Highly precisive digital polymerase chain reaction based on inkjet printer

Abstract

BackgroundTo eliminate the indeterminate droplet partitions that highly obfuscate the distinguishment of the positive and the negative partitions in digital polymerase chain reaction (dPCR), the highly precisive droplets array of dPCR was developed via the inkjet printer coupled with the X-Y stage. ResultsThe highly precisive droplets could be generated by the inkjet printer and the droplets array would be arranged by the movable X-Y stage at the same time. The highly precisive droplets array in a volume of at least 20,000 droplets could be obtained with a coefficients of variation (CV) of 1.57 % for the decided droplets in a diameter of 145.16 μm. The CV of the fluorescence intensities of droplets in array could be lowered to 3.17 % for the droplets of 5 μM fluorescein sodium solution, which guaranteed the clear identification of the positive droplets in the absolute quantification of dPCR. In addition, the thermal amplification in dPCR was designed to implement at the same sheet to avoid the delivery of the droplet partitions. The highly precisive dPCR was demonstrated through the wide linear range of more than 103 in the measurement of the model GAPDH gene with a low limitation of detection (LOD) of 0.60 copies/μL. Finally, the practicality of developed dPCR was confirmed by the measurement of the human mammaglobin (hMAM) gene in MCF-7 cell as well. Significance and noveltyThe successful development of the highly precisive droplet array through the inkjet printer coupled with X-Y stage eliminated the indeterminate positive droplet partitions in dPCR. It paved a way to develop the compact and highly sensitive dPCR.