![131522-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-07/131522-Thumbnail%20Image.png?versionId=d0Jp8DNCqtUHETYcrQIKfqWmKuCe2HrO&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240616/us-west-2/s3/aws4_request&X-Amz-Date=20240616T060921Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=16e3037a6ba1199503ce51c5987a3956408bc56e69e91620764b35dafd16f5de&itok=o9i4p5HP)
![161881-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-11/161881-Thumbnail%20Image.png?versionId=NaU0boY6iU0jlAZQvsmvpNPTMBf4irSY&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240616/us-west-2/s3/aws4_request&X-Amz-Date=20240616T092618Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=b0b55c9ff0b0460b6cc40006b68102169574bfaa3c55f379c352c36b2dfdb9b6&itok=S_PJTgo4)
![127868-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-04/127868-Thumbnail%20Image.png?versionId=65Fhj794hZ4mcOfeMIHX0awPDDxFpyYF&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240616/us-west-2/s3/aws4_request&X-Amz-Date=20240616T042311Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=642af5a7a4b47ed672ff5dd9563b8268a6afdc477a21268012140fd43fe803a5&itok=_ix4B2Yu)
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.