Smart polymers that mimic and even surpass the functionality of natural responsive materials have been actively researched. This study explores the design and characterization of a Single-MOlecule-based material REsponsive to Shear (SMORES) for the targeted release of A1, the platelet binding domain of the blood clotting protein von Willebrand factor (VWF). Each SMORES construct employs an aptamer molecule as the flow transducer and a microparticle to sense and amplify the hydrodynamic force. Within the construct, the aptamer, ARC1172, undergoes conformational changes beyond a shear stress threshold, mimicking the shear-responsive behavior of VWF. This conformational alteration modulates the bioavailability of its target, the VWF-A1 domain, ultimately releasing it at elevated shear. Through optical tweezer-based single-molecule force measurement, ARC1172s role as a force transducer was assessed by examining its unfolding under constant pulling force. We also investigated its refolding rate as a function of force under varied relaxation periods. These analyses revealed a narrow range of threshold forces (3-7 pN) governing the transition between folded and unfolded states. We subsequently constructed the SMORES material by conjugating ARC1172 and a microbead, and immobilizing the other end of the aptamer on a substrate. Single-molecule flow experiments on immobilized SMORES constructs revealed a peak A1 domain release within a flow rate range of (40-70 mu L min-1). A COMSOL Multiphysics model translated these flow rates to total forces of 3.10 pN-5.63 pN experienced by the aptamers, aligning with single-molecule force microscopy predictions. Evaluation under variable flow conditions showed a peak binding of A1 to the platelet glycoprotein Ib (GPIB) within the same force range, confirming released payload functionality. Building on knowledge of aptamer biomechanics, this study presents a new strategy to create shear-stimulated biomaterials based on single biomolecules. (A). Optical tweezers characterized the mechanical properties of the Single-MOlecule-based material REsponsive to Shear (SMORES). (B) Flow experiments demonstrated the release of therapeutic cargo at a threshold flow rate.

Researchers have been working to develop stable and convenient test strips for detecting heavy metals. This paper reports a new portable lead ion test strip based on the exceptional photoluminescence properties of CsPbBr3. The CsBr films deposited on different substrates (rigid, semi-rigid, and flexible substrates) display highly selective luminescent response to Pb2+. For the flexible substrate, CsBr fluorescent probe not only shows lightweight, easy to use in large-scale manufacturing but also exhibits long detection lifetime. In comparison to organometallic perovskite fluorescence sensing of Pb2+, the CsBr fluorescent probe displays better stability and higher detection limit. Moreover, the used test strip can be reused to detect Cl− in solution, the CsBr fluorescent probe also shows a potential for multi-testing in recycling applications.

Quantitation of host cell proteins (HCPs) is essential in the process of preparation of many biological and vaccine products. Common methods of quantitation include the widely applied enzyme-linked immunosorbent assays (ELISAs), mass spectrometry (MS) and other orthogonal assays. Prior to using these techniques, critical reagents need to be evaluated, for example, antibodies need to be assessed for HCP coverage. Percent of HCP coverage is often established by denatured 2D Western blot. However, ELISAs measure the amount of HCP only in a native state. There are limited studies linking reagents validated by 2D-Western to ensure adequate coverage in the final ELISA. ProteinSimple’s newly developed capillary Western blot technology allows for separation, blotting, and detection of proteins in a semi-automated and simplified format. Capillary Westerns are similar to slab Westerns, with the added benefit of being quantitative. Here we outline the capillary Western method that links the 2D Western coverage and ultimately ELISAs for more efficient HCP quantitation. This study describes the development of the capillary Western analytical method to quantitively evaluate HCPs in Vero and Chinese Hamster Ovarian (CHO) cell lines. The amount of CHO HCPs decreases as the sample is purified as expected. Using this approach, we determined that the detected Vero HCPs amount was similar irrespective of denatured (capillary Western) versus native assay format (ELISA). This new method can also be potentially employed to quantitatively assess the anti-HCP antibody reagent coverage used in commercial HCP ELISA kits.

Introduction: Patients implanted with Continuous Flow Ventricular Assisted Devices (CF VADs) exhibit diminished pulsatility and are at a high risk for developing acquired von Willebrand Factor syndrome (AVWS) and non-surgical bleeding. This study aimed to understand how diminished pulsatility due to CF VAD impacts unravelling and patient plasma levels of von Willebrand Factor (vWF). A microfluidic approach was used to study unravelling of vWF under normal pulsatile flow and flow with diminished pulsatility. In addition, vWF levels in CF-VAD patients was measured to determine vWF levels in circulation. Hypothesis: We hypothesized that diminished pulsatility increases vWF unravelling, likely leading to increased vWF degradation and elevated levels of low MW vWF fragments in circulation, this in turn leads to decreased endothelial vWF production in CF-VAD patients. Methods: vWF molecules were immobilized in a microfluidic device and subjected to either normal pulsatile flow or flow with diminished pulsatility (same mean flow). vWF unravelling behavior was observed using total internal reflection fluorescence (TIRF) microscopy. Patient blood samples were collected 1-2 days pre CF-VAD implant and monthly post-implant. Patient plasma vWF levels were measured using an ELISA kit. Results: TIRF imaging showed that vWF molecules undergo unravelling and significantly greater elongation (p<0.05) under diminished pulsatility than with normal physiological pulsatility, despite higher levels of peak shear rates with normal pulsatility ( Fig.1A ). Evaluation of plasma vWF levels in patients (n=9) showed that vWF levels decreased progressively following CF-VAD placement ( Fig.1B ). These results suggest that diminished pulsatility increased unravelling of vWF and exposure of ADAMTS13 binding sites, potentially leading to enhanced cleavage of vWF into low molecular weight (MW) multimers. Review of literature suggests that both low MW multimers and diminished pulsatility cause endothelial dysfunction and decreased endothelial vWF production, which was evident in patient samples. Conclusion: Diminished pulsatility may independently promote vWF degradation and lead to decreased production of vWF, thus contributing to AVWS.

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, vWF level is measured from 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 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.

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.

We perform single-molecule flow experiments using confocal microscopy and a microfluidic device for shear rates up to 20,000 s(-1) 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.

The glycoprotein von Willebrand Factor (vWF) plays a crucial role in forming blood clots upon vascular injury, particularly when the rate of blood flow near a trauma site is high. vWF molecules adopt a compact conformation in typical blood flow but, subject to hydrodynamic forces in regions where flow rate is high, they undergo significant elongation, revealing binding sites for platelets in blood and collagen exposed on damaged vessel walls. In this fashion, vWF molecules initiate clot formation. We have formulated a multiscale simulation platform of vWF, directly coupled to experimental investigation via single molecule force spectroscopy and microfluidic imaging. In addition to experiments, all-atom molecular dynamics simulations of monomer domains are used to further optimize the mechano-biological behavior exhibited by the coarse-grain model. Herein, results from all-atom simulations of the A2 domain in equilibrium, in untethered flow, and subject to external pulling forces will be presented. This domain has been identified as a possible governing unit for vWF binding to both collagen and platelets. Results from molecular dynamics simulations are used to parameterize hydrodynamic drag experienced by monomers in our coarse-grain model as well as the coarse-grain monomer's mechanical behavior. Furthermore, all-atom simulations inform the coarse-grain model's description of binding between vWF and collagen. Following presentation of all-atom simulations, results from Brownian dynamics coarse-grain molecular simulations will be presented of vWF behavior in shear flow and when bound to model collagen. Results in shear flow provide strong evidence that scission of vWF molecules - critical to maintaining a functional size distribution of the protein - occurs preferentially near the center of vWF multimers. Lastly, coarse-grain simulations of vWF-collagen binding in shear flow permit a more detailed description of the possible role played by A2 domains in governing this reaction.

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.

High-tech applications often demand the surface modification of polymeric materials using chemoselective reactions that can proceed under mild conditions. In this paper, a feasible method for the surface modification of polymeric materials with low density polyethylene (LDPE) films as model substrates based on visible light-induced thione-ene cycloaddition reaction is proposed. This strategy includes three steps: 3-((6-Hydroxyhexyl)oxy)-9H-xanthene-9-thione (HXT) containing visible light-reactive thiocarbonyl groups was firstly synthesized; Then thiocarbonyl groups were introduced onto LDPE films previously surface-grafted with poly(styrene-co-maleic anhydride) brushes by the reaction between the hydroxyl groups of HXT and anhydride groups; Functional alkenes were finally fixed onto the surface of LDPE films by thione-ene cycloaddition reaction conducted under visible light at room temperature (r.t.). By FTIR, UV–vis spectroscopy, water contact angle test and XPS, we found that four typical functional alkenes, poly(ethylene glycol) methyl ethermethacrylate, 2-(perflurooctyl)ethyl methacrylate, 2, 3-dibromopropyl acrylate, and diethyl vinylphosphonate, can be successfully ligated, which confirmed the effectiveness and versatility of the present method.