Nucleic acid amplification is a key step in nucleic acid detection assays. The use of digital microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. Unlike polymerase chain reaction (PCR) amplification methods (requiring repeated cycles of three or two temperature-dependent steps during amplification), low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the combined use of digital microfluidics and molecular beacon (MB)-assisted isothermal circular strand displacement polymerization (ICSDP) technique is described for detection of miR-210, the most consistently and predominantly upregulated hypoxia-inducible microRNA. The ability of the droplet ICSDP isothermal method to discriminate between full-matched, single mismatched and unrelated sequences has been demonstrated. The detection of a range of nM/pM miR-210 solutions compartmentalized in nanoliter-sized droplets was performed to demonstrate the method capabilities for detecting as low as 10-18 moles of microRNA target sequences compartmentalized in 20 nL droplets. The method capability to operate with biological samples was tested by detecting miR-210 from transfected K562 cells. When combined with digital microfluidics ICSDP offers a convenient environment for fast, simple and effective DNA and microRNA detection. In addition, droplet ICSDP isothermal procedure simplifies the integration of nucleic acids detection protocols in microfluidic devices. Potentials offered by the method have been demonstrated by designing specific experiments using a total of 200 nL of solutions carrying a total of as low as 6 x 107 molecules. In addition, the method offers possibilities for specifically detecting as low as 3.3 10-18 moles of microRNA target sequences compartmentalized in 20 nL droplets. The new approach represents a promising tool for the development of simpler and more convenient lab-on-chip devices for miRNAs detection.
Digital microfluidic devices for isothermal circular strand displacement polymerization of microRNAs
GASPARELLO, Jessica;
2015
Abstract
Nucleic acid amplification is a key step in nucleic acid detection assays. The use of digital microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. Unlike polymerase chain reaction (PCR) amplification methods (requiring repeated cycles of three or two temperature-dependent steps during amplification), low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the combined use of digital microfluidics and molecular beacon (MB)-assisted isothermal circular strand displacement polymerization (ICSDP) technique is described for detection of miR-210, the most consistently and predominantly upregulated hypoxia-inducible microRNA. The ability of the droplet ICSDP isothermal method to discriminate between full-matched, single mismatched and unrelated sequences has been demonstrated. The detection of a range of nM/pM miR-210 solutions compartmentalized in nanoliter-sized droplets was performed to demonstrate the method capabilities for detecting as low as 10-18 moles of microRNA target sequences compartmentalized in 20 nL droplets. The method capability to operate with biological samples was tested by detecting miR-210 from transfected K562 cells. When combined with digital microfluidics ICSDP offers a convenient environment for fast, simple and effective DNA and microRNA detection. In addition, droplet ICSDP isothermal procedure simplifies the integration of nucleic acids detection protocols in microfluidic devices. Potentials offered by the method have been demonstrated by designing specific experiments using a total of 200 nL of solutions carrying a total of as low as 6 x 107 molecules. In addition, the method offers possibilities for specifically detecting as low as 3.3 10-18 moles of microRNA target sequences compartmentalized in 20 nL droplets. The new approach represents a promising tool for the development of simpler and more convenient lab-on-chip devices for miRNAs detection.
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