Fast Prototyping Microfluidics: Integrating Droplet Digital Lamp for Absolute Quantification of Cancer Biomarkers

• Published in: Sensors (Basel, Switzerland) Switzerland), 2020-03, Vol.20 (6), p.1624
• Main Authors: Oliveira, Beatriz, Veigas, Bruno, Fernandes, Alexandra R, Águas, Hugo, Martins, Rodrigo, Fortunato, Elvira, Baptista, Pedro Viana
• Format: Article
• Language: English
• Subjects: Biomarkers; Biomarkers, Tumor - genetics; Biomarkers, Tumor - isolation & purification; Biosensing Techniques; Cancer; Cell cycle; Deoxyribonucleic acid; Design; digital amplification; Direct laser writing; DNA; DNA, Neoplasm - genetics; DNA, Neoplasm - isolation & purification; Droplets; Gene expression; Humans; integrated microfluidics device; lab-on-a-chip; Lab-On-A-Chip Devices; Limit of Detection; loop-mediated isothermal amplification; Microfluidics; Microfluidics - methods; Molecular Diagnostic Techniques - methods; Multilayers; Mutation; Neoplasms - diagnosis; Neoplasms - genetics; Neoplasms - pathology; Nucleic Acid Amplification Techniques - methods; oncogenes; Pathology, Molecular - methods; Polystyrene resins; Prototyping; Software; Thermal cycling
• ISSN: 1424-8220; 1424-8220
• DOI: 10.3390/s20061624

Microfluidic (MF) advancements have been leveraged toward the development of state-of-the-art platforms for molecular diagnostics, where isothermal amplification schemes allow for further simplification of DNA detection and quantification protocols. The MF integration with loop-mediated isothermal amplification (LAMP) is today the focus of a new generation of chip-based devices for molecular detection, aiming at fast and automated nucleic acid analysis. Here, we combined MF with droplet digital LAMP (ddLAMP) on an all-in-one device that allows for droplet generation, target amplification, and absolute quantification. This multilayer 3D chip was developed in less than 30 minutes by using a low-cost and extremely adaptable production process that exploits direct laser writing technology in "Shrinky-dinks" polystyrene sheets. ddLAMP and target quantification were performed directly on-chip, showing a high correlation between target concentration and positive droplet score. We validated this integrated chip via the amplification of targets ranging from five to 500,000 copies/reaction. Furthermore, on-chip amplification was performed in a 10 µL volume, attaining a limit of detection of five copies/µL under 60 min. This technology was applied to quantify a cancer biomarker, , but it can be further extended to any other disease biomarker.