1. A "quasi" confocal droplet reader based on laser-induced fluorescence (LIF) cytometry for highly-sensitive and contamination-free detection.
- Author
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Zhu X, Liu B, Su S, Wang B, Bai Y, Huang H, Liu X, Cheng X, Wang X, Zhu L, Yang W, Gao N, Jing G, and Guo Y
- Subjects
- Equipment Design, ErbB Receptors genetics, HLA-B27 Antigen genetics, Limit of Detection, Microfluidic Analytical Techniques instrumentation, Mutation, Polymerase Chain Reaction instrumentation, DNA analysis, Lab-On-A-Chip Devices, Microfluidic Analytical Techniques methods, Polymerase Chain Reaction methods
- Abstract
Highly-sensitive and contamination-free droplet digital PCR (ddPCR) is an enabling technology and widely needed for accurate quantification of nucleic acid in clinical applications. In this paper, a novel droplet reader was developed by combining a "quasi" confocal laser-induced fluorescence (LIF) cytometry with a delicate microfluidic chip design. The droplets with a size of 90 μm was illuminated at an out-of-focus position by two aligned laser beams to generate maximum fluorescent signal. Additionally, the lateral offset position of the microfluidic chip should be precisely tuned so that the bandwidth of the FAM and VIC channels were configured at the matching sizes. Then, PMT gain voltages and pneumatic pressures were optimized for better droplet detection efficiencies. An aerosol adsorption experiment was performed to demonstrate that there was no aerosol contamination, and detected copy numbers of both mutants and wild types scaled linearly with the expected input copy numbers (r
2 >0.998) with a LoB of 0.0 copies and LoD of 3.0 copies. The results demonstrated that this droplet reader with the delicate chip is a convenient, highly-sensitive and contamination-free to detect fluorescence signals inside droplets after ddPCR, which is highly promising for broad applications of ddPCR in clinical diagnosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)- Published
- 2020
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