On-Chip Synthesis of RNA Aptamer Microarrays for Multiplexed Protein Biosensing with SPR Imaging Measurements

摘要

Microarrays of RNA aptamers are fabricated in anone-step, multiplexed enzymatic synthesis on gold thin films in anmicrofluidic format and then employed in the detection of proteinnbiomarkers with surface plasmon resonance imaging (SPRI)nmeasurements. Single-stranded RNA (ssRNA) oligonucleotidesnare transcribed on-chip from double-stranded DNA (dsDNA)ntemplates attached to microarray elements (denoted as generatornelements) by the surface transcription reaction of T7 RNAnpolymerase. As they are synthesized, the ssRNA oligonucleotidesndiffuse in the microfluidic channel and are quickly captured by hybridization adsorption onto adjacent single-stranded DNAn(ssDNA) microarray elements (denoted as detector elements) that contain a sequence complementary to 5′-end of the ssRNA.nThe RNA aptamers attached to these detector elements are subsequently used in SPRI measurements for the bioaffinityndetection of protein biomarkers. The microfluidic generator-detector element format permits the simultaneous fabrication ofnmultiple ssRNA oligonucleotides with different capture sequences that can hybridize simultaneously to distinct detector elementsnand thus create a multiplexed aptamer microarray. In an initial set of demonstration experiments, SPRI measurements are used tonmonitor the bioaffinity adsorption of human thrombin (hTh) and vascular endothelial growth factor (VEGF) proteins onto RNAnaptamer microarrays fabricated in situ with this on-chip RNA polymerase synthesis methodology. Additional SPRI measurementsnof the hydrolysis and desorption of the surface-bound ssRNA aptamers with a surface RNase H are used to verify the capture ofnssRNA with RNA−DNA surface hybridization onto the detector elements. The on-chip RNA synthesis described here is annelegant, one-step multiplexed methodology for the rapid and contamination-free fabrication of RNA aptamer microarrays fornprotein biosensing with SPRI.