Measuring and Remediating Nonspecific Modifications of Gold Surfaces Using a Coupled in Situ Electrochemical Fluorescence Microscopic Methodology

Abstract

In surface-based biosensors, the nonspecific or undesired adsorption of the probe is an important characteristic that is typically difficult to measure and therefore to control or eliminate. A methodology for measuring and then minimizing or eliminating this problem on gold surfaces, readily applicable to many common surface modifications is presented. Combining electrochemical perturbation and fluorescence microscopy, we show that the potential at which the adsorbed species is removed can be used as an estimate of the strength of the adsorbate-surface interaction. This desorption potential can be easily measured through an increase in fluorescence intensity as the potential is manipulated. Furthermore, this method can be used to evaluate strategies for preventing or removing nonspecific adsorption. This is demonstrated for a wide variety of surface modifications, from strongly chemisorbed monolayers such as thiol self-assembled monolayers (SAMs) to physisorbed monolayers as well as for complex surface structures like peptide and DNA mixed-component SAMs. The use of a coadsorption strategy or small magnitude potential-step cycles was shown to significantly decrease the amount of nonspecifically or noncovalently bound probe, creating better defined surfaces.

Beyond Simple Cartoons: Challenges in Characterizing Electrochemical Biosensor Interfaces

“…For example, efficient hybridization of bound DNA probes to the complementary strands (targets) in solution requires the tethered DNA probes to be arranged sufficiently far apart. ,, However, local clustering of immobilized probes can exist (Figure b) even on atomically featureless single crystal metal surfaces. These localized areas of high DNA density favor nonspecific adsorption (physisorption) of the probes through base pair interactions and can lead to aggregate formation (Figure c). − In addition, these regions will not effectively hybridize the target strands, reducing sensitivity in the simplest manifestation.…”

Electrochemistry and Optical Microscopy

“…The onset potential for the SAM desorption depends strongly on local crystal orientation, providing an estimate of the adhesion strength of molecules in SAMs, correlated to the underlying atomic arrangement [192]. Besides evaluating the heterogeneity in SAMs surface density [193] and the nonspecific adsorption of molecular clusters, the strategy provides feedback for the preparation of functional SAMs by molecular replacement with the fewest possible defects [194]. Next, the response to CV of diluted SAMs of fluorophore-labelled DNA-tethers shows a potential-modulated elongation/collapse due to electrostatics (Figure 17B).…”

Improved Thermal Stability and Homogeneity of Low Probe Density DNA SAMs Using Potential-Assisted Thiol-Exchange Assembly Methods

“…17−19 This was hypothesized to be a result of heterogeneous assembly of DNA on the surface, 20,21 which could result in cluster formation, and a significant amount of physisorbed DNA, which is not always eliminated by simple rinsing with water or buffer. 7,15,17 The stability of electrochemical sensors has also been highlighted as a limitation resulting in insufficient shelf-life. 8 Biosensor shelf-life can be estimated using accelerated aging methods measured at conditions above room temperature.…”

“…Currently, the most popular method for making DNA SAMs is via formation on a clean gold surface at an uncontrolled potential (open-circuit potential, OCP). ,,, This is followed by backfilling with short alkyl-thiol, such as mecaptohexanol (MCH), to passivate the remaining gold surface and, most importantly, to remove any nonspecifically adsorbed DNA. ,− Codeposition of both DNA and the alkyl-thiol have also been used . These methods are commonly used because of the convenience and ease of the deposition procedure.…”

“…These methods are commonly used because of the convenience and ease of the deposition procedure. However, recently evidence has shown that these conventional strategies for making DNA SAMs resulted in surfaces that demonstrate poor reproducibility and stability. − This was hypothesized to be a result of heterogeneous assembly of DNA on the surface, , which could result in cluster formation, and a significant amount of physisorbed DNA, which is not always eliminated by simple rinsing with water or buffer. ,, …”