Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
7556671 | Analytical Biochemistry | 2018 | 7 Pages |
Abstract
DNA biosensors are attractive tools for genetic analysis as there is an increasing need for rapid and low-cost DNA analysis, primarily driven by applications in personalized pharmacogenomics, clinical diagnostics, rapid pathogen detection, food traceability and forensics. A rapid electrochemical genosensor detection methodology exploiting a combination of modified primers for solution-phase isothermal amplification, followed by rapid detection via hybridization on gold electrodes is reported. Modified reverse primers, exploiting a C18 spacer between the primer-binding site and an engineered single stranded tail, are used in a recombinase polymerase amplification reaction to produce an amplicon with a central duplex flanked by two single stranded tails. These tails are designed to be complementary to a gold electrode tethered capture oligo probe as well as a horseradish peroxidase labelled reporter oligo probe. The time required for hybridization of the isothermally generated amplicons with each of the immobilized and reporter probes was optimised to be 2Â min, in both cases. The effect of amplification time and the limit of detection were evaluated using these hybridization times for both single stranded and double stranded DNA templates. The best detection limit of 70Â fM for a ssDNA template was achieved using 45Â min amplification, whilst for a dsDNA template, just 30Â min amplification resulted in a slightly lower detection limit of 14Â fM, whilst both 20 and 45Â min amplification times were observed to provide detection limits of 71 and 72Â fM, respectively, but 30 and 45Â min amplification resulted in a much higher signal and sensitivity. The genosensor was applied to genomic DNA and real patient and control blood samples for detection of the coeliac disease associated DQB1*02 HLA allele, as a model system, demonstrating the possibility to carry out molecular diagnostics, combining amplification and detection in a rapid and facile manner.
Keywords
dsDNATMBMCHSSPHRPTBSSAMssDNAPCBNTCRPAPBSELONADt16-Mercapto-1-hexanolsingle stranded DNAdouble stranded DNADNASequence-specific primershuman leukocyte antigensHLAenzyme linked immunosorbent assaydeoxyribonucleic acidNo template controlPrinted Circuit BoardTris-buffered salineELISADNA detectionElectrochemical detectionRecombinase polymerase amplificationphosphate buffer salinepolymerase chain reactionPCRhorseradish peroxidiseSelf assembled monolayer
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Sallam Al-Madhagi, Hamdi Joda, Miriam Jauset-Rubio, Mayreli Ortiz, Ioanis Katakis, Ciara K. O'Sullivan,