Quantifying the Performance of Protein-Resisting Surfaces at Ultra-Low Protein Coverages using Kinesin Motor Proteins as Probes**

Surfaces resistant to protein adsorption are very desirable for a variety of applications in biomedical engineering and bionanotechnology, since protein adsorption is often the first step in a cascade of events leading to systems failure. Initial efforts to create adsorption-resistant surfaces succeeded in reducing the adsorption by 80% compared to untreated surfaces to 100 ng cm by employing poly(ethylene glycol) coatings. Recently, optimization of brush density and morphology has reduced adsorption to 1 ng cm or less. These coverages, equal to about one tenth of a percent of a monolayer, represent the detection limit for several characterization techniques, including SPR and radiolabeling. However, biological effects can be observed for protein coverages below this detection limit. For example, adsorption of blood proteins can initiate the intrinsic coagulation cascade at low coverage. Therefore more sensitive techniques for the measurement of protein adsorption are needed. Moreover, if protein adsorption is exceedingly slow, higher sensitivity would permit an accelerated quantification of the performance of the surface (e.g. within hours instead of days). Non-fouling surfaces are also a critical part of hybrid nanodevices, which utilize precisely positioned biomolecules in an artificial environment. For example, controlled adsorption of kinesin, myosin, and F1-ATPase motors has been utilized for the design of molecular shuttles and nanopropellers. Adsorption of motor proteins outside the intended regions at densities down to one motor per square micrometer (0.04 ng cm) can lead to loss of device function, since individual motors can already bind and transport the associated filaments outside their intended tracks. The binding of associated filaments, such as microtubules or actin filaments, is readily observed by fluorescence microscopy, since these filaments are composed of thousands of protein subunits and carry typically at least a thousand covalently linked fluorophores. Howard et al. demonstrated in 1989 that observing the attachment of microtubules from solution to surface-adhered kinesin motors enables the determination of motor densities as low as 2 proteins per lm by measuring the rate of microtubule attachment. Attachment rate measurements have subsequently been adapted to the determination of relative kinesin motor activity on different surfaces and to the evaluation of guiding structures for microtubule transport Since kinesins long tail domain evolved to efficiently connect to cargo, we hypothesize that it can serve as a particularly efficient probe for attachment points on the surface.. Landing rate measurements enable the measurement of absolute coverages of functional kinesins in the range of 0.004 – 1 ng cm, thus enabling to differentiate the performance of even the best non-fouling surfaces. While landing rate measurements in effect count individual proteins, their complexity is low compared to single molecule fluorescence measurements, due to the availability of a kinesin/ microtubule kit (Cytoskeleton Inc.) and the high brightness of fluorescent microtubules which can be imaged with a standard fluorescence microscope. In the following, we demonstrate that the quantification of ultra-low kinesin coverages by landing rate measurements is a valuable tool in determining the performance of novel and established coatings with outstanding resistance to protein adsorption (see Scheme 1 for an example of a designer surface). Specifically, we will describe the adsorption model underlying the method, present experiments which demonstrate the determination of kinesin coverages on fouling and nonfouling surfaces, and discuss the advantages and limitations of the proposed method. Adsorption Model. Microtubule attachment rate measurements are interpreted in the context of a two stage adsorption model: First, kinesin molecules adsorb from solution to the surface, filling a fraction of the available binding sites. Second, the kinesin solution is replaced by a microtubule solution and microtubules adsorb specifically to the kinesin motors bound C O M M U N IC A IO N