Abstract
A reagent-less DNA sensor has been developed exploiting a combination of gold nanoparticles, modified primers, and isothermal amplification. It is applied to the determination ofKarlodinium armiger, a toxic microalgae, as a model analyte to demonstrate this generic platform. Colloidal gold nanoparticles with an average diameter of 14 ± 0.87 nm were modified with a mixed self-assembled monolayer of thiolated 33-mer DNA probes and (6-mercaptohexyl) ferrocene. Modified primers, exploiting a C3 spacer between the primer-binding site and an engineered single-stranded tail, were used in an isothermal recombinase polymerase amplification reaction to produce an amplicon by two single-stranded tails. These tails were designed to be complementary to a gold electrode tethered capture oligo probe, and an oligo probe immobilized on the gold nanoparticles, respectively. The time required for hybridization of the target tailed DNA with the surface immobilized probe and reporter probe immobilized on AuNPs was optimized and reduced to 10 min, in both cases. Amplification time was further optimized to be 40 min to ensure the maximum signal. Under optimal conditions, the limit of detection was found to be 1.6 fM of target dsDNA. Finally, the developed biosensor was successfully applied to the detection of genomic DNA extracted from a seawater sample that had been spiked with K. armiger cells. The demonstrated generic electrochemical genosensor can be exploited for the detection of any DNA sequence and ongoing work is moving towards an integrated system for use at the point-of-need.
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Acknowledgements
This work has been carried out with partial financial support from Spanish Ministerio de Economía y Competitividad (SEASENSING BIO2014-56024-C2-1): “Seasensing: Microsystems for rapid, reliable and cost effective detection of toxic microalgae on-site and in real-time.” S. A-M. acknowledges support from the government of Catalonia (GENCAT) for predoctoral grant 2017FI_B2 00184, and for the training grant that allowed him to visit the University of Ioannina. The authors thank Mònica Campàs and Anna Toldrà for the provision of genomic DNA extracted from seawater samples.
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This work has been carried out with partial financial support from Spanish Ministerio de Economía y Competitividad (SEASENSING BIO2014-56024-C21): “Seasensing: Microsystems for rapid, reliable and cost effective detection of toxic microalgae on-site and in real-time.”
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AL-Madhagi, S., O’Sullivan, C.K., Prodromidis, M.I. et al. Combination of ferrocene decorated gold nanoparticles and engineered primers for the direct reagentless determination of isothermally amplified DNA. Microchim Acta 188, 117 (2021). https://doi.org/10.1007/s00604-021-04771-8
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DOI: https://doi.org/10.1007/s00604-021-04771-8