Label-free, Multiplexed Bioassay on Gold/Silver Striped Nanorods Using Fluorescent Conjugated Polymers.

摘要

Multiplexing techniques allow to obtain high density of biomolecule information with reduced assay time, cost and sample volume. Such multiplexed bioassays have been demonstrated after overcoming the technical hurdles in encoding, functionalizing, decoding, detecting, and improving the limited number of assays to be performed simultaneously. However, challenges in simplifying the tagging process and eliminating the need for pre-labeling reporter molecules for detection still remain. The needs to overcome such a hurdle have motivated research in developing a label-free multiplexed assay system. Fluorescent conjugated polymers, especially, cationic conjugated polythiophene derivatives, offer a unique opportunity as the optical probe for biosensing in a label-free fashion with high sensitivity and specificity.;This dissertation reports the development of a label-free, multiplexed bioassay method by using cationic polythiophene derivatives as optical transducers and Au/Ag striped nanorods as encoded substrates. The background knowledge on current multiplexing techniques and conjugated polymers based biosensing applications is described in Chapter 1.;Chapter 2 describes the synthesis and characterization of metallic striped nanorods and cationic conjugated polythiophene derivatives. Au/Ag stripped nanorods were prepared by templated, sequential electrodeposition of Au and Ag into a mesoporous membrane. In addition, cationic conjugated polythiophene derivatives were successfully synthesized according published procedures. Optimization of synthesized conjugated polymers based DNA detection in aqueous solution was also investigated.;Chapter 3 describes the proof-of-concept experiment for label free, multiplexed DNA detection on Au/Ag stripped nanorods using cationic conjugated polythiophene derivatives. The sensitivity and specificity of conjugated polymer based DNA detection on silica coated nanorods was later studied. To conclude, DNA detection in a triplex fashion was also demonstrated with high selectivity.;Chapter 4 describes the distance-dependent fluorescence quenching study of conjugated polymers on the nanorods, to improve the assay performance of conjugated based bioassay on nanorods. Silica layer with tunable thickness was coated onto the nanorods, and the impact of coating thickness on the fluorescence output of conjugated polymers was investigated. For comparison, distance-dependent quenching of small dye molecules or polymers on metal substrates of different shapes was monitored in parallel.;Chapter 5 describes a detailed study on thermodynamics and kinetics of formation of cationic polymer-DNA complexes. My preliminary finding confirmed the strong influence of electrostatically charged conjugated polymers on the stability and reaction rate of DNA hybridization.;Chapter 6 describes the extension of multiplexing concepts to miRNA detection. I found that hybridization of miRNA to polymer/DNA duplexes produced much lower fluorescence signal than that of DNA due to their conformational difference. A competitive method was developed to compensate the weaker fluorescence responses of polymer-RNA-DNA complexes for miRNA detection. The optimized length of DNA competitor was determined and the assay performance was investigated.;Chapter 7 describes multiplexed detection of protein cancer markers on the metallic striped nanorods using fluorescent conjugated polymers. To use conjugated polymers as transducer for specific protein binding event, DNA was introduced as bridging molecule to protein detection probes. The antigen binding event was revealed by electrostatic binding of cationic polythiophene derivatives onto the negatively charged dsDNA tagged onto a secondary antibody probe prior to antigen recognition. Three cancer markers, prostatespecific antigen (PSA, prostate cancer marker), carcinoembryonic antigen (CEA, colorectal cancer marker), and beta-human chorionic gonadotropin (betahCG, testicular cancer marker), were used as the model molecules to examine assay performance.