BackgroundCells within tissues are subjected to mechanical forces caused by extracellular matrix deformation. Cells sense and dynamically respond to stretching of the matrix by reorienting their actin stress fibers and by activating intracellular signaling proteins, including focal adhesion kinase (FAK) and the mitogen-activated proteins kinases (MAPKs). Theoretical analyses predict that stress fibers can relax perturbations in tension depending on the rate of matrix strain. Thus, we hypothesized stress fiber organization and MAPK activities are altered to an extent dependent on stretch frequency.Principal FindingsBovine aortic endothelial cells and human osteosarcoma cells expressing GFP-actin were cultured on elastic membranes and subjected to various patterns of stretch. Cyclic stretching resulted in strain rate-dependent increases in stress fiber alignment, cell retraction, and the phosphorylation of the MAPKs JNK, ERK and p38. Transient step changes in strain rate caused proportional transient changes in the levels of JNK and ERK phosphorylations without affecting stress fiber organization. Disrupting stress fiber contractile function with cytochalasin D or Y27632 decreased the levels of JNK and ERK phosphorylation. Previous studies indicate that FAK is required for stretch-induced cell alignment and MAPK activations. However, cyclic uniaxial stretching induced stress fiber alignment and the phosphorylation of JNK, ERK and p38 to comparable levels in FAK-null and FAK-expressing mouse embryonic fibroblasts.ConclusionsThese results indicate that cyclic stretch-induced stress fiber alignment, cell retraction, and MAPK activations occur as a consequence of perturbations in fiber strain. These findings thus shed new light into the roles of stress fiber relaxation and reorganization in maintenance of tensional homeostasis in a dynamic mechanical environment.
Point-of-care (POC) device development is a growing field that aims to develop low-cost, rapid, sensitive in-vitro diagnostic testing platforms that are portable, selfcontained, and can be used anywhere -from modern clinics to remote and low resource areas. In this review, surface enhanced Raman spectroscopy (SERS) is discussed as a solution to facilitating the translation of bioanalytical sensing to the POC. The potential for SERS to meet the widely accepted "ASSURED" (Affordable, Sensitive, Specific, Userfriendly, Rapid, Equipment-free, and Deliverable) criterion provided by the World Health Organization is discussed based on recent advances in SERS in vitro assay development. As SERS provides attractive characteristics for multiplexed sensing at low concentration limits with a high degree of specificity, it holds great promise for enhancing current efforts in rapid diagnostic testing. In outlining the progression of SERS techniques over the past years combined with recent developments in smart nanomaterials, high-throughput microfluidics, and low-cost paper diagnostics, an extensive number of new possibilities show potential for translating SERS biosensors to the POC.
Competitive binding assays utilizing concanavalin A (ConA) have the potential to be the basis of improved continuous glucose monitoring devices. However, the efficacy and lifetime of these assays have been limited, in part, by ConA’s instability due to its thermal denaturation in the physiological environment (37 °C, pH 7.4, 0.15 M NaCl) and its electrostatic interaction with charged molecules or surfaces. These undesirable interactions change the constitution of the assay and the kinetics of its behavior over time, resulting in an unstable glucose response. In this work, poly(ethylene glycol) (PEG) chains are covalently attached to lysine groups on the surface of ConA (i.e., PEGylation) in an attempt to improve its stability in these environments. Dynamic light scattering measurements indicate that PEGylation significantly improved ConA’s thermal stability at 37 °C, remaining stable for at least 30 days. Furthermore, after PEGylation, ConA’s binding affinity to the fluorescent competing ligand previously designed for the assay was not significantly affected and remained at ∼5.4 × 106 M–1 even after incubation at 37 °C for 30 days. Moreover, PEGylated ConA maintained the ability to track glucose concentrations when implemented within a competitive binding assay system. Finally, PEGylation showed a reduction in electrostatic-induced aggregation of ConA with poly(allylamine), a positively charged polymer, by shielding ConA’s charges. These results indicate that PEGylated ConA can overcome the instability issues from thermal denaturation and nonspecific electrostatic binding while maintaining the required sugar-binding characteristics. Therefore, the PEGylation of ConA can overcome major hurdles for ConA-based glucose sensing assays to be used for long-term continuous monitoring applications in vivo.
A self-cleaning membrane that periodically rids itself of attached cells to maintain glucose diffusion could extend the lifetime of implanted glucose biosensors. Herein, we evaluate the functionality of thermoresponsive double network (DN) hydrogel membranes based on poly(N-isopropylacrylamide) (PNIPAAm) and an electrostatic co-monomer, 2-acrylamido-2-methylpropane sulfonic acid (AMPS). DN hydrogels are comprised of a tightly crosslinked, ionized first network [P(NIPAAm-co-AMPS)] containing variable levels of AMPS (100:0–25:75 wt% ratio of NIPAAm:AMPS) and a loosely crosslinked, interpenetrating second network [PNIPAAm]. To meet the specific requirements of a subcutaneously implanted glucose biosensor, the volume phase transition temperature is tuned and essential properties, such as glucose diffusion kinetics, thermosensitivity, and cytocompatibility are evaluated. In addition, the self-cleaning functionality is demonstrated through thermally driven cell detachment from the membranes in vitro.
Competitive binding assays based on the lectin Concanavalin A (ConA) have displayed significant potential to serve in continuous glucose monitoring applications. However, to date, this type of fluorescent, affinity-based assay has yet to show the stable, glucose predictive capabilities that are required for such an application. This instability has been associated with the extensive crosslinking between traditionally-used fluorescent ligands (presenting multiple low-affinity moieties) and ConA (presenting multiple binding sites) in free solution. The work herein introduces the design and synthesis of a new type of fluorescent ligand that can avoid this aggregation and allow the assay to be sensitive across the physiologically relevant glucose concentration range. This fluorescent ligand (APTS–MT) presents a single high-affinity trimannose moiety that is recognized by ConA’s full binding site and a fluorophore that can effectively track the ligand’s equilibrium binding via fluorescent anisotropy. This is confirmed by comparing its measured fluorescent lifetime to experimentally-determined rotational correlation lifetimes of the free and bound populations. Using an assay comprised of 200 nM APTS–MT and 1 μM ConA, the fluorescence anisotropy capably tracks the concentration of monosaccharides that are known to bind to ConA’s primary binding site, and the assay displays a MARD of 6.5% across physiologically relevant glucose concentrations. Ultimately, this rationally-designed fluorescent ligand can facilitate the realization of the full potential of ConA-based glucose sensing assays and provide the basis for a new set of competing ligands to be paired with ConA.
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