Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection System for Viral DNA Sensing

Kenneth N Hass; Mengdi Bao; Qian He; Li Liu; Jiacheng He; Myeongkee Park; Peiwu Qin; Ke Du

doi:10.1021/acsomega.0c03917

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection System for Viral DNA Sensing

ACS Omega. 2020 Oct 13;5(42):27433-27441. doi: 10.1021/acsomega.0c03917. eCollection 2020 Oct 27.

Authors

Kenneth N Hass¹, Mengdi Bao¹, Qian He², Li Liu³, Jiacheng He¹, Myeongkee Park⁴, Peiwu Qin², Ke Du^{1

3

5}

Affiliations

¹ Department of Mechanical Engineering, Rochester Institute of Technology, Rochester, New York 14623, United States.
² Center of Precision Medicine and Healthcare, Tsinghua-Berkeley Shenzhen Institute, Shenzhen, Guangdong Province 518055, China.
³ Department of Microsystems Engineering, Rochester Institute of Technology, Rochester, New York 14623, United States.
⁴ Department of Chemistry, Dong-A University, Busan 49315, Republic of Korea.
⁵ School of Chemistry and Materials Science, Rochester Institute of Technology, Rochester, New York 14623, United States.

Abstract

A fully Integrated Micropillar Polydimethylsiloxane Accurate CRISPR deTection (IMPACT) system is developed for viral DNA detection. This powerful system is patterned with high-aspect-ratio micropillars to enhance reporter probe binding. After surface modification and probe immobilization, the CRISPR-Cas12a/crRNA complex is injected into the fully enclosed microchannel. With the presence of a double-stranded DNA target, the CRISPR enzyme is activated and denatures the single-stranded DNA reporters from the micropillars. This collateral cleavage releases fluorescence reporters into the assay, and the intensity is linearly proportional to the target DNA concentration ranging from 0.1 to 10 nM. Importantly, this system does not rely on the traditional dye-quencher-labeled probe, thus reducing the fluorescence background presented in the assay. Furthermore, our one-step detection protocol is performed on-chip at isothermal conditions (37 °C) without using complicated and time-consuming off-chip probe hybridization and denaturation. This miniaturized and fully packed IMPACT chip demonstrates sensitive and accurate DNA detection within 120 min and paves ways to the next-generation point-of-care diagnostics, responding to emerging and deadly pathogen outbreaks.