Covalent Conjugation of Antibodies to Biomolecular-Motor Driven Shuttles

Abstract

Emerging diagnostic needs for the detection of wide arrays of biomarker molecules require the detection of ultralow concentrations of protein in small volumes of biological samples. In past work, a number of microfluidic devices have been developed in attempt to satisfy these needs. One technology incorporates a critical component of the cell's cytoskeletal system, kinesin and microtubules (MT), in engineered device structures to actively collect and concentrate functionalized microtubules into a small concentrator region. The biomolecular motor kinesin hydrolyzes ATP and converts this chemical energy into mechanical work by moving along microtubule filaments. Incorporating MTs coated with antibodies (Ab) into this concentrator device will allow for a large variety of analytes to be captured with high specificity and localized for detection. We use a heterobifunctional, 8.3A long crosslinker to covalently attach a reduced thiol group on the heavy chain of the antibody to a free succinimidyl ester on the outer surface of the microtubule. This conjugation method prevents undesired cross-linked products (MT-MT, Ab-Ab) and is applicable to monoclonal, polyclonal and F(ab)’2 antibodies, specifically anti-BSA and anti-TNFα. The motility of the functionalized microtubule complex was not significantly impaired as the gliding velocity of the functionalized microtubules driven by Kinesin-1 from Neurospora crassa decreased by about 25%, lowering the average velocity of the complex from 2.28 μm/s to 1.71 μm/s. Importantly, image analysis of fluorescent antigens attached to functionalized MTs show that microtubules are conjugated with >100 antibodies/μm of length. Based on these results, we conclude that the high density of antibodies on the microtubules along with the high binding affinity of antigen suggests that these molecular shuttles coupled with fluorescent detection can be incorporated into concentrator devices to detect ultralow concentrations of specific biomolecules.