Double amplified colorimetric detection of DNA using gold nanoparticles, enzymes and a catalytic hairpin assembly
- 99 Downloads
The authors describe an isothermal and ultrasensitive colorimetric DNA assay that consists of two amplification stages using enzymes and a catalytic hairpin assembly (CHA). The first step consists in the selective amplification of DNA using Klenow fragment and nicking enzyme. The second step consists in the amplification of the optical signal by using a catalytic hairpin assembly. After two amplification steps, the DNA reaction induces the aggregation of the red gold nanoparticles to give a blue color shift. The degree of aggregation can be quantified by measurement of the ratio of the UV-vis absorbances of the solutions at 620 and 524 nm which are the wavelengths of the aggregated gold nanoparticles and bare gold nanoparticles. The detection limit is as low as 3.1 fM. Due to the use of a specific enzyme, only the desired DNAs will be detected. The method can be applied to the determination of DNA of various lengths. Despite the presence of large amounts of wildtype DNA, it can readily detect a target DNA. Conceivably, the technique has a large potential because of its high sensitivity and selectivity.
KeywordsKlenow fragment DNA sensor AuNPs UV-vis absorbance CHA Hairpin DNA Liquid biopsy
This study was supported by the National Research Foundation of Korea (NRF) under grant numbers of NRF-2016R1A5A1010148, NRF-2015M3A9D7031026 and funded by the Ministry of Science, ICT & Future Planning. C. Park and H. Park contributed equally to this work.
Compliance with ethical standards
The author(s) declare that they have no competing interests.
- 1.Jang K, Choi J, Park C, Na S (2017) Label-free and high-sensitive detection of Kirsten rat sarcoma viral oncogene homolog and epidermal growth factor receptor mutation using kelvin probe force microscopy. Biosens Bioelectron 87:222–228. https://doi.org/10.1016/j.bios.2016.08.045 CrossRefGoogle Scholar
- 3.Dena M, Kelly B, Anand K, Madelyn SL, Michael M, Franziska B, Katharina V, Daniel L, Jorge N, Lyudmila B, Andrew HK, Korn WM, Ethan S, Michael C, Xing Y, Thomas M, Rachelle L, Meghana H, Craig Y, Joshua K, Yelena P, Devin S, David N, Peter K (2012) Fluid biopsy in patients with metastatic prostate, pancreatic and breast cancers. Phys Biol 9(1):016003CrossRefGoogle Scholar
- 4.Marco W, Lyudmila B, Rogier B, Anand K, Meghana H, Edward HC, Dena M, Ajay S, Anthony P, Patricia T, Kelly B, Jorge N, Michel van den H, Peter K (2012) Fluid biopsy for circulating tumor cell identification in patients with early-and late-stage non-small cell lung cancer: a glimpse into lung cancer biology. Phys Biol 9(1):016005CrossRefGoogle Scholar
- 5.Taly V, Pekin D, Benhaim L, Kotsopoulos SK, Le Corre D, Li X, Atochin I, Link DR, Griffiths AD, Pallier K, Blons H, Bouché O, Landi B, Hutchison JB, Laurent-Puig P (2013) Multiplex Picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal Cancer patients. Clin Chem 59(12):1722–1731. https://doi.org/10.1373/clinchem.2013.206359 CrossRefPubMedGoogle Scholar
- 6.Tuononen K, Mäki-Nevala S, Sarhadi VK, Wirtanen A, Rönty M, Salmenkivi K, Andrews JM, Telaranta-Keerie AI, Hannula S, Lagström S, Ellonen P, Knuuttila A, Knuutila S (2013) Comparison of targeted next-generation sequencing (NGS) and real-time PCR in the detection of EGFR, KRAS, and BRAF mutations on formalin-fixed, paraffin-embedded tumor material of non-small cell lung carcinoma—superiority of NGS. Genes Chromosom Cancer 52(5):503–511. https://doi.org/10.1002/gcc.22047 CrossRefPubMedGoogle Scholar
- 11.Elghanian R, Storhoff JJ, Mucic RC, Letsinger RL, Mirkin CA (1997) Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles. Science 277(5329):1078–1081. https://doi.org/10.1126/science.277.5329.1078 CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Liang C, Chu Y, Cheng S, Wu H, Kajiyama T, Kambara H, Zhou G (2012) Multiplex loop-mediated isothermal amplification detection by sequence-based barcodes coupled with nicking endonuclease-mediated pyrosequencing. Anal Chem 84(8):3758–3763. https://doi.org/10.1021/ac3003825 CrossRefPubMedGoogle Scholar
- 18.Zhao Y, Chen F, Wu Y, Dong Y, Fan C (2013) Highly sensitive fluorescence assay of DNA methyltransferase activity via methylation-sensitive cleavage coupled with nicking enzyme-assisted signalamplification. Biosens Bioelectron 42:56–61. https://doi.org/10.1016/j.bios.2012.10.022 CrossRefPubMedGoogle Scholar
- 25.Xu F, Dong H, Cao Y, Lu H, Meng X, Dai W, Zhang X, Al-Ghanim KA, Mahboob S (2016) Ultrasensitive and multiple disease-related MicroRNA detection based on tetrahedral DNA nanostructures and duplex-specific nuclease-assisted signal amplification. ACS Appl Mater Interfaces 8(49):33499–33505. https://doi.org/10.1021/acsami.6b12214 CrossRefPubMedGoogle Scholar
- 26.Yang W, Zhou X, Zhao J, Xu W (2018) A cascade amplification strategy of catalytic hairpin assembly and hybridization chain reaction for the sensitive fluorescent assay of the model protein carcinoembryonic antigen. Microchim Acta 185(2):100. https://doi.org/10.1007/s00604-017-2620-6 CrossRefGoogle Scholar