TY - JOUR
T1 - Rapid and sensitive detection of miRNA via light scatter-aided emulsion-based isothermal amplification using a custom low-cost device
AU - Hertenstein, Tyler
AU - Tang, Yisha
AU - Day, Alexander S.
AU - Reynolds, Jocelyn
AU - Viboolmate, Patrick V.
AU - Yoon, Jeong Yeol
N1 - Publisher Copyright:
© 2023 Elsevier B.V.
PY - 2023/10/1
Y1 - 2023/10/1
N2 - MicroRNAs are likely to be a next-generation clinical biomarker for many diseases. While gold-standard technologies, e.g., reverse transcription-quantitative polymerase chain reaction (RT-qPCR), exist for microRNA detection, there is a need for rapid and low-cost testing. Here, an emulsion loop-mediated isothermal amplification (eLAMP) assay was developed for miRNA that compartmentalizes a LAMP reaction and shortens the time-to-detection. The miRNA was a primer to facilitate the overall amplification rate of template DNA. Light scatter intensity decreased when the emulsion droplet got smaller during the ongoing amplification, which was utilized to moitor the amplification non-invasively. A custom low-cost device was designed and fabricated using a computer cooling fan, a Peltier heater, an LED, a photoresistor, and a temperature controller. It allowed more stable vortexing and accurate light scatter detection. Three miRNAs, miR-21, miR-16, and miR-192, were successfully detected using the custom device. Specifically, new template and primer sequences were developed for miR-16 and miR-192. Zeta potential measurements and microscopic observations confirmed emulsion size reduction and amplicon adsorption. The detection limit was 0.01 fM, corresponding to 2.4 copies per reaction, and the detection could be made in 5 min. Since the assays were rapid and both template and miRNA + template could eventually be amplified, we introduced the success rate (compared to the 95% confidence interval of the template result) as a new measure, which worked well with lower concentrations and inefficient amplifications. This assay brings us one step closer to allowing circulating miRNA biomarker detection to become commonplace in the clinical world.
AB - MicroRNAs are likely to be a next-generation clinical biomarker for many diseases. While gold-standard technologies, e.g., reverse transcription-quantitative polymerase chain reaction (RT-qPCR), exist for microRNA detection, there is a need for rapid and low-cost testing. Here, an emulsion loop-mediated isothermal amplification (eLAMP) assay was developed for miRNA that compartmentalizes a LAMP reaction and shortens the time-to-detection. The miRNA was a primer to facilitate the overall amplification rate of template DNA. Light scatter intensity decreased when the emulsion droplet got smaller during the ongoing amplification, which was utilized to moitor the amplification non-invasively. A custom low-cost device was designed and fabricated using a computer cooling fan, a Peltier heater, an LED, a photoresistor, and a temperature controller. It allowed more stable vortexing and accurate light scatter detection. Three miRNAs, miR-21, miR-16, and miR-192, were successfully detected using the custom device. Specifically, new template and primer sequences were developed for miR-16 and miR-192. Zeta potential measurements and microscopic observations confirmed emulsion size reduction and amplicon adsorption. The detection limit was 0.01 fM, corresponding to 2.4 copies per reaction, and the detection could be made in 5 min. Since the assays were rapid and both template and miRNA + template could eventually be amplified, we introduced the success rate (compared to the 95% confidence interval of the template result) as a new measure, which worked well with lower concentrations and inefficient amplifications. This assay brings us one step closer to allowing circulating miRNA biomarker detection to become commonplace in the clinical world.
KW - Loop-mediated isothermal amplification
KW - emulsion
KW - light scatter
KW - miR-16
KW - miR-192
KW - miR-21
UR - http://www.scopus.com/inward/record.url?scp=85163793256&partnerID=8YFLogxK
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U2 - 10.1016/j.bios.2023.115444
DO - 10.1016/j.bios.2023.115444
M3 - Article
C2 - 37329805
AN - SCOPUS:85163793256
SN - 0956-5663
VL - 237
JO - Biosensors and Bioelectronics
JF - Biosensors and Bioelectronics
M1 - 115444
ER -