Novel electrochemical xanthine biosensor based on chitosan–polypyrrole–gold nanoparticles hybrid bio-nanocomposite platform

The aim of this study was the electrochemical detection of the adenosine-3-phosphate degradation product, xanthine, using a new xanthine biosensor based on a hybrid bio-nanocomposite platform which has been successfully employed in the evaluation of meat freshness. In the design of the amperometric xanthine biosensor, chitosan–polypyrrole–gold nanoparticles fabricated by an in situ chemical synthesis method on a glassy carbon electrode surface was used to enhance electron transfer and to provide good enzyme affinity. Electrochemical studies were carried out by the modified electrode with immobilized xanthine oxidase on it, after which the biosensor was tested to ascertain the optimization parameters. The Biosensor exhibited a very good linear range of 1–200 μM, low detection limit of 0.25 μM, average response time of 8 seconds, and was not prone to significant interference from uric acid, ascorbic acid, glucose, and sodium benzoate. The resulting bio-nanocomposite xanthine biosensor was tested with fish, beef, and chicken real-sample measurements.     

Glucose decreases extracellular adenosine levels in isolated mouse and rat pancreatic islets

The pancreatic islets of Langerhans are responsible for the regulated release of the endocrine hormones insulin and glucagon that participate in the control of glucose homeostasis. Abnormal regulation of these hormones can result in glucose intolerance and lead to the development of diabetes. Numerous efforts have been made to better understand the physiological regulators of insulin and glucagon secretion. One of these regulators is the purine nucleoside, adenosine. Though exogenous application of adenosine has been demonstrated to stimulate glucagon release and inhibit insulin release, the physiological significance of this pathway has been unclear. We used a novel 7 µm enzyme-coated electrode biosensor to measure adenosine levels in isolated rodent islets. In the mouse islets, basal adenosine levels in the presence of 3 mM glucose were estimated to be 5.7 ± 0.6 µM. As glucose was increased, extracellular adenosine diminished. A 10-fold increase of extracellular KCl increased adenosine levels to 16.4 ± 2.0 µM. This release required extracellular Ca²⁺ suggesting that it occurred via an exocytosis-dependent mechanism. We also found that while rat islets were able to convert exogenous ATP into adenosine, mouse islets were unable to do this. Our study demonstrates for the first time the basal levels of adenosine and its inverse relationship to extracellular glucose in pancreatic islets.     

Label‐free molecular probe based on G‐quadruplex and strand displacement for sensitive and selective detection and naked eye discrimination of exon 2 deletion of AIMP2

Exon 2 deletion of aminoacyl tRNA synthetase complex‐interacting multifunctional protein 2 (AIMP2) is a genetic deletion related to various cancers, for instance ovarian and lung cancers. It can be worked as an indicator of cancer for diagnosis of diseases. Here, we developed a label‐free method based on the formation of split G‐quadruplex in the presence of target DNA combined with strand displacement to detect exon 2 deletion of AIMP2 (DE2) sensitively and selectively. This method is easy‐operating and cost‐saving. Moreover, it has observed discrimination of gene deletion from wild‐types by naked eyes. The results demonstrate that this strategy can be further used for the detection of different gene deletions to achieve early diagnosis of diseases and allow better prognosis.     

Free radical functionalization of surfaces to prevent adverse responses to biomedical devices

Immobilizing a protein, that is fully compatible with the patient, on the surface of a biomedical device should make it possible to avoid adverse responses such as inflammation, rejection, or excessive fibrosis. A surface that strongly binds and does not denature the compatible protein is required. Hydrophilic surfaces do not induce denaturation of immobilized protein but exhibit a low binding affinity for protein. Here, we describe an energetic ion-assisted plasma process that can make any surface hydrophilic and at the same time enable it to covalently immobilize functional biological molecules. We show that the modification creates free radicals that migrate to the surface from a reservoir beneath. When they reach the surface, the radicals form covalent bonds with biomolecules. The kinetics and number densities of protein molecules in solution and free radicals in the reservoir control the time required to form a full protein monolayer that is covalently bound. The shelf life of the covalent binding capability is governed by the initial density of free radicals and the depth of the reservoir. We show that the high reactivity of the radicals renders the binding universal across all biological macromolecules. Because the free radical reservoir can be created on any solid material, this approach can be used in medical applications ranging from cardiovascular stents to heart-lung machines.     

基于氧化石墨烯、适配体DNA及信号放大技术生物传感器的研究及应用

基于核酸适配体DNA和氧化石墨烯(graphene oxide,GO)的光学生物传感器因具有快速、准确及灵敏等优点,已越来越广泛地用于核酸、蛋白质及小分子底物的特异性检测.近年来的研究显示,将信号放大技术与适配体-GO检测方法结合可构建超高灵敏度的荧光光学生物传感器,实现痕量底物的特异性检测.文章综述了近年来氧化石墨烯、适配体DNA及核酸放大技术生物传感器的研究进展,以期为该类生物传感器的深入研究及大规模应用提供参考.     

How to Design a Biosensor

Amperometric sensors for continuous glucose monitoring could prevent acute and chronic complications of diabetes, but research is needed to improve accuracy and stability. In designing sensors, interference from non-glucose analytes can be minimized by use of filtration membranes or electron transfer mediators that allow polarization at low potentials. If oxygen is required for the enzymatic reaction with glucose, then the outer permselective membrane must have substantial oxygen permeability. For this reason, during development of permselective membranes, permeability studies (such as performed by Tipnis and colleagues in this issue) can be used to measure transport of glucose and oxygen and optimize membrane structure. Tipnis and colleagues present a novel biosensor based with separate layers for glucose-oxygen permselectivity, enzymatic conversion, and avoidance of interference. They also address sensor stability, in part by comparing sensor function during ascending vs descending glucose levels. By measuring the difference, they were able to minimize this aspect of instability (hysterisis), which assisted them in selecting a promising permselective membrane based on iron and humic acid.     

Pillared Graphene Structures Supported by Vertically Aligned Carbon Nanotubes as the Potential Recognition Element for DNA Biosensors

The development of electrochemical biosensors is an important challenge in modern biomedicine since they allow detecting femto- and pico-molar concentrations of molecules. During this study, pillared graphene structures supported by vertically aligned carbon nanotubes (VACNT-graphene) are examined as the potential recognition element of DNA biosensors. Using mathematical modeling methods, the atomic supercells of different (VACNT-graphene) configurations and the energy profiles of its growth are found. Regarding the VACNT(12,6)-graphene doped with DNA nitrogenous bases, calculated band structure and conductivity parameters are used. The obtained results show the presence of adenine, cytosine, thymine, and guanine on the surface of VACNT(12,6)-graphene significantly changes its conductivity so the considered object could be the prospective element for DNA biosensing.     

Resting Glutamate Levels and Rapid Glutamate Transients in the Prefrontal Cortex of the Flinders Sensitive Line Rat: A Genetic Rodent Model of Depression

Despite the numerous drugs targeting biogenic amines for major depressive disorder (depression), the search for novel therapeutics continues because of their poor response rates (∼30%) and slow onset of action (2–4 weeks). To better understand role of glutamate in depression, we used an enzyme-based microelectrode array (MEA) that was selective for glutamate measures with fast temporal (2 Hz) and high spatial (15 × 333 μm) resolution. These MEAs were chronically implanted into the prefrontal cortex of 3- to 6-month-old and 12- to 15-month-old Flinders Sensitive Line (FSL) and control Flinders Resistant Line (FRL) rats, a validated genetic rodent model of depression. Although no changes in glutamate dynamics were observed between 3 and 6 months FRL and FSL rats, a significant increase in resting glutamate levels was observed in the 12- to 15-month-old FSL rats compared with the 3- to 6-month-old FSL and age-matched FRL rats on days 3–5 post-implantation. Our MEA also recorded, for the first time, a unique phenomenon in all the four rat groups of fluctuations in resting glutamate, which we have termed glutamate transients. Although these events lasted only for seconds, they did occur throughout the testing paradigm. The average concentration of these glutamate-burst events was significantly increased in the 12- to 15-month-old FSL rats compared with 3- to 6-month-old FSL and age-matched FRL rats. These studies lay the foundation for future studies of both tonic and phasic glutamate signaling in rat models of depression to better understand the potential role of glutamate signaling in depression.     

The regulation of β-adrenergic receptor-mediated PKA activation by substrate stiffness via microtubule dynamics in human MSCs

The mechanical microenvironment surrounding cells has a significant impact on cellular function. One prominent example is that the stiffness of the substrate directs stem cell differentiation. However, the underlying mechanisms of how mechanical cues affect stem cell functions are largely elusive. Here, we report that in human mesenchymal stem cells (HMSCs), substrate stiffness can regulate cellular responses to a β-adrenergic receptor (β-AR) agonist, Isoproterenol (ISO). Fluorescence resonance energy transfer-based A-Kinase Activity Reporter revealed that HMSCs displayed low activity of ISO-induced protein kinase A (PKA) signal on soft substrate, whereas a significantly higher activity can be observed on hard substrate. Meanwhile, there is an increasing ISO-induced internalization of β₂-AR with increasing substrate stiffness. Further experiments revealed that the effects of substrate stiffness on both events were disrupted by interfering the polymerization of microtubules, but not actin filaments. Mechanistic investigation revealed that inhibiting ISO-induced PKA activation abolished β₂-AR internalization and vice versa, forming a feedback loop. Thus, our results suggest that the cellular sensing mechanism of its mechanical environment, such as substrate stiffness, affects its response to chemical stimulation of β-AR signaling and PKA activation through the coordination of microtubules, which may contribute to how mechanical cues direct stem cell differentiation.