Magnetic capture from blood rescues molecular motor function in diagnostic nanodevices

Abstract

BackgroundIntroduction of effective point-of-care devices for use in medical diagnostics is part of strategies to combat accelerating health-care costs. Molecular motor driven nanodevices have unique potentials in this regard due to unprecedented level of miniaturization and independence of external pumps. However motor function has been found to be inhibited by body fluids.ResultsWe report here that a unique procedure, combining separation steps that rely on antibody-antigen interactions, magnetic forces applied to magnetic nanoparticles (MPs) and the specificity of the actomyosin bond, can circumvent the deleterious effects of body fluids (e.g. blood serum). The procedure encompasses the following steps: (i) capture of analyte molecules from serum by MP-antibody conjugates, (ii) pelleting of MP-antibody-analyte complexes, using a magnetic field, followed by exchange of serum for optimized biological buffer, (iii) mixing of MP-antibody-analyte complexes with actin filaments conjugated with same polyclonal antibodies as the magnetic nanoparticles. This causes complex formation: MP-antibody-analyte-antibody-actin, and magnetic separation is used to enrich the complexes. Finally (iv) the complexes are introduced into a nanodevice for specific binding via actin filaments to surface adsorbed molecular motors (heavy meromyosin). The number of actin filaments bound to the motors in the latter step was significantly increased above the control value if protein analyte (50–60 nM) was present in serum (in step i) suggesting appreciable formation and enrichment of the MP-antibody-analyte-antibody-actin complexes. Furthermore, addition of ATP demonstrated maintained heavy meromyosin driven propulsion of actin filaments showing that the serum induced inhibition was alleviated. Detailed analysis of the procedure i-iv, using fluorescence microscopy and spectroscopy identified main targets for future optimization.ConclusionThe results demonstrate a promising approach for capturing analytes from serum for subsequent motor driven separation/detection. Indeed, the observed increase in actin filament number, in itself, signals the presence of analyte at clinically relevant nM concentration without the need for further motor driven concentration. Our analysis suggests that exchange of polyclonal for monoclonal antibodies would be a critical improvement, opening for a first clinically useful molecular motor driven lab-on-a-chip device.

Synthetic Systems Powered by Biological Molecular Motors

“…While Bachand and Bachand found that several common environmental contaminants are well tolerated by kinesins and microtubules, Korten et al demonstrated that specifically blood, plasma, and serum even at hundred-fold dilution interfere with the operation of kinesin- and myosin-driven systems . Kumar et al solved this problem by capturing analytes from serum, exchanging the buffer, and then introducing the motors . Clearly, protein engineering to mitigate adverse effects, protected environments or effective packaging solutions are needed.…”

Sensing protein antigen and microvesicle analytes using high-capacity biopolymer nano-carriers

“…8,18 This may be further improved using enhanced fluorescence detection approaches 20,[60][61][62] and elimination of background fluorescence as well as magnetic pre-separation steps. 63…”

Regeneration of Assembled, Molecular-Motor-Based Bionanodevices

“…Protein adsorption to surfaces is a ubiquitous and extremely important phenomenon. − Whereas it may be unwanted in several instances, it has also been widely exploited in biotechnology as well as in fundamental biophysical and biochemical investigations . For instance, antibodies, or other protein-based recognition molecules, are adsorbed to detector surfaces in biosensing − and proteomics , applications. In addition, fundamental functional studies often rely on surface adsorption of receptors and protein nanomachines. − One widely used assay of the latter type is the in vitro gliding motility assay.…”

“…Modified versions of the assay may also be applied to noncytoskeletal motors, such as processive DNA enzymes . In vitro gliding motility assays have also enabled a variety of nanotechnological applications, for example, in nanoseparation and biosensing ,− and more recently, parallel biocomputation . Many of these applications require nano/microstructured surfaces for physically and chemically confined filament transport along predefined paths, involving widely differing surface materials and chemistries.…”