Quantification of DNA and protein adsorption by optical phase shift

Abstract

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 ∼1 ng/mm² 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.

Introduction

Macromolecule interactions involving proteins and DNA are at the core of our understanding of cell biology and disease. The importance of techniques to interrogate these interactions via in vitro binding to a solid support, especially those that are high-throughput such as in arrayed systems, has been firmly established in both biological research and medicine (Stears et al., 2003, Predki, 2004, Macbeath and Schreiber, 2000, Zhu et al., 2001). Commonly, probe molecules are immobilized on a solid support using surface chemistry, target molecules are introduced in solution, and ensemble binding interactions between targets and probes are detected by fluorescently labeled secondary reagents (Espina et al., 2004). Although very successful for qualitative analyses, labeled fluorescence detection is an indirect measure of binding and molecular accumulation. Generating quantitative and real-time data with labeled techniques is difficult due to surface- and self-quenching at high densities (Ramdas et al., 2001), non-linear responses due to bleaching, lifetime and energy transfer issues (Song et al., 1995), and problems associated with quantifying responses between different fluorophores, assays, and measurement equipment. Additionally, the fluorescent molecules may alter the natural binding properties of the target molecules. As a result, complex calibrations are required for quantification of labeled signal response (Haab et al., 2001).

Label-free detection techniques have been developed for dynamic and quantitative sensing and have provided the advantages of simplicity and cost effectiveness (Ramachandran et al., 2005, Zhu and Snyder, 2003, Yu et al., 2006, Mitchell, 2002, Cooper, 2002). A leading technology in label-free sensing is the surface plasmon resonance (SPR) technique which has demonstrated quantitative and dynamic measurement capabilities (Jung et al., 1998, Campbell et al., 2002). Optical interferometric techniques that use layered substrates as solid supports for immobilized probes have gained attention as well because of their simplicity and sensitivity (Schmitt et al., 1997, Nikitin et al., 2005, Zhao et al., 2007, Bergstein et al., 2008, Özkumur et al., 2008). In interferometric approaches, the signal is created by an additional phase shift or optical path length difference (OPD) caused by the adsorbed biolayer. The dielectric solid support materials are optically homogenous and have a uniform refractive index at the wavelengths used, implying a simple model where the optical path length that creates the phase delay is the product of the distance traveled and the refractive index. Here we validate this model for adsorbed layers of DNA and protein using an interferometric detection technique.

A similar investigation was conducted previously by De Feijter et al. They modeled adsorbed protein layers as having a fixed height based on the dimensions of the molecule with a variable refractive index based on surface concentration, and used ellipsometric measurements to validate their model (De Feijter et al., 1978). More recently, other investigators have made the approximation that adsorbed protein layers can be modeled as having a fixed index and a variable height (Schmitt et al., 1997, Nikitin et al., 2005). The aim of the present work is to validate the fixed-index model and to calibrate measured signals against known surface concentrations for DNA and protein.

We have recently introduced an interferometric technique termed Spectral Reflectance Imaging Biosensor (SRIB) that can detect label-free and dynamic biomolecular interactions in a high-throughput format (Özkumur et al., 2008). A silicon chip with a top layer of thick uniform oxide is used as the solid support for arrayed biomolecules. A tunable laser is incident on the array and reflection spectra are recorded for hundreds of spots simultaneously. The mass at each spot is found by measuring the phase difference between the biolayer and buried mirror reflections in the collected reflection spectra. A key advantage of SRIB over evanescent wave techniques such as SPR or waveguide sensors is that SRIB is highly insensitive to changes in temperature or buffer refractive index because the two reflections causing the phase shift are co-propagating. We have also recently shown that SRIB is insensitive to changes in molecular conformation on the surface, a topic that will be revisited in the discussion.

In our experiments, we detected the OPD for adsorbed protein and DNA using SRIB and calculated the height of the layers by assuming a uniform refractive index of n = 1.45. The height values were then compared to the known mass deposited on the surface. The ability to quantify mass by this method was assessed for oligomeric single-stranded DNA (ssDNA), bovine serum albumin (BSA), and immunoglobulin G (IgG) and results were compared with fluorescent measurements.