Surface enzyme kinetics and enzymatically amplified biosensing of nucleic acid arrays studied by surface plasmon resonance imaging and surface plasmon fluorescence spectroscopy.

摘要

The application of surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS) to surface enzyme kinetics studies and ultrasensitive detection of microRNAs (miRNAs) is presented, A surface enzyme reaction was described using a simple reaction model that coupled the contributions of both enzyme adsorption and surface enzyme catalysis. The overall enzymatic reaction was found to follow classical Langmuir adsorption and Michaelis-Menten concepts using three rate constants: enzyme adsorption (ka), enzyme desorption (k d) and enzyme catalysis (kcat). Experiments using the 3' → 5' exodeoxyribonuclease activity of exonuclease III (Exo III) on double-stranded DNA microarrays as a function of temperature and enzyme concentrations were used to demonstrate how this reaction model can be applied to quantitatively analyze the SPRI data.; This reaction model was further employed for the kinetic studies of ribonuclease H (RNase H) surface hydrolysis of RNA-DNA heteroduplexes formed on DNA microarrays. A combination of real-time SPRI and SPFS measurements was used in order to obtain a complete and independent set of surface coverage data for each surface species. The surface enzyme kinetics of RNase H is significantly different from that observed for Exo III. The time-dependent SPRI and SPFS data at various enzyme concentrations were quantitatively analyzed and their rate constants were determined from fitting all of the data sets.; Finally, a novel surface amplification methodology was developed for the ultrasensitive detection of miRNAs with SPRI in a microarray format. In this process, miRNAs were first adsorbed onto locked nucleic acid (LNA) modified DNA oligonucleotide arrays of complementary sequences. The surface bound miRNAs were then polyadenylated by the enzyme poly(A) polymerase, followed by exposure of the array to T30-coated gold nanoparticles that was finally detected with SPRI. This methodology was first used to detect synthetic miRNA targets that has reduced the detection limit of SPRI to a concentration of 10 fM. The utility of this methodology was finally demonstrated by the direct detection and quantitation of multiple miRNA sequences from mouse total RNA samples.