On-chip synthesis of protein microarrays from DNA microarrays via coupled in vitro transcription and translation for surface plasmon resonance imaging biosensor applications

Abstract

Protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). A microarray of dsDNA elements (denoted as generator elements) that encode either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein is utilized to create multiple copies of mRNA (mRNA) in a surface RNA polymerase reaction; the mRNA transcripts are then translated into proteins by cell-free protein synthesis in a microfluidic format. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements (denoted as detector elements) and are specifically adsorbed. The net result is the on-chip, cell-free synthesis of a protein microarray that can be used immediately for SPRI protein biosensing. The dual element format greatly reduces any interference from the nonspecific adsorption of enzyme or proteins. SPRI measurements for the detection of the antibodies anti-GFP and antiluciferase were used to verify the formation of the protein microarray. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRI biosensing measurements in both clinical and research applications.

Figure 1

Schematic diagram of the on-chip synthesis of protein microarray from DNA microarray via surface in vitro transcription-translation. On the generator elements, the encoding dsDNA was covalently attached to the gold surface and mRNA was transcribed with T7 RNA polymerase. Translated His-tagged protein diffused to the adjacent detector elements and was captured by Cu(II)-NTA surface.

Figure 2

Real-time SPRI synthesis/adsorption kinetics measurements of newly synthesized GFP His-tagged protein onto detector, generator and control elements. The inset plot shows the initial SPRI Reflectivity changes at times less than 600 s.

Figure 3

a) A five-component, 16-element GFP-luciferase DNA microarray in a dual-channel microfluidic cell. b) Spatial diagram of the five components on the DNA microarray. c) Schematic diagram of antibody binding onto synthesized protein microarray. d) The SPRI difference images taken before and after the exposure to anti-GFP solutions showing the antibody binding onto corresponding element D1. e) The SPRI difference images taken before and after the exposure to anti-luciferase solutions showing the antibody binding onto corresponding element D2. f) Real-time SPRI adsorption kinetics measurements of anti-GFP specific binding onto D1. g) Real-time SPRI adsorption kinetics measurements of anti-luciferase specific binding onto D2.