On-chip synthesis of protein microarrays from DNA microarrays via coupled in vitro transcription and translation for surface plasmon resonance imaging biosensor applications.
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.
Department of Chemistry, University of California-Irvine, Irvine, California 92697, United States.
Ting H
TH
Seefeld
Aaron R
AR
Halpern
Robert M
RM
Corn
D000327:Adsorption; D017123:Bacteriophage T7 / Q000201:enzymology; D003300:Copper / Q000737:chemistry; D004247:DNA / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006639:Histidine / Q000032:analysis; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D009842:Oligopeptides / Q000032:analysis; D040081:Protein Array Analysis / Q000295:instrumentation*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D020349:Surface Plasmon Resonance / Q000295:instrumentation; D014158:Transcription, Genetic
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D006639:Histidine; D003300:Copper; D004247:DNA; D008156:Luciferases; D012321:DNA-Directed RNA Polymerases