Article pubs.acs.org/ac
Temperature Gradient Approach for Rapidly Assessing Sensor Binding Kinetics and Thermodynamics Caleb E. Wagner, Lucyano J. A. Macedo, and Aric Opdahl* Department of Chemistry and Biochemistry, University of Wisconsin−La Crosse, La Crosse, Wisconsin 54601, United States S Supporting Information *
ABSTRACT: We report a highly resolved approach for quantitatively measuring the temperature dependence of molecular binding in a sensor format. The method is based on surface plasmon resonance (SPR) imaging measurements made across a spatial temperature gradient. Simultaneous recording of sensor response over the range of temperatures spanned by the gradient avoids many of the complications that arise in the analysis of SPR measurements where temperature is varied. In addition to simplifying quantitative analysis of binding interactions, the method allows the temperature dependence of binding to be monitored as a function of time, and provides a straightforward route for calibrating how temperature varies across the gradient. Using DNA hybridization as an example, we show how the gradient approach can be used to measure the temperature dependence of binding kinetics and thermodynamics (e.g., melt/denaturation profile) in a single experiment.
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chemical labeling, by not providing quantitative results, or by being based on instrumentation that is not commonly available. Of these tools, spatially resolved SPR imaging has several desirable features for a general analysis tool, particularly that it is a label-free and quantitative technique that is capable of multiplex analysis. The difficulty of performing variable temperature SPR measurements comes from its nonspecific nature, as the reflectivity technique essentially monitors refractive index changes in the region extending a few hundred nanometers from the gold sensor surface.15,16 If the temperature of the sensor is continuously incremented, reflectivity will be influenced by both the analytical response from molecular association/dissociation events on the sensor surface, and a continuously increasing or decreasing background response from the surrounding aqueous buffer solution, whose refractive index changes with temperature.5,8 Reproducible and quantitative separation of the two contributions in measurements where temperature is ramped is challenging. Using DNA hybridization as an example, we report here how this problem is avoided by using a differential heating system that creates a spatial temperature gradient across the SPR sensor surface (Scheme 1). The temperature gradient eliminates the need to account for a changing background, because the temperature, and thus refractive index of the buffer solution, at any specific location on the sensor surface remains constant during the experiment. This simplifies analysis,
easuring the thermodynamics of interactions involving nucleic acids and peptides is a vital component of biomolecular studies. Conventionally, these properties are analyzed in solution by temperature dependent spectroscopic measurement (e.g., UV−vis, CD, fluorescence) or thermal methods (ITC, DSC). 1,2 For example, DNA thermal denaturation profiles obtained by monitoring changes in UV absorption as the temperature of the solution is incremented have provided the empirical data used to generate robust models for predicting the stabilities of DNA structures in solutions. Surface-based diagnostics, such as microarrays, offer the benefit of high throughput analysis of binding kinetics and affinities of biomolecular interactions,3 but are conventionally analyzed at a single temperature, limiting the thermodynamic information they can provide. Accordingly, there are substantial benefits that could be realized by integrating variable temperature capability into routine biosensor measurements. Several surface-based instrumental methods have demonstrated the ability to obtain measurements of binding interactions as temperature is varied. For measurements involving DNA hybridization on metal and glass substrates, the list includes surface plasmon resonance (SPR) imaging,4−8 fluorescence,9,10 electrochemical analysis,11,12 second harmonic generation,13 and SERs.14 Results from these methods have provided fundamental information regarding how DNA hybridization at interfaces differs from hybridization in solution, however, the methods themselves have not been widely adopted. Each technique has restrictive attributes, for example by yielding measurements at discrete temperatures instead of continuously over a range of temperatures, by requiring © XXXX American Chemical Society
Received: April 22, 2015 Accepted: July 3, 2015
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DOI: 10.1021/acs.analchem.5b01518 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry Scheme 1. SPR Instrument Schematica
The sample handling system consists of a custom stainless steel two-channel microfluidic cell (channel volume < 20 μL) that provided uniform flow of degassed (Elite, Alltech) solutions at flow rates of
