart Systems and Emerging Technologies Unit, Department of Life Science Technologies, Kapeldreef 75, 3001 Leuven, Belgium.
authors.forename: Wim
authors.initials: W
authors.lastname: Van Roy
authors.affiliation: Nanobiotechnology and Bioanalysis Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007 Tarragona, Spain; Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys, 23, 08010 Barcelona, Spain. Electronic address: ckosulli@etse.urv.es.
authors.forename: Ciara K
authors.initials: CK
authors.lastname: O'Sullivan
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D057930:Click Chemistry; D003199:Computer Systems; D004247:DNA / Q000032:analysis*; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D004800:Enzymes, Immobilized; D004867:Equipment Design; D005604:Francisella tularensis / Q000235:genetics; D061925:Immobilized Nucleic Acids; D036103:Nanotechnology; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D045522:Recombinases; D012825:Silicon; D060327:Solid-Phase Synthesis Techniques
chemical_list: D015342:DNA Probes; D004269:DNA, Bacterial; D004800:Enzymes, Immobilized; D061925:Immobilized Nucleic Acids; D045522:Recombinases; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D012825:Silicon
keywords: Genosensor; Recombinase polymerase amplification; Ring resonator