TY - JOUR
T1 - Digital Microfluidics-Powered Real-Time Monitoring of Isothermal DNA Amplification of Cancer Biomarker
AU - Coelho, Beatriz Jorge
AU - Veigas, Bruno
AU - Bettencourt, Luís
AU - Águas, Hugo
AU - Fortunato, Elvira
AU - Martins, Rodrigo
AU - Baptista, Pedro V.
AU - Igreja, Rui
N1 - Funding Information:
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDP%2F04378%2F2020/PT#
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F04378%2F2020/PT#
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDP%2F50025%2F2020/PT#
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F50025%2F2020/PT#
info:eu-repo/grantAgreement/FCT/3599-PPCDT/PTDC%2FBTM-SAL%2F31201%2F2017/PT#
info:eu-repo/grantAgreement/FCT/OE/SFRH%2FBD%2F132904%2F2017/PT#
LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy?i4HB. Additional funding was received from FCT under the scope of the project reference LA/P/0037/2020, Beatriz J. Coelho and grant COVID/BD/152453/2022.
Publisher Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2022/3/28
Y1 - 2022/3/28
N2 - We introduce a digital microfluidics (DMF) platform specifically designed to perform a loop-mediated isothermal amplification (LAMP) of DNA and applied it to a real-time amplification to monitor a cancer biomarker, c-Myc (associated to 40% of all human tumors), using fluorescence microscopy. We demonstrate the full manipulation of the sample and reagents on the DMF platform, resulting in the successful amplification of 90 pg of the target DNA (0.5 ng/µL) in less than one hour. Furthermore, we test the efficiency of an innovative mixing strategy in DMF by employing two mixing methodologies onto the DMF droplets—low frequency AC (alternating current) actuation as well as back-and-forth droplet motion—which allows for improved fluorescence readouts. Fluo-rophore bleaching effects are minimized through on-chip sample partitioning by DMF processes and sequential droplet irradiation. Finally, LAMP reactions require only 2 µL volume droplets, which represents a 10-fold volume reduction in comparison to benchtop LAMP.
AB - We introduce a digital microfluidics (DMF) platform specifically designed to perform a loop-mediated isothermal amplification (LAMP) of DNA and applied it to a real-time amplification to monitor a cancer biomarker, c-Myc (associated to 40% of all human tumors), using fluorescence microscopy. We demonstrate the full manipulation of the sample and reagents on the DMF platform, resulting in the successful amplification of 90 pg of the target DNA (0.5 ng/µL) in less than one hour. Furthermore, we test the efficiency of an innovative mixing strategy in DMF by employing two mixing methodologies onto the DMF droplets—low frequency AC (alternating current) actuation as well as back-and-forth droplet motion—which allows for improved fluorescence readouts. Fluo-rophore bleaching effects are minimized through on-chip sample partitioning by DMF processes and sequential droplet irradiation. Finally, LAMP reactions require only 2 µL volume droplets, which represents a 10-fold volume reduction in comparison to benchtop LAMP.
KW - cancer biomarker
KW - digital microfluidics
KW - fluorescence detection
KW - loop-mediated isothermal amplification
KW - real-time nucleic acid amplification monitoring
UR - http://www.scopus.com/inward/record.url?scp=85128011730&partnerID=8YFLogxK
U2 - 10.3390/bios12040201
DO - 10.3390/bios12040201
M3 - Article
C2 - 35448261
AN - SCOPUS:85128011730
SN - 2079-6374
VL - 12
JO - Biosensors
JF - Biosensors
IS - 4
M1 - 201
ER -