Sensitive microarray-based biomarker detection using photonic crystal enhanced fluorescence and label-free imaging

Optical biosensors;Photonic crystals;Enhanced fluorescence

George, Sherine

关键词: Optical biosensors;    Photonic crystals;    Enhanced fluorescence;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/44753/Sherine_George.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Fluorescence-based biomarker detection in serum, saliva, urine, or breath condensate samples holds great promise for disease diagnostics and therapy monitoring because of its non-invasive nature. The microarray format, for its high-throughput and low analyte consumption characteristics, is particularly well suited for such applications since testing with acceptable levels of clinical sensitivity and specificity requires the use of a panel of atleast four to ten biomarkers in part due to inherent genetic diversity. While there exist reports on novel optical and electrochemical approaches capable of detection biomarkers at concentrations in the subpicomolar range, currently no platforms for the multiplexed cancer biomarker detection are in widespread use in point-of-care (POC) settings. This work presents the application of photonic crystal (PC) surfaces a platform for fluorescence enhancement utilized to improve detection sensitivity and as a platform for label free (LF) detection utilized for microspot quality analysis and anticipated to complement the fluorescence modality. First, a demonstration of the PC enabled LF imaging is applied to the quality analysis of printed spots in a DNA microarray. The first generation of polymer-based PCs is utilized alongside a high resolution label-free imaging detection instrument. By automating the process of identify missed spots and the assignment of a spot quality score for remaining features based on the acquired quantitative LF images, the variability in microarray data was capturedreduced and accuracy improved. Next, the fluorescent enhancement properties of a novel second generation of quartz-based PCs as a function of resonant angle were characterized in the context of DNA microarrays using a commercial microarray scanner. For the first time, the impact of multi-wavelength PC fluorescence enhancement was characterized in a two-color DNA microarray using Cyanine-5 (Cy5) and Cyanine-3 (Cy3). Observed enhancements in spot signal intensity and spot signal-to-noise ratio resulted in a 2x increase in the number of genes that were detected on the PC relative to standard glass microscope slides for each color. Meaningful enhancements in signal intensities and more importantly signal-to-noise ratios at shorter excitation wavelengths (as is the case for Cy3) were made possible only due to the negligible substrate fluorescence of the quartz-based PCs. Finally, a third generation of PCs based in silicon was successfully pursued to address the need to combine the negligible substrate fluorescence characteristics of the quartz-based PCs with an inexpensive and high-throughput fabrication process. The fluorescence enhancement afforded by silicon PCs combined with a compact detection instrument for optimal coupling incident light to PC resonant modes was characterized in the context of two types of biomarker assays. Using only 10 µL of analyte solutions, protein and miRNAFluorescence-based biomarker detection in serum, saliva, urine, or breath condensate samples holds great promise for disease diagnostics and therapy monitoring because of its non-invasive nature. The microarray format, for its high-throughput and low analyte consumption characteristics, is particularly well suited for such applications since testing with acceptable levels of clinical sensitivity and specificity requires the use of a panel of atleast four to ten biomarkers in part due to inherent genetic diversity. While there exist reports on novel optical and electrochemical approaches capable of detection biomarkers at concentrations in the subpicomolar range, currently no platforms for the multiplexed cancer biomarker detection are in widespread use in point-of-care (POC) settings. This work presents the application of photonic crystal (PC) surfaces a platform for fluorescence enhancement utilized to improve detection sensitivity and as a platform for label free (LF) detection utilized for microspot quality analysis and anticipated to complement the fluorescence modality. First, a demonstration of the PC enabled LF imaging is applied to the quality analysis of printed spots in a DNA microarray. The first generation of polymer-based PCs is utilized alongside a high resolution label-free imaging detection instrument. By automating the process of identify missed spots and the assignment of a spot quality score for remaining features based on the acquired quantitative LF images, the variability in microarray data was capturedreduced and accuracy improved. Next, the fluorescent enhancement properties of a novel second generation of quartz-based PCs as a function of resonant angle were characterized in the context of DNA microarrays using a commercial microarray scanner. For the first time, the impact of multi-wavelength PC fluorescence enhancement was characterized in a two-color DNA microarray using Cyanine-5 (Cy5) and Cyanine-3 (Cy3). Observed enhancements in spot signal intensity and spot signal-to-noise ratio resulted in a 2x increase in the number of genes that were detected on the PC relative to standard glass microscope slides for each color. Meaningful enhancements in signal intensities and more importantly signal-to-noise ratios at shorter excitation wavelengths (as is the case for Cy3) were made possible only due to the negligible substrate fluorescence of the quartz-based PCs. Finally, a third generation of PCs based in silicon was successfully pursued to address the need to combine the negligible substrate fluorescence characteristics of the quartz-based PCs with an inexpensive and high-throughput fabrication process. The fluorescence enhancement afforded by silicon PCs combined with a compact detection instrument for optimal coupling incident light to PC resonant modes was characterized in the context of two types of biomarker assays. Using only 10 µL of analyte solutions, protein and miRNA biomarkers implicated in cancer were detected at concentrations as low as 0.1pM and 0.6pM, respectively. This work lays the foundation for the development of a PC enhanced fluorescence (PCEF) based detection platform for POC diagnostics. biomarkers implicated in cancer were detected at concentrations as low as 0.1pM and 0.6pM, respectively. This work lays the foundation for the development of a PC enhanced fluorescence (PCEF) based detection platform for POC diagnostics.