High Affinity Polyvalent Nanoether SAMs on Gold: Why More is Better

By: Dr. Brenda D. Spangler, Dr. E. Scott Tarter, Dr. Benjamin D. Reeves, Dr. Zhiyong Suo, Material Matters 2006, 1.2, 15.

SensoPath Technologies, Inc.

Association of Sulfur with Gold

Self-assembled monolayers or SAMs of thiols and organic disulfides on gold surfaces were described by Nuzzo and Allara in 19831 as a considerable improvement over difficult to maintain Langmuir-Blodgett monolayers. Extensive studies by these authors and by the Whitesides group at Harvard University led to a generally accepted conclusion that the bonding state of an adsorbed thiol or disulfide on gold was an Au-thiolate (Au0-S-) with a binding energy somewhere between the binding energy for electrostatic interaction and a covalent bond, based on X-ray-photoelectron spectroscopy (XPS), Auger electron spectroscopy, temperature-programmed desorption and high-resolution electron energy loss spectroscopy 2–5. There is also indication that the disulfide association is much faster and more stable than a simple monothiol association with the gold surface, a point we will return to later in this article, and that the S-S bond of a disulfide may be cleaved during adsorption to the Au(111) surface5 to yield the thiolate. On the other hand, an in-depth X-ray diffraction study by Fentner et al.6 suggests that the monolayer spacing (2.2 A°) of alkane thiols on Au(111) is close enough to imply the existence of a disulfide bond between adjacent alkane thiols. It is then possible to hypothesize that the sulfur atoms may intercalate between the gold atoms, thus accounting for the deprotonation of the thiol to thiolate or disulfide and concomitant presence of Au0, as well as enhanced binding energy and enhanced stability of the sulfurgold association beyond a simple electrostatic interaction.

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More is Better

Polyvalent interactions are one of the basic concepts of biological molecular recognition. Multiple ligand binding, represented by polyvalent antibodies or oligomeric binding subunits is characteristic of many bacterial toxins, as well as several enzyme systems and the well known example in molecular assembly, the tetrameric strepavidin interaction with biotin. Multiple interactions provide a far stronger association than the affinity (KA) of single ligand for its target substrate. This point has been extensively explored7 and in fact, discussed in terms of stability of alkane thiol monolayers, which spontaneously desorbed in solvent under ambient conditions from gold, silver, platinum, or copper. Both desorption and self-exchange were observed although residual thiols that could not be desorbed were observed8. The resulting patchy surface, however, left significant areas of metal exposed. The desorption-resorption phenomenon has been proposed as a mechanism for stochastic switching in wired molecules that are part of an alkanemonothiol self-assembled monolayer9. It is clear that stability of the monolayer could be improved significantly by the simple construction of divalent or polyvalent thiols. The reasoning is as follows: for uncorrelated desorption of widely spaced adsorbing groups such as those on a dithiolalkane phenyl ring, the adsorption can be treated statistically. The probability of a polyvalent molecule, for example, a Nanotether™, having all thiols off the gold surface simultaneously (total desorption) is a power series. That is, if the association constant ka = x for a monothiol, then ka = x2 for a dithiolalkane phenyl group. Imagine a group of monkeys hanging by only one hand from a tree branch, reaching for a banana in a wind flow. Any monkey who lets go will be blown away without the banana. If the monkeys were hanging by both hands while reaching for a banana with their tails, then they would be exponentially more stable and much less likely to be blown off the branch. Monkeys hanging by both hands and their tails would, of course, be ka 3 more stable.

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Monothiol vs Dithiol Monolayers

Figure 1 shows that aromatic dithiol tethers (Aldrich Prod. No. 674338) do in fact adsorb more quickly and provide more surface coverage than aromatic monothiols (Aldrich Prod. No. 673560). The data were recorded on a Reichert 7000 Surface Plasmon Resonance (SPR) instrument by flowing the nanotethersTM (1 mM in 100% ethanol) over a clean gold SPR slide then washing with 100% ethanol at a preset time.

Figure 1. Monolayer formation by dithiol aromatic NanotetherTM vs monothiol aromatic NanotetherTM followed by solvent wash

Figure 1. Monolayer formation by dithiol aromatic NanotetherTM (red) vs monothiol aromatic NanotetherTM (blue) followed by solvent wash. Arrow indicates introduction of 100% ethanol wash.(674338, 673560)

The dithiol adsorption is significantly faster and after washing, the resulting dithiol monolayer has a higher refractive index (higher pixel number) than the monothiol dendron monolayer suggesting better coverage. We believe the sharp drop upon initiation of ethanol wash is due to loss of unadsorbed dendrons accumulated on the saturated monolayer surface. Non-specifically adsorbed films have been previously observed when slides are pulled from solutions containing thiols after set-up of the self-assembled monolayer. Desorption and loss of the non-specifically adsorbed material can be directly observed on a quartz crystal microbalance or a surface plasmon resonance slide during washing of the freshly prepared slide mounted in the appropriate instrument. For 1:10 molar ratio of mixed self assembled monolayers consisting of carboxyl-terminated and hydroxyl-terminated thiol or dithiol nanotethersTM (see Figure 2), unpublished AFM data (Suo, Z. Montana State University, Bozeman, personal communication) indicated that monothiol alkane PEG mixed self-assembled monolayers showed patches of bare gold while dithiol aromatic PEG mixed monolayers appeared to be homogeneous. XPS data for the same slides indicated significantly more sulfur-gold interaction for the dithiol over the monothiol mixed self-assembled monolayers.

Figure 2. Monothiol and dithiol mixed self-assembled monolayers with PEGolated spacers.

Figure 2. Monothiol and dithiol mixed self-assembled monolayers with PEGolated spacers.

The apparent stability of the dithiol mixed self-assembled monolayer may account for the observation that the detection ratio of Staphylococcus aureus binding compared to E. coli binding for a dithiol surface ratio was 2.3, while for the monothiol surface was 1.29, indicating greater selectivity for the pathogen on the dithiol surface. In the same study, nonspecific binding response was 133.3 for the monothiol surface and 39 for the dithiol surface10.

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Immobilization Strategies

Self-assembled monolayers provide the opportunity for a variety of chemical strategies to be employed for immobilization of specific ligands, for construction of a non-fouling surface or for electrochemical applications. The hydroxyl terminated dithiolalkane aromatic rigid-rod tether (Nanotether BPA, Aldrich Prod. No. 674346) can be used as a non-fouling SAM or for further functionalization. The carboxylterminated Nanotether BPA-HA (Aldrich Prod. No. 674354) in Figure 3 can be coupled to proteins, amino-terminated DNA oligomers or any other amine-containing molecule using standard EDC/NHS coupling chemistry11. It could be used to provide a hydrophilic, negatively charged surface, or further functionalized chemically. Hydrazide-terminated tethers (Nanotether™ BPA-HH, Aldrich Prod. No. 674370) in Figure 4 couple easily with any aldehyde, including aldehydes generated from glycosylated antibodies by sodium periodate11,12 resulting in specific orientation of the antibody without impairing antibody activity, rather than the random orientation that results from EDC/NHS coupling to carboxylterminated tethers.

Figure 3. A typical rigid-rod dithiol tether Figure 3. A typical rigid-rod dithiol tether (Nanotether™ BPA-HA Aldrich Prod. No. 674354).

Figure 4. Tethering an oxidized antibody to a hydrazide-terminated tetherFigure 4. Tethering an oxidized antibody to a hydrazide-terminated tether

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Future Outlook

Monodisperse macromolecules including flexible linear, rigidrod and dendritic segments offer unique design capabilities that allow positioning of a variety of ligands through multivalent attachment functionalities while controlling the ultimate flexibility of the tether. These types of constructs can be functionalized for attachment to various surfaces and, on their other terminus, functionalized for covalent coupling of proteins, peptides, and small organic ligands, all of which can be varied independent of one another and be connected to each other through spacers with varying degrees of rigidity. A hydroxyl terminus, for example Nanotether BPA (Aldrich Prod. No. 674346) either alone or as part of a poly(ethylene glycol) module, provides an excellent antifouling surface.

The rigid-rod (bisphenolA) modular construct shown in Figure 3 contains a flexible alkane spacer with functional terminus for attachment of ligand. In effect, it becomes a “nano fishing rod” for many different assay applications in which the ligand may be presented as if it were on a cell membrane. A typical application is shown in Figure 4 in which antibody capture ligand (the “bait”) has been covalently coupled to immobilized hydrazide-terminated Nanotether BPA-HH (Aldrich Prod. No. 674370). With a PEGolated spacer module, the “line” would swing free in aqueous solvent, to mimic solution-phase binding.

The rigidity of the bisphenol A module on the tether makes it an interesting choice for use with a quartz crystal microbalance where it could be used to probe viscoelastic effects in a flow cell configuration. Atomic force microscopy applications are another potential use for rigid-rod tethers, to prevent the coupled (or “bait”) ligand from swinging back onto the AFM tip where it may become ensnared. Considering other modular approaches, it is possible to modify the thiol terminus by substituting orthopyridinium disulfide or other coupling reagents, linking the Nanotether™ to carbon nanotubes through the carboxyl terminus, or through a triethoxysilane terminus to provide a 3-point attachment for coupling to glass. Applications are limited only by a researcher’s imagination.

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  12. Spangler, B.D.; Tyler, B.J., Anal. Chim. Acta 1999, 399, 51.

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