title: Multiplex PCB-based electrochemical detection of cancer biomarkers using MLPA-barcode approach.
abstract: Asymmetric multiplex ligation-dependent probe amplification (MLPA) was developed for the amplification of seven breast cancer related mRNA markers and the MLPA products were electrochemically detected via hybridization. Seven breast cancer genetic markers were amplified by means of the MLPA reaction, which allows for multiplex amplification of multiple targets with a single primer pair. Novel synthetic MLPA probes were designed to include a unique barcode sequence in each amplified gene. Capture probes complementary to each of the barcode sequences were immobilized on each electrode of a low-cost electrode microarray manufactured on standard printed circuit board (PCB) substrates. The functionalised electrodes were exposed to the single-stranded MLPA products and following hybridization, a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the amplified strand completed the genocomplex, which was electrochemically detected following substrate addition. The electrode arrays fabricated using PCB technology exhibited an excellent electrochemical performance, equivalent to planar photolithographically-fabricated gold electrodes, but at a vastly reduced cost (>50 times lower per array). The optimised system was demonstrated to be highly specific with negligible cross-reactivity allowing the simultaneous detection of the seven mRNA markers, with limits of detections as low as 25pM. This approach provides a novel strategy for the genetic profiling of tumour cells via integrated "amplification-to-detection".
authors.affiliation: Universitat Rovira i Virgili, Departament de Enginyeria Química, Av. Països Catalans 26, 43007 Tarragona, Spain. Electronic address: joseplluis.acero@urv.cat.
authors.forename: J L Acero
authors.initials: JL
authors.lastname: Sánchez
authors.affiliation: Universitat Rovira i Virgili, Departament de Enginyeria Química, Av. Països Catalans 26, 43007 Tarragona, Spain.
authors.forename: O Y F
authors.initials: OY
authors.lastname: Henry
authors.affiliation: Universitat Rovira i Virgili, Departament de Enginyeria Química, Av. Països Catalans 26, 43007 Tarragona, Spain.
authors.forename: H
authors.initials: H
authors.lastname: Joda
authors.affiliation: KTH Royal Institute of Technology, Science for Life Laboratory (SciLifeLab Stockholm), School of Biotechnology, Division of Gene Technology, SE-171 65 Solna, Sweden.
authors.forename: B Werne
authors.initials: BW
authors.lastname: Solnestam
authors.affiliation: KTH Royal Institute of Technology, Science for Life Laboratory (SciLifeLab Stockholm), School of Biotechnology, Division of Gene Technology, SE-171 65 Solna, Sweden.
authors.forename: L
authors.initials: L
authors.lastname: Kvastad
authors.affiliation: KTH Royal Institute of Technology, Science for Life Laboratory (SciLifeLab Stockholm), School of Biotechnology, Division of Gene Technology, SE-171 65 Solna, Sweden.
authors.forename: E
authors.initials: E
authors.lastname: Johansson
authors.affiliation: KTH Royal Institute of Technology, Science for Life Laboratory (SciLifeLab Stockholm), School of Biotechnology, Division of Gene Technology, SE-171 65 Solna, Sweden.
authors.forename: P
authors.initials: P
authors.lastname: Akan
authors.affiliation: KTH Royal Institute of Technology, Science for Life Laboratory (SciLifeLab Stockholm), School of Biotechnology, Division of Gene Technology, SE-171 65 Solna, Sweden.
authors.forename: J
authors.initials: J
authors.lastname: Lundeberg
authors.affiliation: MRC Holland, Willem Schoutenstraat 6, 1057 DN Amsterdam, The Netherlands.
authors.forename: N
authors.initials: N
authors.lastname: Lladach
authors.affiliation: Labman Automation Ltd., Seamer Hill, Seamer, Stokesley, North Yorkshire TS9 5NQ, United Kingdom.
authors.forename: D
authors.initials: D
authors.lastname: Ramakrishnan
authors.affiliation: Labman Automation Ltd., Seamer Hill, Seamer, Stokesley, North Yorkshire TS9 5NQ, United Kingdom.
authors.forename: I
authors.initials: I
authors.lastname: Riley
authors.affiliation: Universitat Rovira i Virgili, Departament de Enginyeria Química, Av. Països Catalans 26, 43007 Tarragona, Spain; Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys 23, 08010 Barcelona, Spain. Electronic address: ciara.osullivan@urv.cat.
authors.forename: C K
authors.initials: CK
authors.lastname: O'Sullivan
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics; D015374:Biosensing Techniques / Q000295:instrumentation; D001940:Breast / Q000473:pathology; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics*; D055664:Electrochemical Techniques / Q000295:instrumentation*; D005260:Female; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000295:instrumentation*; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D059467:Transcriptome
chemical_list: D014408:Biomarkers, Tumor; D012333:RNA, Messenger
keywords: Barcodes; Breast cancer markers; Electrochemical detection; Multiplex ligation-dependent probe amplification system (MLPA); PCB genosensor arrays