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Rationale: Cell-free protein microarrays display naturally-folded proteins based on just-in-time in situ synthesis, and have made important contributions to basic and translational research. However, the risk of spot-to-spot cross-talk from protein diffusion during expression has limited the feature density of these arrays.
Methods: In this work, we developed the Multiplexed Nucleic Acid Programmable Protein Array (M-NAPPA), which significantly increases the number of displayed proteins by multiplexing as many as five different gene plasmids within a printed spot.
Results: Even when proteins of different sizes were displayed within the same feature, they were readily detected using protein-specific antibodies. Protein-protein interactions and serological antibody assays using human viral proteome microarrays demonstrated that comparable hits were detected by M-NAPPA and non-multiplexed NAPPA arrays. An ultra-high density proteome microarray displaying > 16k proteins on a single microscope slide was produced by combining M-NAPPA with a photolithography-based silicon nano-well platform. Finally, four new tuberculosis-related antigens in guinea pigs vaccinated with Bacillus Calmette-Guerin (BCG) were identified with M-NAPPA and validated with ELISA.
Conclusion: All data demonstrate that multiplexing features on a protein microarray offer a cost-effective fabrication approach and have the potential to facilitate high throughput translational research.
- Yu, Xiaobo (Author)
- Song, Lusheng (Author)
- Petritis, Brianne (Author)
- Bian, Xiaofang (Author)
- Wang, Haoyu (Author)
- Viloria, Jennifer (Author)
- Park, Jin (Author)
- Bui, Hoang (Author)
- Li, Han (Author)
- Wang, Jie (Author)
- Liu, Lei (Author)
- Yang, Liuhui (Author)
- Duan, Hu (Author)
- McMurray, David N. (Author)
- Achkar, Jacqueline M. (Author)
- Magee, Mitch (Author)
- Qiu, Ji (Author)
- LaBaer, Joshua (Author)
- Biodesign Institute (Contributor)
Yu, X., Song, L., Petritis, B., Bian, X., Wang, H., Viloria, J., . . . Labaer, J. (2017). Multiplexed Nucleic Acid Programmable Protein Arrays. Theranostics, 7(16), 4057-4070. doi:10.7150/thno.20151
- 2018-02-22 03:44:05
- 2021-12-07 11:13:39
- 2 years 11 months ago