An integrated E-Tube cap for sample preparation, isothermal amplification and label-free electrochemical detection of DNA.

A simple, disposable, and integrated electronic-tube cap (E-tube cap) for DNA detection at the point-of-care was designed, fabricated, and tested. The E-tube cap contains a 3D printed electrode substrate for DNA extraction and label-free pH sensing detection. One Flinders Technology Associates (Whatman FTA) membrane was incorporated into the 3D printed electrode substrate for the isolation, concentration, and purification of DNA. The E-tube cap with captured DNA by the membrane was inserted directly into a reaction tube for loop-mediated isothermal amplification (LAMP). The isothermal amplification process was monitored in real-time by a 3D printed electrochemical electrode coated with pH-sensitive material (carbon/iridium oxide layer). The pH sensing electrode showed an excellent linear response within the pH range of 6-9 with a slope of -31.32 ± 0.5 mV/pH at room temperature. The utility of the integrated E-tube cap was demonstrated by detecting the presence of lambda DNA spiked in saliva samples with a sensitivity of 100 copies per mL sample within 30 min. Such a simple, rapid, and affordable diagnostic device is particularly suitable for point-of-care molecular diagnostics of infectious diseases.

Mechanical stress compromises multicomponent efflux complexes in bacteria.

Physical forces have a profound effect on growth, morphology, locomotion, and survival of organisms. At the level of individual cells, the role of mechanical forces is well recognized in eukaryotic physiology, but much less is known about prokaryotic organisms. Recent findings suggest an effect of physical forces on bacterial shape, cell division, motility, virulence, and biofilm initiation, but it remains unclear how mechanical forces applied to a bacterium are translated at the molecular level. In Gram-negative bacteria, multicomponent protein complexes can form rigid links across the cell envelope and are therefore subject to physical forces experienced by the cell. Here we manipulate tensile and shear mechanical stress in the bacterial cell envelope and use single-molecule tracking to show that octahedral shear (but not hydrostatic) stress within the cell envelope promotes disassembly of the tripartite efflux complex CusCBA, a system used by Escherichia coli to resist copper and silver toxicity. By promoting disassembly of this protein complex, mechanical forces within the cell envelope make the bacteria more susceptible to metal toxicity. These findings demonstrate that mechanical forces can inhibit the function of cell envelope protein assemblies in bacteria and suggest the possibility that other multicomponent, transenvelope efflux complexes may be sensitive to mechanical forces including complexes involved in antibiotic resistance, cell division, and translocation of outer membrane components. By modulating the function of proteins within the cell envelope, mechanical stress has the potential to regulate multiple processes required for bacterial survival and growth.

Large-scale polymeric carbon nanotube membranes with sub-1.27-nm pores.

We report the first characterization study of commercial prototype carbon nanotube (CNT) membranes consisting of sub-1.27-nm-diameter CNTs traversing a large-area nonporous polysulfone film. The membranes show rejection of NaCl and MgSO4 at higher ionic strengths than have previously been reported in CNT membranes, and specific size selectivity for analytes with diameters below 1.24 nm. The CNTs used in the membranes were arc discharge nanotubes with inner diameters of 0.67 to 1.27 nm. Water flow through the membranes was 1000 times higher than predicted by Hagen-Poiseuille flow, in agreement with previous CNT membrane studies. Ideal gas selectivity was found to deviate significantly from that predicted by both viscous and Knudsen flow, suggesting that surface diffusion effects may begin to dominate gas selectivity at this size scale.

Elevated solute transport at sites of diffuse matrix damage in cortical bone: Implications on bone repair.

Diffuse matrix damage in rat cortical bone has been observed to self-repair efficiently in 2 weeks without activating bone remodeling, and unlike the case with linear cracks, the local osteocytes at the sites of diffuse damage remain healthy. However, the reason(s) for such high efficiency of matrix repair remains unclear. We hypothesized that transport of minerals and other compounds essential for damage repair is enhanced at the damaged sites and further increased by the application of tensile loading. To test our hypothesis, diffuse damage was introduced in notched bovine wafers under cyclic tensile loading and unloading. Using the Fluorescence Recovery After Photobleaching (FRAP) approach, we measured the transport of a small fluorescent tracer (sodium fluorescein, 376 Da) in damaged versus undamaged regions and under varying tensile load magnitudes (0.2 N, 10 N, 20 N, and 30 N), which corresponded to nominal strains of 12.5, 625, 1,250, and 1,875 microstrains, respectively. We found a 37% increase in transport of fluorescein in damaged regions relative to undamaged regions and a further ∼18% increase in transport under 20 N and 30 N tension compared to the non-loaded condition, possibly due to the opening of the cracking surfaces. The elevated transport of minerals and other adhesive proteins may, at least partially, account for the highly effective repair of diffuse damage observed in vivo. CLINICAL SIGNIFICANCE: Diffuse damage adversely affects bone's fracture resistance and this study provided quantitative data on elevated transport, which may be involved in repairing diffuse damage in vivo. 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:692-698, 2018.

Seeing is believing, PLGA microsphere degradation revealed in PLGA microsphere/PVA hydrogel composites.

The aim of this study was to understand the polymer degradation and drug release mechanism from PLGA microspheres embedded in a PVA hydrogel. Two types of microspheres were prepared with different molecular weight PLGA polymers (approximately 25 and 7 kDa) to achieve different drug release profiles, with a 9-day lag phase and without a lag phase, respectively. The kinetics of water uptake into the microspheres coincided with the drug release profiles for both formulations. For the 25 kDa microspheres, minimal water uptake was observed in the early part of the lag phase followed by substantial water uptake at the later stages and in the drug release phase. For the 7 kDa microspheres, water uptake occurred simultaneously with drug release. Water uptake was approximately 2-3 times that of the initial microsphere weight for both formulations. The internal structure of the PLGA microspheres was evaluated using low temperature scanning electron microscopy (cryo-SEM). Burst drug release occurred followed by pore forming from the exterior to the core of both microspheres. A well-defined hydrogel/microsphere interface was observed. For the 25 kDa microspheres, internal pore formation and swelling occurred before the second drug release phase. The surface layer of the microspheres remained intact whereas swelling, and degradation of the core continued throughout the drug release period. In addition, microsphere swelling reduced glucose transport through the coatings in PBS media and this was considered to be a as a consequence of the increased thickness of the coatings. The combination of the swelling and microdialysis results provides a fresh understanding on the competing processes affecting molecular transport of bioanalytes (i.e. glucose) through these composite coatings during prolonged exposure in PBS.

A microfluidic platform for profiling biomechanical properties of bacteria.

The ability to resist mechanical forces is necessary for the survival and division of bacteria and has traditionally been probed using specialized, low-throughput techniques such as atomic force microscopy and optical tweezers. Here we demonstrate a microfluidic technique to profile the stiffness of individual bacteria and populations of bacteria. The approach is similar to micropipette aspiration used to characterize the biomechanical performance of eukaryotic cells. However, the small size and greater stiffness of bacteria relative to eukaryotic cells prevents the use of micropipettes. Here we present devices with sub-micron features capable of applying loads to bacteria in a controlled fashion. Inside the device, individual bacteria are flowed and trapped in tapered channels. Less stiff bacteria undergo greater deformation and therefore travel further into the tapered channel. Hence, the distance traversed by bacteria into a tapered channel is inversely related to cell stiffness. We demonstrate the ability of the device to characterize hundreds of bacteria at a time, measuring stiffness at 12 different applied loads at a time. The device is shown to differentiate between two bacterial species, E. coli (less stiff) and B. subtilis (more stiff), and detect differences between E. coli submitted to antibiotic treatment from untreated cells of the same species/strain. The microfluidic device is advantageous in that it requires only minimal sample preparation, no permanent cell immobilization, no staining/labeling and maintains cell viability. Our device adds detection of biomechanical phenotypes of bacteria to the list of other bacterial phenotypes currently detectable using microchip-based methods and suggests the feasibility of separating/selecting bacteria based on differences in cell stiffness.

Osteoblasts detect pericellular calcium concentration increase via neomycin-sensitive voltage gated calcium channels.

The mechanisms underlying the detection of critically loaded or micro-damaged regions of bone by bone cells are still a matter of debate. Our previous studies showed that calcium efflux originates from pre-failure regions of bone matrix and MC3T3-E1 osteoblasts respond to such efflux by an increase in the intracellular calcium concentration. The mechanisms by which the intracellular calcium concentration increases in response to an increase in the pericellular calcium concentration are unknown. Elevation of the intracellular calcium may occur via release from the internal calcium stores of the cell and/or via the membrane bound channels. The current study applied a wide range of pharmaceutical inhibitors to identify the calcium entry pathways involved in the process: internal calcium release from endoplasmic reticulum (ER, inhibited by thapsigargin and TMB-8), calcium receptor (CaSR, inhibited by calhex), stretch-activated calcium channel (SACC, inhibited by gadolinium), voltage-gated calcium channels (VGCC, inhibited by nifedipine, verapamil, neomycin, and ω-conotoxin), and calcium-induced-calcium-release channel (CICRC, inhibited by ryanodine and dantrolene). These inhibitors were screened for their effectiveness to block intracellular calcium increase by using a concentration gradient induced calcium efflux model which mimics calcium diffusion from the basal aspect of cells. The inhibitor(s) which reduced the intracellular calcium response was further tested on osteoblasts seeded on mechanically loaded notched cortical bone wafers undergoing damage. The results showed that only neomycin reduced the intracellular calcium response in osteoblasts, by 27%, upon extracellular calcium stimulus induced by concentration gradient. The inhibitory effect of neomycin was more pronounced (75% reduction in maximum fluorescence) for osteoblasts seeded on notched cortical bone wafers loaded mechanically to damaging load levels. These results imply that the increase in intracellular calcium occurs by the entry of extracellular calcium ions through VGCCs which are sensitive to neomycin. N-type and P-type VGCCs are potential candidates because they are observed in osteoblasts and they are sensitive to neomycin. The calcium channels identified in this study provide new insight into mechanisms underlying the targeted repair process which is essential to bone adaptation.

Novel mechanical bioreactor for concomitant fluid shear stress and substrate strain.

The two main types of mechanical stimuli used in cellular-level bone mechanotransduction studies are substrate strain and flow-induced shear stress. A subset of studies has investigated which of these stimuli induces the primary mechanotransduction effect on bone cells. The shortcomings of these experiments are twofold. First, in some experiments the magnitude of one loading type is able to be quantitatively measured while the other loading mode is only estimated. Second, the two loading modes are compared using different bioreactors, representing different cellular environments and substrates to which the cells are attached. In addition, none of these studies utilized bioreactors which apply controlled magnitudes of substrate strain and flow-induced shear stress differentially and simultaneously. This study presents the design of a multimodal loading device which can apply substrate stretch and fluid flow simultaneously while allowing for real-time cell imaging. The mechanical performance of the bioreactor is validated in this study by correlating the output levels of flow-induced shear stress and substrate strain with the input levels of displacement and displacement rate. The magnitudes of cross-talk loading (i.e. flow-induced strain, and strain-induced fluid flow) are also characterized and shown to be magnitudes lower than physiological levels of loading estimated to occur in bone in vivo.

Mechanical stretch induced calcium efflux from bone matrix stimulates osteoblasts.

The mechanisms by which bone cells sense critically loaded regions of bone are still a matter of ongoing debate. Animal models to investigate response to microdamage involve post mortem immunohistological analysis and do not allow real-time monitoring of cellular response during the emergence of the damage in bone. Most in vitro mechanical stimulation studies are conducted on non-bone substrates, neglecting the damage-related alterations in the pericellular niche and their potential effects on bone cells. The current study reports spontaneous efflux of calcium ions (Ca(2+)) (1.924±0.742 pmol cm(-2)s(-1)) from regions of devitalized bone matrix undergoing post-yield strains, induced by a stress concentrator. When these samples are seeded with MC3T3-E1 osteoblasts, the strain-induced Ca(2+) efflux from bone elicits cell response at the stress concentration site as manifested by activation of intracellular calcium signaling (increase in fluorescence by 52%±27%). This activity is associated with extracellular calcium because the intracellular calcium signaling in response to mechanical loading subsides when experiments are repeated using demineralized bone substrates (increase in fluorescence by 6%±10%). These results imply a novel perspective where bone matrix acts as an intermediary mechanochemical transducer by converting mechanical strain into a chemical signal (pericellular calcium) to which cells respond. Such a mechanism may be responsible for triggering repair at locations of bone matrix undergoing critical deformation levels.

Tenogenic differentiation of human MSCs induced by the topography of electrochemically aligned collagen threads.

Topographical cues from the extracellular microenvironment can influence cellular activity including proliferation and differentiation. Information on the effects of material topography on tenogenic differentiation of human mesenchymal stem cells (human MSCs) is limited. A methodology using the principles of isoelectric focusing has previously been developed in our laboratory to synthesize electrochemically aligned collagen (ELAC) threads that mimics the packing density, alignment and strength of collagen dense connective tissues. In the current study, human MSCs were cultured on ELAC and randomly oriented collagen threads and the effect of collagen orientation on cell morphology, proliferation and tenogenic differentiation was investigated. The results indicate that higher rates of proliferation were observed on randomly oriented collagen threads compared to ELAC threads. On the other hand, tendon specific markers such as scleraxis and tenomodulin, were significantly increased on ELAC threads compared to randomly oriented collagen threads. Additionally, osteocalcin, a specific marker of bone differentiation was suppressed on ELAC threads. Previous studies have reported that BMP-12 is a key growth factor to induce tenogenic differentiation of MSCs. To evaluate the synergistic effect of BMP-12 and collagen orientation, human MSCs were cultured on ELAC threads in culture medium supplemented with and without BMP-12. The results revealed that BMP-12 did not have an additional effect on the tenogenic differentiation of human MSCs on ELAC threads. Together, these results suggest that ELAC induces tenogenic differentiation of human MSCs by presenting an aligned and dense collagen substrate, akin to the tendon itself. In conclusion, ELAC has a significant potential to be used as a tendon replacement and in the development of an osteotendinous construct towards the regeneration of bone-tendon interfaces.

Random lasing in bone tissue.

Owing to the low-loss and high refractive index variations derived from the basic building block of bone structure, we, for the first time to our knowledge, demonstrate coherent random lasing action originated from the bone structure infiltrated with laser dye, revealing that bone tissue is an ideal biological material for random lasing. Our numerical simulation shows that random lasers are extremely sensitive to subtle structural changes even at nanoscales and can potentially be an excellent tool for probing nanoscale structural alterations in real time as a novel spectroscopic modality.

Visualization of a phantom post-yield deformation process in cortical bone.

A prominent opacity is evident in the process zone of notched thin wafers of bone loaded in tension. Being recoverable upon unloading, this opaque zone can be stained only when the sample is under load, unlike the classically reported forms of damage which take up the stain in the unloaded state. Furthermore, despite the stain uptake, microcracks are absent in the stained area examined by high magnification optical microscopy and atomic force microscopy (AFM). Therefore, the size scale and the electric charge of the features involved in the process zone were probed at the submicron level by using a wide range of fluorescent dyes of different molecular weights and charges. It was observed that negatively charged dyes penetrate the process zone and that dyes greater than 10 kDa (about 10-20 nm in size) were unable to label the process zone. Digital image correlation (DIC) measurements indicated that the opacity initiates at about 1% principal strain and the strain accumulates up to 14%. While the opacity was largely recoverable upon unloading, the core regions which experienced large strains had permanent residual strains up to 2%, indicating that the observed deformation phenomenon can be interlocked within bone matrix without the formation of microcracks. Based on the similarity of size and their known affinity for negatively charged species, exposure of mineral nanoplatelets is proposed as prime candidates. Therefore, the deformation process reported here may be associated with debonding of mineral crystals from the neighboring collagen molecules. Overall, post-yield deformation of bone at the micron scale takes place by large strain events which are accommodated in bone matrix by the generation of nanoscale positively charged interfaces.

Spectroscopic visualization of nanoscale deformation in bone: interaction of light with partially disordered nanostructure.

Given that bone is an intriguing nanostructured dielectric as a partially disordered complex structure, we apply an elastic light scattering-based approach to image prefailure deformation and damage of bovine cortical bone under mechanical testing. We demonstrate that our imaging method can capture nanoscale deformation in a relatively large area. The unique structure, the high anisotropic property of bone, and the system configuration further allow us to use the transfer matrix method to study possible spectroscopic manifestations of prefailure deformation. Our sensitive yet simple imaging method could potentially be used to detect nanoscale structural and mechanical alterations of hard tissue and biomaterials in a fairly large field of view.

Detection of nanoscale structural changes in bone using random lasers.

We demonstrate that the unique characteristics of random lasing in bone can be used to assess nanoscale structural alterations as a mechanical or structural biosensor, given that bone is a partially disordered biological nanostructure. In this proof-of-concept study, we conduct photoluminescence experiments on cortical bone specimens that are loaded in tension under mechanical testing. The ultra-high sensitivity, the large detection area, and the simple detection scheme of random lasers allow us to detect prefailure damage in bone at very small strains before any microscale damage occurs. Random laser-based biosensors could potentially open a new possibility for highly sensitive detection of nanoscale structural and mechanical alterations prior to overt microscale changes in hard tissue and biomaterials.