The thermal stabilities of double-stranded DNA hybrids immobilized on gold surfaces are shown to be significantly affected by the conformation of the hybrid. To analyze this behavior, DNA probes were immobilized using attachment strategies where the nucleotides within the strand had varying levels of interactions with the gold substrate. The abilities of these probes to form double-stranded hybrids with solution DNA targets were evaluated by surface plasmon resonance (SPR) over a temperature range 25-60 °C. The measurements were used to construct thermal stability profiles for hybrids in each conformation. We observe that DNA hybrids formed with probe strands that interact extensively with the gold surface have stability profiles that are shifted lower by 5-10 °C compared to hybrids formed with end-tethered probes that have fewer interactions with the surface. The results provide an understanding of the experimental conditions in which these weaker DNA hybrids can form and show the additional complexity of evaluating denaturation profiles generated from DNA on surfaces.

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.

Commonly used lactose assays [enzymatic spectrophotometric absorbance (EZA) and HPLC] for dairy ingredients are relatively expensive and time consuming. A blood glucose meter (BGM)-based method has successfully been documented as a rapid lactose assay in milk. However, the BGM-based method has not been evaluated in dairy ingredients. The objective of this study was to evaluate the BGM-based lactose analysis method in whey-derived (WD) and skim milk-derived (SMD) ingredients. The study was carried out in 4 phases. In phase 1, the effect of pH and lactose concentrations on the BGM reading was investigated using a factorial design with 2 factors: pH (6.02-7.50) and lactose (0.2 or 0.4%). We found that BGM readings were significantly affected by lower pH values at both lactose levels. In phase 2, the effect of total solids and ingredient type was investigated using a factorial design with 2 factors: ingredient type (WD or SMD) and total solids (0-8%). It was observed that the BGM reading was significantly affected by ingredient type and total solids. Phase 3 involved developing a linear relationship between the BGM reading and the EZA reference method to ascertain the accuracy of the proposed BGM method. Different ingredient types (WD or SMD) and non-lactose solids (0.5-27%) model ingredient dilutions prepared over a range of lactose contents (0.08-0.62%) were measured using the BGM and EZA methods. The average absolute percentage bias difference between the BGM method and EZA reference method results for these model dilutions was found to be between 2.2 and 7.3%. In phase 4, 15 samples procured from commercial sources ranging from 0.01 to 81.9% lactose were evaluated using the BGM method and EZA reference method. The average absolute percentage bias difference for lactose results between the 2 methods ranged from 3.6 to 5.0% and 5.3 to 9.7% for well-performing and poorly performing meters, respectively. Overall, the BGM method is a promising tool for rapid and low-cost analysis of lactose in both high-lactose and low-lactose dairy ingredients.

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