Enzymatic Amplification for Ultrasensitive Biomarker Detection Using Surface Plasmon Resonance Imaging.

摘要

The field of clinical diagnostics calls for novel, ultrasensitive detection of biomarkers at femtomolar concentrations, which requires beyond the typical limit of florescence-based enzyme-linked immunosorbent assays (ELISA). Surface plasmon resonance imaging (SPRI), in conjunction with surface enzymatic amplification, serves as a highly suitable detection method of biomolecules at such low concentration levels.;The fundamental but crucial step of surface enzymatic amplification on both planar substrates and nanoparticle surfaces is to develop a bioactive surface attachment chemistry with the properties of high fidelity, reliability and reproducibility; hereby a newly developed pGlu surface attachment used for both DNA microarrays and DNA-coated silica nanoparticles is mentioned in all chapters and in Appendix A. The application of using this pGlu attachment on DNA microarrays is combined with the piezoelectrically-driven micromixer as demonstrated in Chapter 2. In Chaper 3-5, the dissertation is focused on using surface enzymatic methods in order to enhance the sensitivity, specificity and applicability of multiplexed biomarker detections, such as microRNA and biomarker proteins. One technique involves an enzymatic capture reaction (T4 ligation) on DNA-modified silica nanoparticles, which drives multiplexed microRNA detection by SPRI toward higher sensitivity as described in Chapter 3. On-chip multiplexed ssRNA synthesis by T7 RNA polymerase, in order to generate self-adsorbed RNA aptamer microarrays for protein biosensing, is demonstrated in Chapter 4. In Chapter 5, another approach used to advance SPRI detection utilizes the concept of "DNAzyme footprinting", in conjugation with transcription amplification and nanoparticle-enhanced measurements in order to reach a SPRI detection limit of human throbin as low as 100 femtomolar.