CBST researchers led by Prof. Xiangdong Zhu have developed an optical method for reading DNA, protein, and small molecule microarrays that does require the use of fluorescent labels as is used in reading most conventional microarrays. The advantage of label-free detection is the simplicity, low-cost, and elimination of possible binding interferences from the fluorescent labels. Recent efforts have resulted in the award of $2M grant (High-Speed Label-Free Optical Detection System for Small-Molecule Microarrays, 1R01HG003827-01) from the National Human Genome Research Institute of the NIH.

Biomolecular microarrays or increasingly high-density are becoming indispensable tools in genomic and proteomic research and biomarker discovery. The need for label-free microarray detection methods that are complementary to fluorescence-based methods is also becoming very important. We have developed a label-free oblique-incidence reflectivity difference (OI-RD) optical microscope for detecting DNA-DNA, protein-protein, and protein-small molecule reactions in microarray format through ellipsometry measurements of changes in density, thickness, and conformation of surface-bound proteins on solid supports. This can be done without extrinsic labeling molecules (e.g. organic fluorophores or quantum dots), which are costly and potentially intrusive. Of particular interest are microarrays of small molecules that may be used for high-throughput screening for protein binding.

The goal of the new NIH project is to develop a robust, sensitive and reliable label-free detector that can evaluate the real-time binding kinetics of 10,000 spot small molecule chemical microarrays against various single component or multi-component analytes. We envision that we can easily print thousands of replicates of high density small molecule microarrays (10,000 spots/ slide) and use them to probe a variety of biological analytes such as serum, cell extracts or pure proteins. We expect that by combining our novel combinatorial chemistry and microarray platforms with a highly sensitive and reliable 2-D label-free optical detector that can efficiently measure real-time binding kinetics, we will be able to rapidly and accurately study the binding kinetics of a large number of samples (e.g., individual protein or complex samples such as whole blood serum) against a large number of immobilized small molecules, peptides, DNA molecules or proteins.

These two images of a 3 x 3 microarray section of DNA spots show a proof-of-principle demonstration of the effectiveness of label-free optical detection. The top image is made using an oblique-incidence reflectivity difference (OI-RD) optical measurement. The left column shows hybridization (binding) with a label-free DNA complement. The center column shows very weak hybridization with non-complementary DNA, and the right column shows hybridization with a Cy5 fluorescent labeled DNA complement. Shown at the bottom is a conventional fluorescent image of the Cy5 labeled DNA complement.

 

 

 

 

Schematic of the instrument used for oblique-incidence reflectivity difference (OI-RD) measurements.

References:

1. Landry JP, Gray J, O'Toole MK, Zhu XD., Incidence-angle dependence of optical reflectivity difference from an ultrathin film on solid surface, Opt Lett 31 (2006) 531

2. Zhu XD, Comparison of two optical techniques for label-free detection of biomolecular microarrays on solids, Opt. Comm. 259 (2006) 751

3. Landry JP, Zhu XD, Gregg JP., Label-free detection of microarrays of biomolecules by oblique-incidence reflectivity difference microscopy, Opt Lett. 29 (2004) 581

4. US Patent US6917428, Methods and apparatus for measuring refractive index and optical absorption differences

For more information about this article, please contact Prof. Xiangdong Zhu (xdzhu@physics.ucdavis.edu).