Quantification of DNA by a Thermal-Durable Biosensor Modified with Conductive Poly(3,4-ethylenedioxythiophene).

The general clinical procedure for viral DNA detection or gene mutation diagnosis following polymerase chain reaction (PCR) often involves gel electrophoresis and DNA sequencing, which is usually time-consuming. In this study, we have proposed a facile strategy to construct a DNA biosensor, in which the platinum electrode was modified with a dual-film of electrochemically synthesized poly(3,4-ethylenedioxythiophene) (PEDOT) resulting in immobilized gold nanoparticles, with the gold nanoparticles easily immobilized in a uniform distribution. The DNA probe labeled with a SH group was then assembled to the fabricated electrode and employed to capture the target DNA based on the complementary sequence. The hybridization efficiency was evaluated with differential pulse voltammetry (DPV) in the presence of daunorubicin hydrochloride. Our results demonstrated that the peak current in DPV exhibited a linear correlation the concentration of target DNA that was complementary to the probe DNA. Moreover, the electrode could be reused by heating denaturation and re-hybridization, which only brought slight signal decay. In addition, the addition of the oxidized form of nicotinamide adenine dinucleotide (NAD⁺) could dramatically enhance the sensitivity by more than 5.45-fold, and the limit-of-detection reached about 100 pM.

Lower-than-normal glycemic levels to achieve optimal reduction of diabetes risk among individuals with prediabetes: A prospective cohort study.

AIMS/HYPOTHESIS: To determine whether lower than currently accepted glycemic levels could lead to optimal risk reduction of incident diabetes among individuals with prediabetes. METHODS: We enrolled 9903 individuals with prediabetes and 16,902 individuals with normoglycemia from a prospective cohort participating health check-ups between 2006 and 2017. While classifying fasting glucose into <5.0, 5.0-5.5, and 5.6-6.9 mmol/L and postprandial glucose into <6.7, 6.7-7.7, and 7.8-11.0 mmol/L, we grouped fasting/postprandial glucose into five categories (<5.0/<6.7, <5.0/6.7-7.7, 5.0-5.5/<6.7, 5.0-5.5/6.7-7.7 mmol/L, 5.6-6.9/7.8-11.0 mmol/L). The primary outcome was incident diabetes. RESULTS: In individuals with prediabetes, the presence of a baseline fasting glucose <5.0 mmol/L or a postprandial glucose <6.7 mmol/L led to a greater risk reduction of incident diabetes with hazard ratios of 0.34 (95% confidence interval, 0.27-0.42) and 0.47 (0.41-0.54), respectively, relative to a fasting glucose 5.6-6.9 mmol/L and a postprandial glucose 7.8-11.0 mmol/L. For individuals with prediabetes having fasting/postprandial glucose <5.0/<6.7 mmol/L, the incidence of 6.4 (4.7-8.8) per 1000 person-years corresponded to that of 5.8 (4.2-8.0) per 1000 person-years for individuals with normoglycemia having 5.0-5.5/6.7-7.7 mmol/L. CONCLUSIONS/INTERPRETATION: Given that lower-than-normal glycemic levels were plausible for optimal risk reduction of diabetes, stringent glycemic management could be beneficial for diabetes prevention among individuals with prediabetes.

Tethered fibronectin liposomes on supported lipid bilayers as a prepackaged controlled-release platform for cell-based assays.

A biomimetic construct containing an extracellular matrix protein-liposome composite tethered on supported lipid bilayers (SLBs) was formed with fibronectin (FN), and polyethylene glycol (PEG) and cholesterol-containing liposomes. The construct can serve as a multifunctional platform for cell attachment and drug release. The successful fabrication of the FN-liposome-SLB model platform was analyzed in situ with a quartz crystal microbalance with dissipation. The long-term stability of the surface tethered liposomes was measured via an encapsulated fluorescent probe. Less than 20% of the fluorescent probe content was released in 8 days, which compared favorably to the release of 90% of the probe content in one day from a similar construct made without PEG and cholesterol. HeLa cells were used to study the cellular interactions with the model platform. The extracellular matrix composition, FN, was found to be essential to promote HeLa cell adhesion on the liposome-SLB surfaces. Upon cell adhesion, the liposomes were spatially reorganized and absorbed by the cells. The rate of HeLa cell apoptosis was correlated with the surface density of doxorubicin-loaded liposomes, confirming the effective drug delivery through liposomes. The multifunctional model platform could be useful as preadministered, controlled-release platforms for cell- and tissue-based assays.

Type I collagen-functionalized supported lipid bilayer as a cell culture platform.

The supported phospholipid bilayer serves as an important biomimetic model for the cell membrane in both basic and applied scientific research. We have constructed a biomimetic platform based on a supported phospholipid bilayer that is functionalized with type I collagen to serve as a substrate for cell culture. To create the type I collagen-functionalized lipid bilayer assembly, a simple chemical approach was employed: lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (DP-NGPE), a carboxylic acid-functionalized phospholipid, were prepared and then fused onto an SiO(2) substrate to form a supported lipid bilayer. Subsequently, type I collagen molecules were introduced to form stable collagen-lipid conjugates via amide linkages with activated DP-NGPE lipids. The binding kinetics of the conjugation process and the resultant changes in film thickness and viscoelasticity were followed using the quartz crystal microbalance with dissipation (QCM-D) monitoring. The morphology of the conjugated collagen adlayer was investigated with atomic force microscopy (AFM). We observed that the adsorbed collagen molecules tended to self-assemble into fibrillar structures. Fluorescence recovery after photobleaching (FRAP) was utilized to estimate lateral lipid mobility, which was reduced by up to 20% after the coupling of type I collagen to the underlying lipid bilayer. As a cell culture platform, the collagen-conjugated supported lipid bilayer showed promising results. Smooth muscle cells (A10) retained normal growth behavior on the collagen-functionalized platform, unlike the bare POPC lipid bilayer and the POPC/DG-NGPE bilayer without collagen. The biomimetic functionalized lipid system presented here is a simple, yet effective approach for constructing a cell culture platform to explore the interactions between extracellular matrix components and cells.

Patellar malalignment treated with modified knee extension training: An electromyography study.

BACKGROUND: Patellar malalignment (PM) in most patients is ascribed to an imbalance of peripatellar soft tissue tension. RESEARCH QUESTION: Conservative treatment of PM initially with enforced training of the vastus medialis obliquus (VMO) has been widely applied. Non-operative techniques for treatment of PM require continuing development. METHODS: Thirty healthy young adults participated in the study. Two surface electromyography (EMG) electrodes were placed on the skin of the dominant lower thigh in each subject: one on the center of the muscle belly of the VMO and the other on the symmetric location of the vastus lateralis (VL). Maximum of knee extension action (from various angles of knee flexion to full extension) was initiated. Tests were conducted with knee flexion decreasing by 10° at each step. Each action was repeated three times, and the average value was calculated. The root mean square value of excited muscles in the EMG was recorded. The ratio of the VMO to the VL (VMO/VL) was used to indicate the effectiveness of the treatment. The knee position varied from 90° flexion initially, decreasing by 10° at each step. RESULTS: Nine sets of values were obtained. All extension actions were effective (VMO/VL >1; range, 1.23-1.35). The maximal value was observed at 60° flexion (VMO/VL = 1.35). Differences were not significant among the nine groups (p = 0.08, ANOVA). SIGNIFICANCE: Using the described knee extension training for conservative treatment of PM may be an effective alternative. The technique is simple, and the results of our experimental tests are encouraging. This method may become another popular and effective technique for treating PM.

Catalytic efficiency of a thrombomodulin-functionalized membrane-mimetic film in a flow model.

The protein C anticoagulant pathway generates an "on demand" physiologic anticoagulant response, which is initiated when thrombin binds to thrombomodulin (TM), a transmembrane protein constitutively expressed by endothelial cells. A stable, protein C activating membrane-mimetic film was produced on a polyelectrolyte multilayer (PEM) by in situ photopolymerization of a phospholipid assembly containing TM. The monoacrylated phospholipid monomer was initially synthesized and prepared as unilamellar vesicles with varying molar concentrations of TM. Membrane-mimetic films were constructed on planar substrates with defined surface concentrations of catalytically active TM. 125I-labeled radiolabeling demonstrated little change in TM surface concentration over periods of up to 4 weeks. We utilized a parallel plate flow system to investigate the effects of simulated arterial (500 s(-1)) and venous (50 s(-1)) shear rates and TM surface concentration (0-1400 fmol cm(-2)) on the rate and extent of activation of protein C. The rate of production of activated protein C increased with shear rate and TM surface content. However, in agreement with an analysis of reaction kinetics and mass transfer, experimental results demonstrate that reaction rates become saturated at TM surface densities greater than or equal to 800 fmol cm(-2). We believe that the design of membrane-mimetic films that have the capacity to activate the protein C pathway will provide a useful strategy for generating "actively" antithrombogenic surfaces.

Fabrication and characterization of heparin functionalized membrane-mimetic assemblies.

A membrane-mimetic assembly incorporating surface bound heparin was fabricated as a system to improve the hemocompatibility of blood-contacting devices. As a model system, heparin was chemically modified by end-point conjugation to biotin and immobilized onto membrane-mimetic thin films via biotin-streptavidin interactions. Heparin surface density, determined by radiochemical titration, confirmed that surface density was directly related to the molar concentration of biotinylated lipid within the assembled membrane-mimetic film. The capacity of surface bound heparin to promote ATIII-mediated thrombin inactivation was investigated in a parallel plate flow chamber under simulated venous and arterial wall shear rates of 50 and 500 s(-1), respectively. Significantly, we observed that the rate of thrombin inactivation approached a maximum at a heparin surface concentration greater than 4.4 pmol/cm(2) (61 ng/cm(2)). In the process, mass transport limited regimes were identified for heparin potentiated thrombin inactivation under both simulated venous and arterial conditions.

Membrane-mimetic films containing thrombomodulin and heparin inhibit tissue factor-induced thrombin generation in a flow model.

Membrane-mimetic thin films containing thrombomodulin (TM) and/or heparin were produced and their capacity to inhibit thrombin generation evaluated in a continuous flow system. Tissue factor (TF) along with TM and heparin were immobilized in spatially restricted zones as components of a membrane-mimetic film. Specifically, TF was positioned as an upstream trigger for thrombin generation and TM and/or heparin positioned over the remaining downstream portion of test films. Peak and steady-state levels of thrombin were decreased by antithrombin III (ATIII), as well as by surface bound heparin and TM. Although physiologic concentrations of ATIII have the capacity to significantly inhibit thrombin activity, surface bound TM and heparin nearly abolished steady-state thrombin responses. In particular, surface bound TM appears to be superior to heparin in reducing local thrombin concentrations. These studies are the first to demonstrate the additive effect of surface bound heparin and TM as a combined interactive strategy to limit TF-induced thrombin formation.

High Mobility of Graphene-Based Flexible Transparent Field Effect Transistors Doped with TiO2 and Nitrogen-Doped TiO2.

Graphene with carbon atoms bonded in a honeycomb lattice can be tailored by doping various species to alter the electrical properties of the graphene for fabricating p-type or n-type field-effect transistors (FETs). In this study, large-area and single-layer graphene was grown on electropolished Cu foil using the thermal chemical vapor deposition method; the graphene was then transferred onto a poly(ethylene terephthalate) (PET) substrate to produce flexible, transparent FETs. TiO2 and nitrogen-doped TiO2 (N-TiO2) nanoparticles were doped on the graphene to alter its electrical properties, thereby enhancing the carrier mobility and enabling the transistors to sense UV and visible light optically. The results indicated that the electron mobility of the graphene was 1900 cm(2)/(V·s). Dopings of TiO2 and N-doped TiO2 (1.4 at. % N) lead to n-type doping effects demonstrating extremely high carrier mobilities of 53000 and 31000 cm(2)/(V·s), respectively. Through UV and visible light irradiation, TiO2 and N-TiO2 generated electrons and holes; the generated electrons transferred to graphene channels, causing the FETs to exhibit n-type electric behavior. In addition, the Dirac points of the graphene recovered to their original state within 5 min, confirming that the graphene-based FETs were photosensitive to UV and visible light. In a bending state with a radius of curvature greater than 2.0 cm, the carrier mobilities of the FETs did not substantially change, demonstrating the application possibility of the fabricated graphene-based FETs in photosensors.

Antibody conjugated supported lipid bilayer for capturing and purification of viable tumor cells in blood for subsequent cell culture.

Interest in the identification and isolation of circulating tumor cells (CTCs) has been growing since the introduction of CTCs as an alternative to the tumor tissue biopsy, which can potentially be important indices for prognosis and cancer treatment. However, the contamination of non-specific binding of normal hematologic cells makes high purity CTCs detection problematic. Furthermore, preserving the viability of CTCs remains a challenge. In this study, we proposed to construct an anti-EpCAM functionalized supported lipid bilayer (SLB), a biomimetic and non-fouling membrane coating, for CTCs capturing, purification and maintaining the viability. Healthy human blood spiked with pre-stained colorectal cancer cell lines, HCT116 and colo205, were used to investigate interaction of cells with the anti-EpCAM functionalized SLB surfaces. Over 97% of HCT116, and 72% of colo205 were captured and adhered by the surface anti-EpCAM; conversely, the majority of blood cells were easily removed by gentle buffer exchange, with the overall purity of cancer cells exceeding 95%. The bound cancer cells were subsequently detached for cell culture. Both HCT116 and colo205 continued to proliferate over 2-week observation period, indicating that the anti-EpCAM functionalized SLB platform providing a simple strategy for capturing, purifying, and releasing viable targeted rare cells.

Effects of extracellular matrix protein functionalized fluid membrane on cell adhesion and matrix remodeling.

In order to study cellular responses and extracellular matrix protein remodeling mediated by biomaterials coating, we proposed a biomimetic construct containing protein-conjugated supported lipid bilayers (SLBs) as a cell culture platform. Single or multi-component proteins-bound SLBs were fabricated by conjugating type I collagen and/or fibronectin on the N-hydroxysulfosuccinimide-functionalized SLBs. The proposed protein-conjugated systems were quantitatively characterized by the quartz crystal microbalance with dissipation. NIH 3T3 fibroblasts were cultured on the model constructs and on oxygen plasma pretreated polystyrene (PSo) for parallel comparison. The retards of mobility of SLB after protein conjugation and cell culture were estimated by fluorescence recovery after photobleaching. The resulting cell morphology, adsorption kinetics and somatic dynamics were examined microscopically. We found that, on the SLB based cultures, the largest spreading size and cell number counts of 3T3 fibroblasts were found on the fibronectin containing surfaces. However, on the protein-coated PSo surfaces, no such distinguishable differences can be observed on all protein contents. Immunofluorescent staining results revealed that adsorption of endogenously produced fibronectin by 3T3 cells on PSo based surfaces is significantly more than that on SLB based surfaces. This suggests that the anti-fouling nature of underneath SLBs have played an important role in preventing 3T3 cells from effectively remodeling their microenvironment, whereas cells can easily remodel the nonspecific adsorption prone surfaces such as PSo based platforms. In summary, the protein conjugated SLB surfaces can serve as a platform for determining and regulating cell specific binding and subsequent signaling events with extracellular environments.