We perform single-molecule flow experiments using confocal microscopy and a microfluidic device for shear rates up to 20,000 s À1 and present results for the shear-induced unraveling and elongation of tethered von Willebrand factor (VWF) multimers. Further, we employ companion Brownian dynamics simulations to help explain details of our experimental observations using a parameterized coarse-grained model of VWF. We show that global conformational changes of tethered VWF can be accurately captured using a relatively simple mechanical model. Good agreement is found between experimental results and computational predictions for the threshold shear rate of extension, existence of nonhomogenous fluorescence distributions along unraveled multimer contours, and large variations in extensional response behaviors. Brownian dynamics simulations reveal the strong influence of varying chain length, tethering point location, and number of tethering locations on the underlying unraveling response. Through a complex molecule like VWF that naturally adopts a wide distribution of molecular size and has multiple binding sites within each molecule, this work demonstrates the power of tandem experiment and simulation for understanding flow-induced changes in biomechanical state and global conformation of macromolecules.

Nanomaterial‐based drug delivery systems (DDSs) increase the efficacy of various therapeutics, and shear stress has been shown to be a robust modulator of payload release. In the past few decades, a deeper understanding has been gained of the effects of flow in the body and its alteration in pathological microenvironments. More recently, shear‐responsive nanomaterial DDSs have been developed. Studies on this subject mainly from the last decade are reviewed here, focusing on innovations of the material design and mechanisms of the shear response. The two most popular shear‐controlled drug carriers distinguished by different release mechanisms, that is, shear‐deformable nanoparticles (NPs) and shear‐dissociated NP aggregates (NPAs), are surveyed. The influence of material structures on their properties such as drug loading, circulation time, and shear sensitivity are discussed. The drug development stages, therapeutic effects, limitations, and potential of these DDSs are further inspected. The reviewed research emphasizes the advantages and significance of nanomaterial‐based shear‐sensitive DDSs in the field of targeted drug delivery. It is also believed that efforts to rationally design nanomaterial DDSs responsive to shear may prompt a new class of diagnostics and therapeutics for signaling and rectifying pathological flows in the body.

Background: Patients with continuous flow ventricular assist devices (CF-VADs) are at high risk for non-surgical bleeding, speculated to associate with the loss of pulsatility following CF-VAD placement. It has been hypothesized that continuous shear stress causes elongation and increased enzymatic degradation of von Willebrand Factor (vWF), a key player in thrombus formation at sites of vascular damage. However, the role of loss of pulsatility on the unravelling behavior of vWF has not been widely explored.Methods: vWF molecules were immobilized on the surface of microfluidic devices and subjected to various pulsatile flow profiles, including continuous flow and pulsatile flow of different magnitudes, dQ/dt (i.e., first derivative of flow rate) of pulsatility and pulse frequencies to mimic in vivo shear flow environments with and without CF-VAD support. VWF elongation was observed using total internal reflection fluorescence (TIRF) microscopy. Besides, the vWF level is measured from the patients' blood sample before and after CF-VAD implantation from a clinical perspective. To our knowledge, this work is the first in providing direct, visual observation of single vWF molecule extension under controlled-pulsatile shear flow. Results: Unravelling of vWF (total sample size n ~ 200 molecules) is significantly reduced under pulsatile flow (p < 0.01) compared to continuous flow. An increase in the magnitude of pulsatility further reduces unravelling lengths, while lower frequency of pulsatility (20 vs. 60 pulses per min) does not have a major effect on the maximum or minimum unravelling lengths. Evaluation of CF-VAD patient blood samples (n = 13) demonstrates that vWF levels decreased by ~40% following CF-VAD placement (p < 0.01), which correlates to single-molecule observations from a clinical point of view. Conclusions: Pulsatile flow reduces unfolding of vWF compared to continuous flow and a lower pulse frequency of 20 pulses/minute yielded comparable vWF unfolding to 60 pulses/minute. These findings could shed light on non-surgical bleeding associated with the loss of pulsatility following CF-VAD placement.

Microfluidic devices that allow biological particle separation and concentration have found wide applications in medical diagnosis. Here we present a viral separation polydimethylsiloxane (PDMS) device that combines tangential flow microfiltration and affinity capture to enrich HIV virus in a single flow-through fashion. The set-up contains a filtration device and a tandem resistance channel. The filtration device consists of two parallel flow channels separated by a polycarbonate nanoporous membrane. The resistance channel, with dimensions design-guided by COMSOL simulation, controls flow permeation through the membrane in the filtration device. A flow-dependent viral capture efficiency is observed, which likely reflects the interplay of several processes, including specific binding of target virus, physical deposition of non-specific particles, and membrane cleaning by shear flow. At the optimal flow rate, nearly 100% of viral particles in the permeate are captured on the membrane with various input viral concentrations. With its easy operation and consistent performance, this microfluidic device provides a potential solution for HIV sample preparation in resource-limited settings.

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while Förster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.