Fast Prototyping Microfluidics: Integrating Droplet Digital Lamp for Absolute Quantification of Cancer Biomarkers
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 a...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2020-03-01
|
| Series: | Sensors |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1424-8220/20/6/1624 |
| _version_ | 1828120649779904512 |
|---|---|
| author | Beatriz Oliveira Bruno Veigas Alexandra R. Fernandes Hugo Águas Rodrigo Martins Elvira Fortunato Pedro Viana Baptista |
| author_facet | Beatriz Oliveira Bruno Veigas Alexandra R. Fernandes Hugo Águas Rodrigo Martins Elvira Fortunato Pedro Viana Baptista |
| author_sort | Beatriz Oliveira |
| collection | DOAJ |
| description | 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, <i>c-MYC</i>, but it can be further extended to any other disease biomarker. |
| first_indexed | 2024-04-11T14:08:33Z |
| format | Article |
| id | doaj.art-ccef6e916ca544f8bf5f8ccbb195e735 |
| institution | Directory Open Access Journal |
| issn | 1424-8220 |
| language | English |
| last_indexed | 2024-04-11T14:08:33Z |
| publishDate | 2020-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Sensors |
| spelling | doaj.art-ccef6e916ca544f8bf5f8ccbb195e7352022-12-22T04:19:48ZengMDPI AGSensors1424-82202020-03-01206162410.3390/s20061624s20061624Fast Prototyping Microfluidics: Integrating Droplet Digital Lamp for Absolute Quantification of Cancer BiomarkersBeatriz Oliveira0Bruno Veigas1Alexandra R. Fernandes2Hugo Águas3Rodrigo Martins4Elvira Fortunato5Pedro Viana Baptista6UCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Campus de Caparica, 2829-516 Caparica, PortugalUCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Campus de Caparica, 2829-516 Caparica, PortugalUCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugali3N|CENIMAT, Departamento de Ciência dos Materiais, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugali3N|CENIMAT, Departamento de Ciência dos Materiais, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugali3N|CENIMAT, Departamento de Ciência dos Materiais, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Campus de Caparica, 2829-516 Caparica, PortugalUCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Campus de Caparica, 2829-516 Caparica, PortugalMicrofluidic (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, <i>c-MYC</i>, but it can be further extended to any other disease biomarker.https://www.mdpi.com/1424-8220/20/6/1624digital amplificationintegrated microfluidics deviceloop-mediated isothermal amplificationoncogeneslab-on-a-chip |
| spellingShingle | Beatriz Oliveira Bruno Veigas Alexandra R. Fernandes Hugo Águas Rodrigo Martins Elvira Fortunato Pedro Viana Baptista Fast Prototyping Microfluidics: Integrating Droplet Digital Lamp for Absolute Quantification of Cancer Biomarkers Sensors digital amplification integrated microfluidics device loop-mediated isothermal amplification oncogenes lab-on-a-chip |
| title | Fast Prototyping Microfluidics: Integrating Droplet Digital Lamp for Absolute Quantification of Cancer Biomarkers |
| title_full | Fast Prototyping Microfluidics: Integrating Droplet Digital Lamp for Absolute Quantification of Cancer Biomarkers |
| title_fullStr | Fast Prototyping Microfluidics: Integrating Droplet Digital Lamp for Absolute Quantification of Cancer Biomarkers |
| title_full_unstemmed | Fast Prototyping Microfluidics: Integrating Droplet Digital Lamp for Absolute Quantification of Cancer Biomarkers |
| title_short | Fast Prototyping Microfluidics: Integrating Droplet Digital Lamp for Absolute Quantification of Cancer Biomarkers |
| title_sort | fast prototyping microfluidics integrating droplet digital lamp for absolute quantification of cancer biomarkers |
| topic | digital amplification integrated microfluidics device loop-mediated isothermal amplification oncogenes lab-on-a-chip |
| url | https://www.mdpi.com/1424-8220/20/6/1624 |
| work_keys_str_mv | AT beatrizoliveira fastprototypingmicrofluidicsintegratingdropletdigitallampforabsolutequantificationofcancerbiomarkers AT brunoveigas fastprototypingmicrofluidicsintegratingdropletdigitallampforabsolutequantificationofcancerbiomarkers AT alexandrarfernandes fastprototypingmicrofluidicsintegratingdropletdigitallampforabsolutequantificationofcancerbiomarkers AT hugoaguas fastprototypingmicrofluidicsintegratingdropletdigitallampforabsolutequantificationofcancerbiomarkers AT rodrigomartins fastprototypingmicrofluidicsintegratingdropletdigitallampforabsolutequantificationofcancerbiomarkers AT elvirafortunato fastprototypingmicrofluidicsintegratingdropletdigitallampforabsolutequantificationofcancerbiomarkers AT pedrovianabaptista fastprototypingmicrofluidicsintegratingdropletdigitallampforabsolutequantificationofcancerbiomarkers |