National Science Foundation     |     Directorate for Engineering  (ENG)
Division of Chemical, Bioengineering, Environmental, & Transport Systems  (CBET)
 
CBET Award Achievements
Notable Accomplishments from CBET Awards
 
 
Photonic Crystal Enhanced Fluorescence
 
Brian T. Cunningham    University of Illinois at Urbana-Champaign

Background:  There is a critical need for developing techniques that can describe altered gene expression and cellular protein profiles and relate these to human pathologies.  It is therefore of key importance to identify and evaluate the performance in the generation, processing, and interpretation of microarray data to determine the extent to which each part of the process contributes to data variability.  Currently, however, there is no way to measure the quality of the immobilized DNA spots in terms of their density, repeatability, and uniformity.  Published studies indicate that site-related effects should be taken into account when combining data from multiple sites, and that the root cause of variability arises from the density of the deposited spots.
 
Drug companies are pursuing research to develop reliable biomarkers of efficacy and toxicity, often supported by DNA microarray data.  Although DNA microarrays represent one of the core technologies for gene expression analysis, concerns have been raised regarding the reliability and consistency of microarray data and its application in the clinical and regulatory setting.
 
Although it is of paramount importance to identify and evaluate the performance of each individual step in the generation, processing, and interpretation of microarray data to determine the extent to which each part of the process contributes to data variability, there is currently no way to measure the quality of the immobilized DNA spots in terms of their density, repeatability, and uniformity.
 
While pin-based printing is relatively simple and inexpensive, several aspects of the slide preparation, printing, and drying processes result in intraspot and interspot nonuniformities both within a single array and across multiple arrays produced in the same batch.  For example, pin-to-pin variation can result in 10-25% variation in spot density, and problems with pin clogging and gradual deformation due to wear have been well-documented.  The accumulation of mechanical wear in spotting pins occurs at different rates for each pin, resulting in defects such as missing, irregular sized, and irregular shaped spots.  In addition to the pins themselves, spotting buffer composition, surface chemistry uniformity, and lack of environmental control can all lead to variability in deposited spot density, or spot drying characteristics that lead to nonuniform distribution of DNA either around the periphery or in the center of spots.

Results:  Currently, few if any objective metrics or established quality control standards are used to evaluate the quality of microarray studies, and several sources of error and variability have been identified in addition to the quality of the microarray spots.  Often, the assessment of microarray data quality requires running replicates and making intra-sample comparisons to determine reproducibility, which is particularly expensive for large translational studies involving large patient populations.  Using replicate arrays is an expensive strategy and cannot be routinely applied where quantities of precious biological samples are limited.  Building in a quality control assessment of the spotted array and an incorporated report of quality metrics would be enormously useful for comparing data across different experiments.
 
One of the specific objectives of the research project is to combine Photonic Crystal (PC) label-free detection with PC-enhanced fluorescence as a means for providing quantitative measurements of DNA capture probes on microarray surfaces.  The Cunningham Team has designed and constructed a microscope that is capable of providing high resolution label-free images of DNA or protein capture probes on a PC surface, that is also capable of gathering fluorescent images of the same spots, and overlaying the two images.
 
 
Brian Cunningham 1-1
 
Figure 1.  Using a Perkin-Elmer Piezorray, ~300 pL spots of strepavidin-cyanine5 (SA-Cy5) at 50mg/ml and two-fold dilutions were deposited onto a photonic crystal functionalized with an epoxysilane surface chemistry.  After a 24 hour incubation and subsequent wash, label-free and fluorescence images of the arrayed spots were taken.  The label-free image (a) demonstrates sensitive detection of the surface-bound molecules and excellent spatial resolution.  An enhanced fluorescence image (b) precisely registered to the label-free image provides 315x intensity enhancement, 45x signal-to-noise enhancement, and a detection limit reduced by nearly two orders of magnitude compared to a glass slide control
 
 
Brian Cunningham 1-2
Figure 2.  A custom combined label-free and enhanced fluorescence microscope enables the excellent sensitivity, resolution and registration, and is illustrated schematically in (c).  It consists of an expanded Helium-Neon excitation source, a precision motorized optical gimbal mount, an Olympus BX51 fluorescence microscope, and custom control and acquisition software.  A scanning electron micrograph of the photonic crystal used in this study is shown in (d).
 
Credit for Figures 1 & 2:  Brian T. Cunningham, University of Illinois at Urbana-Champaign
 
Scientific Uniqueness:  This research project combines Photonic Crystal (PC) label-free detection with PC-enhanced fluorescence as a means for providing quantitative measurements of DNA capture probes on microarray surfaces.  Using this innovative technique this powerful system is capable of providing high resolution label-free images of DNA or protein capture probes on a Photonic Crystal surface, and is also capable of gathering fluorescent images of the same spots, and overlaying the two images.

This project addresses the NSF Strategic Outcome Goals, as described in the NSF Strategic Plan 2006-2011, as follows:
 
Primary Strategic Outcome Goal:      (1) Discovery:  Fluorescent labels are among the most powerful means by which biomolecules and cells are studied, quantified, and imaged.  Nearly all fluorescent-labeled assays and imaging work is currently performed upon glass surfaces, plastic surfaces, or in solution where fluorescent dyes are excited by a narrow wavelength light source (such as a laser), and a sensor captures only a small fraction of the emitted light.  This research project is focused on design, fabrication, and testing of photonic crystal surfaces can enhance the detected output of any fluorescent dye by 2-3 orders of magnitude.
 
                                                                   (1) Discovery Category:
                                                                          - Biological Sciences
                                                                          - Engineering Research
                                                                          - Mathematical & Physical Sciences
 
Secondary Strategic Outcome Goal:  (2) Learning:  The education program linked to the research program involves undergraduate interns, incorporation of the topic into the PI's undergraduate Biosensors course (which includes hands-on laboratory exposure to the technology), and graduate research assistants.  The PI has a strong track record of involving undergraduates and students with diverse backgrounds into his laboratory research through the Junior/Senior Thesis Research Program and the NSF Research Experience for Undergraduates (REU) summer program.
 
                                                                   (2) Learning Category:
                                                                          - Graduate Education and Graduate Student Research

 
In terms of Intellectual Merit, this outcome is notable.  In this NSF-funded project, the Cunningham Team applies PC surfaces that incorporate optical resonances for both label-free detection and EF to spotted gene expression microarrays for the first time.  The label-free resonance will be utilized to quantify the density variability of deposited DNA spots, thereby providing a quality-control tool that is not currently available to researchers using spotted arrays.  The label-free images of DNA spots will be used to quantify interspot and intraspot density variability, providing information that will be used to eliminate defective spots from further analysis or as a means for normalizing the detected signal from subsequent fluorescent measurements.  The enhanced fluorescence resonance will be applied to enhance the output of Cy5-labeled hybridized DNA, enabling gene expression analysis to be conducted with lower sample concentrations and the ability to observe gene expression at lower levels than has previously been possible.

In terms of Broader Impacts, this outcome is notable.  The developed capability will have broad implications for gene expression analysis (the ability to observe the presence of genes that are expressed at lower concentrations than currently possible), molecular diagnostics (the ability to detect the presence of disease biomarkers at lower concentrations), cell microscopy (the ability to observe cells labeled with low efficiency fluorescent proteins with improved time resolution and relaxation of the requirement for expensive cooled CCD imaging cameras), among many other fields.

This research is Transformative because the PI has developed a system that increases the microarray sensitivity, by almost 3 orders of magnitude, of tagged molecules that will greatly enhance reproducibility and accuracy of microarray data.

Existing or potential Societal Benefits of this research:  When fully developed, photonic crystal enhanced fluorescence can be incorporated into any surface that supports a fluorescent assay or measurement, and designed to match the excitation/emission profile of any fluorophore.  Depending on the design of the detection instrument, researchers will be able to take advantage of enhanced excitation, enhanced emission or both effects simultaneously.  The developed capability will have broad implications for gene expression analysis (the ability to observe the presence of genes that are expressed at lower concentrations than currently possible), molecular diagnostics (the ability to detect the presence of disease biomarkers at lower concentrations), cell microscopy (the ability to observe cells labeled with low efficiency fluorescent proteins with improved time resolution and relaxation of the requirement for expensive cooled CCD imaging cameras), among many other fields.


 
Program Director:
 
 
 
Leon Esterowitz
CBET Program Director - Biophotonics, Advanced Imaging, and Sensing for Human Health
     
NSF Award Number:   0754122
     
Award Title:   Photonic Crystal Enhanced Fluorescence
     
PI Name:   Brian Cunningham
     
Institution Name:   University of Illinois at Urbana-Champaign
     
Program Element Code:   7236
     
NSF Investments:
 
 
 
  - American Competitiveness Initiative (ACI)
- National Nanotechnology Initiative (NNI)
- Understanding Complex Biological Systems (including the
      interfaces of life, physical, and computational sciences)
     
CBET Nugget:

  FY 2009


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This Nugget was Updated on 25 February 2011.