Ayca Yalcin

Optical sensing of biomolecules using microring resonators

A biosensor application of vertically coupled glass microring resonators with Q/spl sim/12 000 is introduced. Using balanced photodetection, very high signal to noise ratios, and thus high sensitivity to refractive index changes (limit of detection of 1.8/spl times/10/sup -5/ refractive index units), are achieved. Ellipsometry and X-ray photoelectron spectroscopy results indicate successful modification of biosensor surfaces. Experimental data obtained separately for a bulk change of refractive index of the medium and for avidin-biotin binding on the ring surface are reported. Excellent repeatability and close-to-complete surface regeneration after binding are experimentally demonstrated.

Quantification of DNA and protein adsorption by optical phase shift

A primary advantage of label-free detection methods over fluorescent measurements is its quantitative detection capability, since an absolute measure of adsorbed material facilitates kinetic characterization of biomolecular interactions. Interferometric techniques relate the optical phase to biomolecular layer density on the surface, but the conversion factor has not previously been accurately determined. We present a calibration method for phase shift measurements and apply it to surface-bound bovine serum albumin, immunoglobulin G, and single-stranded DNA. Biomolecules with known concentrations dissolved in salt-free water were spotted with precise volumes on the array surface and upon evaporation of the water, left a readily calculated mass. Using our label-free technique, the calculated mass of the biolayer was compared with the measured thickness, and we observed a linear dependence over 4 orders of magnitude. We determined that the widely accepted conversion of 1 nm of thickness corresponds to approximately 1 ng/mm(2) surface density held reasonably well for these substances and through our experiments can now be further specified for different types of biomolecules. Through accurate calibration of the dependence of thickness on surface density, we have established a relation allowing precise determination of the absolute number of molecules for single-stranded DNA and two different proteins.

Label-free microarray imaging for direct detection of DNA hybridization and single-nucleotide mismatches

A novel method is proposed for direct detection of DNA hybridization on microarrays. Optical interferometry is used for label-free sensing of biomolecular accumulation on glass surfaces, enabling dynamic detection of interactions. Capabilities of the presented method are demonstrated by high-throughput sensing of solid-phase hybridization of oligonucleotides. Hybridization of surface immobilized probes with 20 base pair-long target oligonucleotides was detected by comparing the label-free microarray images taken before and after hybridization. Through dynamic data acquisition during denaturation by washing the sample with low ionic concentration buffer, melting of duplexes with a single-nucleotide mismatch was distinguished from perfectly matching duplexes with high confidence interval (>97%). The presented technique is simple, robust, and accurate, and eliminates the need of using labels or secondary reagents to monitor the oligonucleotide hybridization.

Direct observation of conformation of a polymeric coating with implications in microarray applications.

The conformation of a three-dimensional polymeric coating (copoly(DMA-NAS-MAPS)) and immobilization and hybridization of DNA strands on the polymer coated surface are investigated. A conformational change, specifically the swelling of the surface adsorbed polymer upon hydration, is quantified in conjunction with the application of this polymer coating for DNA microarray applications. Fluorescently labeled short DNA strands (23mers) covalently linked to the functional groups on the adsorbed polymer are used as probes to measure the swelling of the polymer. A fluorescence microscopy technique, Spectral Self-Interference Fluorescence Microscopy (SSFM), is utilized to directly measure the change in axial position of fluorophores due to swelling with subnanometer accuracy. Additionally, immobilization characteristics of single stranded DNA (ssDNA) and double stranded DNA (dsDNA) probes, as well as hybridization of ssDNA with target strands have been studied. The results show that ssDNA further away from the surface is hybridized more efficiently, which strengthens the earlier analysis of this polymeric coating as a simple but highly efficient and robust DNA microarray surface chemistry.

Platform for in situ real-time measurement of protein-induced conformational changes of DNA

A platform for in situ and real-time measurement of protein-induced conformational changes in dsDNA is presented. We combine electrical orientation of surface-bound dsDNA probes with an optical technique to measure the kinetics of DNA conformational changes. The sequence-specific Escherichia coli integration host factor is utilized to demonstrate protein-induced bending upon binding of integration host factor to dsDNA probes. The effects of probe surface density on binding/bending kinetics are investigated. The platform can accommodate individual spots of microarrayed dsDNA on individually controlled, lithographically designed electrodes, making it amenable for use as a high throughput assay.

Spectral Reflectance Imaging for a Multiplexed, High-Throughput, Label-Free, and Dynamic Biosensing Platform

There are a number of emerging optical biosensing techniques utilizing interferometric and resonant characteristics of light. We have recently demonstrated an interferometric technique, the spectral reflectance imaging biosensor (SRIB) that uses optical wave interference to detect changes in the optical path length as a result of capture of biological material on the microarray surface without the need for labels and secondary reagents. In this paper, we review the principles and performance of the SRIB technique in the context of label-free biosensors and demonstrate its high-throughput, quantitative and calibrated, versatile, and dynamic (kinetic) capabilities. A unique aspect of the SRIB system is that the measurement technique is independent of surface conformation and allows for utilization of novel polymeric coatings for surface binding, thus providing a versatile and high-density platform. We present experimental results on multiplexed antibody/antigen arrays and DNA hybridization in real time, as well as specific binding of whole virus particles. The simplicity of the overall system, its high sensitivity and compatibility with glass surface chemistries, and a linear dynamic range of nearly four orders of magnitude makes SRIB a promising platform for multiplexed detection of different biological analytes in a complex sample, with potential impact in research and clinical applications.

TATA Binding Proteins Can Recognize Nontraditional DNA Sequences

We demonstrate an accurate, quantitative, and label-free optical technology for high-throughput studies of receptor-ligand interactions, and apply it to TATA binding protein (TBP) interactions with oligonucleotides. We present a simple method to prepare single-stranded and double-stranded DNA microarrays with comparable surface density, ensuring an accurate comparison of TBP activity with both types of DNA. In particular, we find that TBP binds tightly to single-stranded DNA, especially to stretches of polythymine (poly-T), as well as to the traditional TATA box. We further investigate the correlation of TBP activity with various lengths of DNA and find that the number of TBPs bound to DNA increases >7-fold as the oligomer length increases from 9 to 40. Finally, we perform a full human genome analysis and discover that 35.5% of human promoters have poly-T stretches. In summary, we report, for the first time to our knowledge, the activity of TBP with poly-T stretches by presenting an elegant stepwise analysis of multiple techniques: discovery by a novel quantitative detection of microarrays, confirmation by a traditional gel electrophoresis, and a full genome prediction with computational analyses.

Synergetic chemiluminescence and label-free dual detection for developing a hepatitis protein array.

A dual detection system for protein arrays is presented that combines label-free detection by optical interference with chemiluminescence. A planar protein array that targets hepatitis B surface antigen is developed. Surface densities for individual antibody spots are quantitated using optical interference prior to use. Target binding (10 ng/ml) is detected label-free. Target binding (1 ng/ml) is detected by both optical interference and chemiluminescence with the inclusion of secondary antibodies. Binding results using both methods are found to be directly proportion to the capture probe density measured initially. The dual detection system provides the analytical utility of optical interference detection with the established clinical utility of chemiluminescence detection.

Fluorescence enhancement on reflecting substrates for microarray applications

In this study, we report on the utilization of layered substrates for increased performance of fluorescent-based detection schemes. Through optimization of layer thicknesses, we demonstrate that enhancement with respect to commonly used microscope slides is achieved for the collected fluorescence signal.

A platform for in situ real-time measurement of protein induced conformational changes of DNA

A platform for in situ and real-time measurement of protein induced conformational changes in dsDNA is presented. We demonstrate protein induced bending upon sequence specific binding of Integration Host Factor (IHF) to dsDNA probes.