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Development of a DNA multiplexing system utilizing encoded porous silica photonic crystal particles


A DNA multiplexing system utilizing encoded porous silica photonic crystal particles has been developed. The critical components necessary to achieve this were the development of (1) a high capacity encoding methodology, (2) instrumentation and methods for decoding and assay readout, (3) a microfabrication technique and a means to stabilize the microparticles in assay conditions as well as allow for the use of existing surface chemistries for probe immobilization, and (4) a model system multiplexed DNA assay. Once, these components were in place an experiment was carried out that demonstrated multiplexed detection. The encoding method produces a one dimensional photonic structure that exhibits optical reflectivity spectra with multiple peaks. The height of a given peak can be modulated, or the peak can be deleted all together. The resultant spectral peaks can be used as a spectral barcode, where the number of codes possible is the number of resolvable peak intensity levels (states) raised to the maximum number of wavelength resolved peaks (bits) present in the spectral code. The capacity to encode over 1 million codes has been demonstrated using the method. Instrumentation and methods for high-throughput decoding and assay readout of the encoded microparticles were developed. The readout system is comprised of a fluorescence microscope modified for high resolution spectral imaging. A decoding method, which identifies codes based on the number of peaks present, the relative distances between neighbor peaks, and thresholds of the peak intensity, was also developed. A method for the microfabrication of freestanding porous silica particles containing spectral barcodes was developed and shown to produce uniform populations of particles, approximately 25 [mu]m in diameter. Additionally, thermal oxidation of the porous silicon to porous silica was demonstrated not to have a detrimental effect on the optical codes, thus creating an encoded particle with increased chemical resistance and compatibility with common glass-based immobilization chemistries. A model system multianalyte assay based on 50-mer oligonucleotide probes and perfect match fluorescently labeled targets was used as a means to demonstrate multiplexed detection and act as a benchmark for the accuracy of the photonic encoding method. Essentially, a set three particle types, each with a unique photonic code, were each immobilized with a specific oligonucleotide probe and reacted with a pool of fluorescently labeled targets. Each target was dyed a different color. The results agreed with the experimental design, thus demonstrating both multiplexed detection and validation of the photonic encoding method. Finally, the successful decoding and assay readout demonstrated chemical stability of both the photonic codes and the immobilized probes through the biological assay conditions

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