High density diffusion-free nanowell arrays

J Proteome Res. 2012 Aug 3;11(8):4382-91. doi: 10.1021/pr300467q. Epub 2012 Jul 13.


Proteomics aspires to elucidate the functions of all proteins. Protein microarrays provide an important step by enabling high-throughput studies of displayed proteins. However, many functional assays of proteins include untethered intermediates or products, which could frustrate the use of planar arrays at very high densities because of diffusion to neighboring features. The nucleic acid programmable protein array (NAPPA) is a robust in situ synthesis method for producing functional proteins just-in-time, which includes steps with diffusible intermediates. We determined that diffusion of expressed proteins led to cross-binding at neighboring spots at very high densities with reduced interspot spacing. To address this limitation, we have developed an innovative platform using photolithographically etched discrete silicon nanowells and used NAPPA as a test case. This arrested protein diffusion and cross-binding. We present confined high density protein expression and display, as well as functional protein-protein interactions, in 8000 nanowell arrays. This is the highest density of individual proteins in nanovessels demonstrated on a single slide. We further present proof of principle results on ultrahigh density protein arrays capable of up to 24000 nanowells on a single slide.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Diffusion
  • Humans
  • Lab-On-A-Chip Devices*
  • Protein Array Analysis / instrumentation*
  • Protein Biosynthesis
  • Protein Interaction Mapping
  • Proteome / biosynthesis
  • Proteome / genetics
  • Proteomics
  • Silicon / chemistry


  • Proteome
  • Silicon