A signal amplification strategy via enzymatic cascade reactions for ultrasensitive DNA detection

Abstract

Event Abstract Back to Event A signal amplification strategy via enzymatic cascade reactions for ultrasensitive DNA detection Eunjung Kim1*, Philip D. Howes1 and Molly M. Stevens1, 2 1 Imperial College London, Materials, United Kingdom 2 Imperial College London, Bioengineering and Institute of Biomedical Engineering, United Kingdom Introduction: The field of molecular diagnostics is in dire need of fast and easy-to-use diagnostic tools that detect and measure the presence of genetic materials or proteins associated with a specific health condition or disease. The development of such techniques would provide a greater depth of information that can be derived from genetic profiles and molecular signatures of disease compared to traditional diagnostic approaches. While some diagnostic tests such as pregnancy tests and diabetes test strips have been successfully implemented into the health care system, the ability to detect DNA, RNA, and other biomolecules has been hampered by lack of sensitivity and selectivity[1]. Herein, we developed a simple mix-and-read fluorescent assay for the detection of pathogen-derived DNA from Neisseria meningitides, which leads to meningococcal diseases, by using highly sensitive and selective fluorescence resonance energy transfer (FRET) signals from a quantum dot (QD)-based detection system. This assay is based on an enzymatic cascade signal amplification composed of flap endonuclease-1 (FEN-1)-assisted massive flap generation in the presence of a target DNA and exonuclease III (Exo III)-mediated flap recycling and exponential signal amplification. The synergistic generation and recycling of flap DNA by combining two enzymatic reactions are interpreted to be a contributing factor for the high detection sensitivity. Therefore, our approach enabled a simple and robust diagnostic tool that operates in complex media, detecting an extremely low abundance of targets. Materials and Methods: The first amplification assay was tested by mixing synthetic probe DNA with a target, followed by incubation with FEN-1 at 40oC for 1 hr, and polyacrylamide gel analysis was carried out to determine the target-dependent flap accumulation in a solution. Next, the Exo III-catalysed sensing was tested using the amplified flaps and a dye-functionalised QD. The kinetics of the flap binding to the capture probe on the surface of QD were also determined by controlling enzyme concentrations and the surface density of DNA. Results and Discussion: We used synthetic targets with different lengths of 50 and 200 nt respectively by selecting a target region of Neisseria meningitides genomic DNA in our assay[2]. In the first cleavage reaction, we showed the positive response of flaps to the concentration of both targets, meaning that our assay has the ability to detect DNA targets of any length including extremely long ones (Fig 1). We then examined whether an increase in the FRET signals could be initiated by the introduction of the flap DNA and a distinct dose-response profile between the concentration of flap and the QD fluorescence was observed. Conclusions: In summary, we designed a novel DNA detection assay by coupling FEN-1-based signal amplification with Exo III-based QD signal enhancement. Our assay does not require the target of interest to have an endonuclease recognition sequence or specific lengths, therefore, there is not any significant restriction in the choice of targets. Furthermore, due to the isothermal reaction and simple process consisting of stepwise addition of reagents into each well in the microplate, our detection method would open up the potential application into in situ simple and rapid diagnosis of infectious diseases. This project was funded by Imperial Confidence in Concept Scheme, Imperial NIHR BRC/Imperial Innovations Funds, and NIHR BRC at The Royal Marsden and The Institute of Cancer Research Funds 2014.