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

Abstract

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 -2 by employing poly(ethylene glycol) coatings.[1]Recently, optimization of brush density and morphology has reduced adsorption to 1 ng cm -2 or less.[2] These coverages, equal to about one tenth of a percent of a monolayer, represent the detection limit for several characterization techniques, including SPR [2,3] and radiolabeling.[4] 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.[5] 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.[6] For example, controlled adsorption of kinesin, [7][8][9][10][11][12][13][14] myosin, [15][16][17] and F1-ATPase motors [18] 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 -2) 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. [19,20] 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 2 by measuring the rate of microtubule attachment. [21,22] Attachment rate measurements have subsequently been adapted to the determination of relative kinesin motor activity on different surfaces [9] and to the evaluation of guiding structures for microtubule transport [10,23] 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. [24,25] . Landing rate measurements enable the measurement of absolute coverages of functional kinesins in the range of 0.004 -1 ng cm -2 , thus enabling to differentiate the performance of even the best non-fouling surfaces. While landing rate measurements in effect count individual proteins, their complexity ...

Controlling self-assembly of microtubule spools via kinesin motor density

“…Based on landing rate measurements, 24 the concentration of this kinesin solution is 730±180nM in this stock solution.…”

In Pursuit of Zero: Polymer Brushes that Resist the Adsorption of Proteins

“…The protein resistance of POEGMA brushes has been confirmed by a number of groups, including Katira et al, who found that adsorption of kinesin was 20-fold lower on POEGMA surfaces compared to OEG-SH SAMs on gold. [55] SI-ATRP of OEGMA (and other monomers) is also compatible with soft-lithography; an ATRP initiator with a terminal thiol group was patterned on gold by microcontact printing (mCP), followed by SI-ATRP to yield micropatterns of POEGMA (Fig. 3A).…”

Ultrasmall implantable composite microelectrodes with bioactive surfaces for chronic neural interfaces

“…This polymer provides initiator groups for subsequent atom transfer radical polymerization 28 (ATRP). By ATRP, a ~200-nm-thick poly(ethylene glycol) methacrylate (PEGMA) top layer was deposited, which rendered the neuronal probe devices protein-resistant 28,29 (Fig. 1c).…”