Global Medical Discovery: June 2016

Friday, June 17, 2016

Global Medical Discovery features paper: Highly specific quantification of microRNA by coupling probe-rolling circle amplification and Förster resonance energy transfer

Significance Statement

The first microRNA (miRNA) was discovered in C. elegans in 1993. Subsequently, miRNAs have been identified as a class of small non-coding RNAs containing around 21 nucleotides and virtually exist in all mammalian cells. Currently, miRNAs have been regarded as a class of potential diagnostic markers or therapeutic targets for cancer, cardiovascular and others diseases.

The short length, the similarity of nucleotide sequence and extremely low level in living cells have seriously limited the detection of miRNAs with ultimate sensitivity, high specificity and superior accuracy. Up to date, such a limitation has not been adequately overcome by reported methods. In this paper, the authors presented a novel approach (pRCA-FRET) for quantitative detection of miRNA based on a sequential combination of padlock probe-rolling circle amplification (p-RCA) and förster resonance energy transfer (FRET). First, the padlock probe is specifically ligated and circularized with the target miRNA to produce a long ssDNA by p-RCA in the presence of a DNA polymerase. In this step, p-RCA can superiorly distinguish the mismatch between miRNA and padlock probe and increase the specificity towards target miRNAs. Then, FRET probes labeled with florescent groups Cy3 and Cy5 are used to hybridize the ssDNA from p-RCA. After the excitation of the donor fluorophore Cy3, detectable readouts from the fluorescence emission of Cy5 are generated by FRET. Consequently, the target miRNA can be quantified by analyzing the intensity of fluorescent emission. Compared to previous methods, the sensitivity and operational simplicity have obviously been improved in the step of FRET without complex PCR amplification, accompanying a further enhancement of specificity for miRNA detection.

Using pRCA-FRET, the detection limit of miRNA quantification is markedly reduced from fM level to 103 aM, and remarkable specificity is exemplified to differentiate single-base mismatch between target miRNA and similar miRNAs. Accordingly, this pRCA-FRET has the potential to quantify low amount of miRNA with excellent sensitivity and specificity for the exploration of the biological functions of miRNAs and their clinical applications.

About The Author

Prof. Dr. Yijun Chen received a B.S. degree in pharmaceutical science at China Pharmaceutical University, Nanjing, China, in 1982 and completed his Ph.D. degree in medicinal and natural products chemistry at University of Iowa, Iowa, USA, in 1996 under the guidance of Professor John P. N. Rosazza. After his postdoctoral research with Professor Edward A. Dennis at University of California, San Diego, he worked as a Senior Scientist at MicroGenomics, Inc., California, USA from 1998 to 1999. From 1999 to 2006, he served as a Senior Research Investigator at Lead Discovery and Enzyme Technology Departments of Bristol-Myers Squibb Pharmaceutical Research Institute in New Jersey, USA. Since 2007, he has been appointed as an Endowed Professor and Director of Laboratory of Chemical Biology at China Pharmaceutical University, and he presently is also an Adjunct Professor of Department of Chemical Biology, Rutgers University, USA. Currently, he has published over 80 peer-reviewed papers and invented more than 40 patents. The research of Dr. Chen’s group focuses on discovery and validation of novel antitumor targets using chemical proteomics approach, elucidation and manipulation of biosynthetic pathways for microbial secondary metabolites and development of biocatalytic routes for chiral drug intermediates.

Journal Reference

Anal Biochem. 2016 Jun 1;502:16-23.

Wu X¹, Zhu S¹, Huang P¹, Chen Y².

Show Affiliations

State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, Nanjing, Jiangsu Province, 210009, People’s Republic of China.
State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, Nanjing, Jiangsu Province, 210009, People’s Republic of China. Electronic address: yjchen@cpu.edu.cn.

Abstract

MicroRNA (miRNA) plays vital roles in various biological processes. In general, sensitivity and specificity are the major parameters for the quantification of miRNA. In this study, padlock probe-rolling circle amplification and Förster resonance energy transfer (pRCA-FRET) were coupled for specific and quantitative detection of miRNA. pRCA-FRET showed superior specificity to differentiate single-base mismatch and excellent sensitivity with a detection limit of 103 aM. The current method has the potential to quantify low amounts of miRNA in the same family for studies on their biological functions.

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Global Medical Discovery features paper: Diagnostic Utility of Flow Cytometry Analysis of Reactive T Cells in Nodular Lymphocyte-Predominant Hodgkin Lymphoma

About The Author

James Huang, M.D.

Oakland University William Beaumont School of Medicine, Rochester, MI, USA

James Huang undertook his early medical studies at Hunan Medical University, Changsha, China, completing a research fellowship at Harvard Medical School, a pathology residency at Dartmouth-Hitchcock Medical Center, a hematopathology fellowship in University of Nebraska Medical Center. He is currently an Associate Professor of Pathology, Co-Director of Diagnostic Medicine Clerkship, and Director of Clinical Pathology Elective at Oakland University William Beaumont School of Medicine. He is also a senior staff hematopathologist at Beaumont Health.

Dr. Huang is on the Editorial Board of a number of journals including International Journal of Pathology and Clinical Research, the Clinics in Oncology, and Pathology- Remedy Open Access.

About The Author

James David, M.D.

Oakland University William Beaumont School of Medicine, Rochester, MI, USA

James David graduated from Oakland University William Beaumont School of Medicine. This research was his capstone project for which he received a competitive scholarship award form Oakland University William Beaumont School of Medicine. He is currently in residency program in ophthalmology at Louisiana State University-Ochsner, Louisiana.

Journal Reference

Am J Clin Pathol. 2016 Jan;145(1):107-15.

David JA¹, Huang JZ².

Show Affiliations

From the Department of Pathology, Oakland University William Beaumont School of Medicine, Rochester, MI;
From the Department of Pathology, Oakland University William Beaumont School of Medicine, Rochester, MI; Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, MI. james.huang@beaumont.edu.

Abstract

OBJECTIVES:

This study aims to define the diagnostic utility of flow cytometric features of T cells in nodular lymphocyte- predominant Hodgkin lymphoma (NLPHL).

METHODS:

Cases were retrospectively identified based on diagnosis with NLPHL (n = 30 samples), classic Hodgkin lymphoma (CHL; n = 33), and reactive lymphoid hyperplasia (RLH; n = 43). Pathology slides were reviewed. Flow cytometry list mode data were reanalyzed.

RESULTS:

The mean proportion of CD4 + CD8 + T cells (8.4%) in cases of NLPHL was significantly higher than seen in CHL (1.0%) or RLH (0.6%). Of the T cells, 28.4% were CD57 + in NLPHL, significantly higher (P < .05) than in CHL (3.2%) or RLH (3.2%). Based on receiver operating characteristic curve analysis, when using a cutoff of 3.0% of CD4 + CD8 + T cells, the diagnostic sensitivity for NLPHL is 83.3% with a specificity of 97.4%. The diagnostic sensitivity was 96.7% with a specificity of 98.7% when using a cutoff of 12% for CD57 + T cells.

CONCLUSIONS:

Increased portions of CD57 + T cells and CD4 + CD8 + T cells are highly suggestive of the possibility of NLPHL. In addition, NLPHL diagnosis appears unlikely if neither CD57 + T cells nor CD4 + CD8 + T cells are increased. Future prospective studies including cases of progressive transformation of germinal center and T-cell/histiocyte-rich large B-cell lymphoma will further define the utility of flow cytometry of T cells in NLPHL.

Go To Am J Clin Pathol.

Global Medical Discovery features paper: Kir3 channels undergo arrestin-dependant internalization following delta opioid receptor activation

Significance Statement

Protein-protein interactions are of great importance for virtually all biological processes and whether transient or stable, they support the formation of multimeric complexes. Monitoring such interactions may help us characterize signaling and trafficking behaviours of these complexes and sometimes allows us to elucidate new signaling pathways for a known protein. A better and detailed knowledge of these different aspects of complex function is essential not only to understand the majority of physiological processes but also for the development of new therapeutic ligands.

In this article, we characterized protein-protein interactions within a complex involved in opioid analgesia which is formed by delta opioid receptors (DORs), heterotrimeric G protein (Gαoβ1γ2) and their effector, the G protein-gated inwardly rectifying potassium channel (GIRK/Kir3). It is well established that sustained DOR stimulation by an agonist triggers a series of adaptive changes that reduce receptor ability to signal and this desensitization may contribute to analgesic tolerance. Although there is considerable information of how desensitization reduces receptor ability to interact and activate the G protein, much less is known on how desensitization modifies the channel standing in the complex. We therefore focused on how interactions between the channel and other complex components were modified by sustained receptor activation.

Our results show that DORs, G protein and Kir3 channels form a constitutive complex at the plasma membrane. This complex undergoes rapid conformational rearrangements upon acute DOR stimulation and maintains its integrity over more prolonged periods of receptor activation. During this time, the DOR/G protein/Kir3 complex undergoes additional conformational changes imposed by βarrestin 2 (βarr2) recruitment and association with receptors and channels. This interaction not only induces DOR removal from the membrane but also that of the channel. Both signaling partners are concomitantly internalized via a clathrin and dynamin-dependent mechanism.

Conclusion: Taken together, these data show that DORs and Kir3 channels form a constitutive complex which is recognized and internalized as a signaling unit by βarr2.

Contribution to the advancement of knowledge: Kir3 channels removal from the membrane represents an additional level of regulation of opioid receptor signaling that had not been previously described. Moreover, given active Kir3 channels participation in opioid analgesia, their removal from the membrane may constitute an additional and powerful mechanism of tolerance. Thus, it is reasonable to expect that development of DOR ligands that activate the channel but could prevent complex interaction with βarr2 could lead to the production of opioid analgesics that preserve their therapeutic efficacy.

Highlights

– Kir3.1/3.2 channels, G proteins and DORs form a complex.

– The complex maintains its integrity over prolonged periods of receptor stimulation.

– βarr2 is recruited to DORs and channels mediating their internalization as a unit.

– DOR-Kir3 channel internalization is clathrin/dynamin dependent.

Figure Legend. Mechanism of analgesia induced by Kir3 channels at the synaptic cleft.

When released into the synaptic cleft, neurotransmitters such as endogenous opioids (pink) activate the DOR receptor (red) of the postsynaptic neuron, which in turn activate the Kir3 channel (green). Activation of Kir3 channels produces hyperpolarization at the postsynaptic membrane thereby reducing the transmission of nociceptive impulses.

About The Author

Dr. Karim Nagi received his Bachelor’s degree in Biology from the Lebanese University, Tripoli, Lebanon (2005-2008). He then moved to Canada where he completed one year in basic research in Molecular Cardiology and Genetics at Sacré-Cœur Hospital’s Research Center, Montreal, Canada. In 2010, he entered the MSc program at the Department of Pharmacology, University of Montreal under the supervision of Prof. Pineyro at Sainte-Justine Hospital Research Center. By the end of his first year, he was offered accelerated switch to the PhD program which he completed in 2015. During this time Dr Nagi applied a variety of approaches in biochemistry and neuroscience to investigate the network properties of GPCR signaling with particular focus in the analgesic actions of opioid receptor ligands and their potential to induce tolerance.

Dr. Nagi has authored 7 publications in peer-reviewed journals and 50+ presentations. In addition, he received eight awards for the best oral and poster presentations in scientific conferences, two travel awards to international congresses and his studies were supported by a number of fellowship awards including CHU Sainte-Justine and Foundation of Stars fellowship, fellowship from the Department of Pharmacology and a fellowship from the Faculty of Graduate and Postdoctoral Studies, University of Montreal. He was also granted a Recognition Award for the Best Scientific Contribution of the year (2014-2015) among students in the Department of Pharmacology.

After completing graduate studies, Dr. Nagi continued his training as a postdoctoral fellow in the Department of Cellular Biology, Duke University, Durham, USA under the supervision of Prof. Marc G. Caron. His current research focuses on characterizing different biased receptors signaling and regulation.

Working at these different institutions with a world-renowned reputation in pharmacological research, Dr. Nagi has developed expertise in GPCRs pharmacology, BRET-based biosensor development for drug screening, molecular biology and biased signaling.

About The Author

Graciela Pineyro, MD, Ph.D.

Prof. Graciela Pineyro is a Full Professor at the Department of Psychiatry, University of Montreal, Montreal, Canada. In 1991, she received her medical degree with specialty in Pharmacology from the Faculty of Medicine, National University, Uruguay. She then moved to Canada where she obtained a Ph.D. in Neurosciences from McGill University, Montreal, Canada (1997) followed by postdoctoral training in molecular pharmacology in the Department of Biochemistry, University of Montréal (1997-2001). During her career she was supported by different fellowships and awards including McGill Major Fellowships (Canada), Fogarty-NIH International Fellowship (USA) and Postdoctoral Fellowships from Medical Research Council of Canada and Heart and Stroke Foundation of Canada.

Today, she is head of a pharmacology laboratory with research focus on molecular determinants of analgesic efficacy of opioids, as well as cellular and molecular bases of analgesic tolerance. She has substantially contributed to the notion of biased signaling showing that delta opioid receptors adopt ligand-specific conformations with distinct signaling and trafficking properties. Insights from her research have provided the basis for the rational development of novel opioid analgesics with a reduced side effects profile.

Prof. Pineyro has authored 37 peer-reviewed publications, 7 book chapters and 100+ presentations, and holds 2 licensed patents.

As an independent investigator, she has received the New Investigator Award from Fond de Recherche en Santé du Québec and her research has been continuously funded by Canadian Institutes of Health Research and Natural Sciences and Engineering Research Council of Canada.

About The Author

Iness Charfi, MSc

Iness Charfi received her Bachelor in Pharmacy in 2008 at Monastir University (Tunisia). In 2009, she joined Prof. Graciela Pineyro’s Lab as an MSc student in Neuropsychopharmacology at the Sainte-Justine Hospital Research Center, through the Department of Pharmacology, University of Montreal (Canada). After graduating in 2012, she started a PhD in the same laboratory. Throughout her training, she focused on the mechanistic understanding of the molecular basis of delta opioid receptor post-endocytic trafficking, in order to better understand the development of analgesic tolerance to opioids. Iness has been awarded a number of prizes and fellowships, including two presentation awards for best oral and poster presentations at scientific meetings. Her fellowship awards include a CHU Sainte-Justine and Fondation of Stars fellowship, a recruitment fellowship from the Department of Pharmacology, University of Montreal and a fellowship award from the FRSQ.

Journal Reference

Cell Mol Life Sci. 2015 Sep;72(18):3543-57.

Karim Nagi^1,2, Iness Charfi^1,2 and Graciela Pineyro^1,2,3

Show Affiliations

Sainte-Justine Hospital Research Center, Montreal, Quebec, H3T 1C5, Canada.
Department of Pharmacology, Faculty of Medicine, University of Montreal, Montreal, Quebec, H3T 1J4, Canada.
Department of Psychiatry, Faculty of Medicine, University of Montreal, Montreal, Quebec, H3T 1J4, Canada.

Abstract

Kir3 channels control excitability in the nervous system and the heart. Their surface expression is strictly regulated but mechanisms responsible for channel removal from the membrane remain incompletely understood. Using transfected cells, we show that Kir3.1/3.2 channels and delta opioid receptors (DORs) associate in a complex which persists during receptor activation, behaving as a scaffold that allows beta-arrestin (βarr) to interact with both signaling partners. This organization favored co-internalization of DORs and Kir3 channels in a βarr-dependent manner via a clathrin/dynamin-mediated endocytic path. Taken together, these findings identify a new way of modulating Kir3 channel availability at the membrane and assign a putatively novel role for βarrs in regulating canonical effectors for G protein-coupled receptors.

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Global Medical Discovery features paper: RASGRF2 controls nuclear migration in postnatal retinal cone photoreceptors

Journal Reference

J Cell Sci. 2016 Feb 15;129(4):729-42. doi: 10.1242/jcs.180919. Epub 2016 Jan 7.

Jimeno D¹, Gómez C¹, Calzada N¹, de la Villa P², Lillo C³, Santos E⁴.

Show Affiliations

Centro de Investigación del Cáncer-Instituto de Biología Molecular y Celular del Cáncer (CSIC – Universidad de Salamanca), Salamanca 37007, Spain.
Departamento de Fisiología, Universidad Alcalá, Alcalá de Henares 28871, Spain, Spain.
INCYL, IBSAL (Universidad de Salamanca), Salamanca 37006, Spain.
Centro de Investigación del Cáncer-Instituto de Biología Molecular y Celular del Cáncer (CSIC – Universidad de Salamanca), Salamanca 37007, Spain esantos@usal.es.

Abstract

Detailed immunocytochemical analyses comparing wild-type (WT), GRF1-knockout (KO), GRF2-KO and GRF1/2 double-knockout (DKO) mouse retinas uncovered the specific accumulation of misplaced, ‘ectopic’ cone photoreceptor nuclei in the photoreceptor segment (PS) area of retinas from GRF2-KO and GRF1/2-DKO, but not of WT or GRF1-KO mice. Localization of ectopic nuclei in the PS area of GRF2-depleted retinas occurred postnatally and peaked between postnatal day (P)11 and P15. Mechanistically, the generation of this phenotype involved disruption of the outer limiting membrane and intrusion into the PS layer by cone nuclei displaying significant perinuclear accumulation of signaling molecules known to participate in nuclear migration and cytoskeletal reorganization, such as PAR3, PAR6 and activated, phosphorylated forms of PAK, MLC2 and VASP. Electroretinographic recordings showed specific impairment of cone-mediated retinal function in GRF2-KO and GRF1/2-DKO retinas compared with WT controls. These data identify defective cone nuclear migration as a novel phenotype in mouse retinas lacking GRF2 and support a crucial role of GRF2 in control of the nuclear migration processes required for proper postnatal development and function of retinal cone photoreceptors.

Go To J Cell Sci

Friday, June 10, 2016

Global Medical Discovery features paper: Direct Electron Transfer of Enzymes in a Biologically Assembled Conductive Nanomesh Enzyme Platform

Significance Statement

Recently, wearable biomedical sensors that enable non-invasive monitoring of health condition have been attracting drastically increasing interest. In particular, a non-invasive device that can measure glucose levels in bio-fluid such as tear, saliva, and sweat is regarded as one of the most promising biosensor platforms for future healthcare application. Bio-fluids have been known to contain informative indicators (i.e., glucose). Moreover, they are naturally and continuously excreted from the human body. Therefore, a wearable glucose sensor that is placed or attached on a body could continuously measure glucose levels in a non-invasive manner and real rime. Thus, patients do not have to prick their fingers for blood collection every time, and it would ultimately reduce the pain of diabetes patients and prevent the possible secondary infection.

Current blood glucometers best work for measuring glucose level in blood. Meanwhile, the glucose concentration in tear, saliva, or sweat, are about 20~100 times lower than that in blood glucose. In addition, at this low glucose level, the interfering redox species, such as ascorbic acid, uric acid or acetaminophen, greatly influence the sensor performance. Therefore, to realize non-invasive and wearable monitoring of glucose levels, the flexible biosensor platform must own much more enhanced sensitivity and selectivity than current blood glucometers. Furthermore, the biosensor has to be non-toxic for wearable purpose. Considering all these requirements for wearable biomedical sensors, the 3^rd generation biosensor platform that employs the direct-electron-transfer (DET) mechanism of enzymes would be a promising candidate. Owing to the direct electrical communication with enzymes at the negative potential range, being apart from the oxidation potential of interfering redox species by -600 mV, the sensor can achieve both high sensitivity at sub-millimolar level and excellent selectivity on target analytes. Moreover, since the electrode and the enzyme directly communicate, no chemical mediator is required. Therefore, DET obviates the toxicity issues involved in the chemicals.

To achieve efficient DET of enzymes, realizing intimate electrical contacts with redox biomolecules at nanoscale is of crucial importance. In this work, we report a new approach that enables highly facilitated biomolecular electron transfer with unprecedented versatility and applicability. Enzymes are intimately interfaced with a conductive nanomesh made of single-walled carbon nanotubes (SWNTs) via a rationally designed interlayer. SWNTs are hydro-dynamically assembled into a conductive nanomesh by using a filamentous phage that shows a strong binding affinity toward SWNTs. The nanostructure of the nanomesh is extremely well controlled over large area. The filamentous phage controls the nanostructure and at the same time serves as a biological glue to stabilize the nanomesh structure in solution. The conductive nanomesh is then transferred onto metallic electrodes to produce a nanostructured electrical platform for biomolecular direct electron transfer (DET). The conductive nanomesh is then rationally interfaced with enzymes by using a polyelectrolyte layer with appropriate charge characteristics. This interfacial layer enables both intimate nanoscale electrical contacts to biomolecules for facilitated electron transfer and highly porous nanostructures for high sensitivity.

Using this layered conductive nanomesh enzyme platform, we successfully achieve DET for eight different enzymes (including glucose oxidase and lactate oxidase) with various types of catalytic activities. The direct electrical communication of enzymes on the conductive nanomesh enables not only the high sensitivity at sub-millimolar level of target (i.e, glucose or lactate), but also the high selectivity of the sensor due to the operation at negative potential range. We further demonstrate a flexible DET-based glucose-biosensor, for the first time, to the best of our knowledge. The electron transfer efficiency and sensitivity of these flexible integrated biosensors are comparable to those obtained using commercialized screen printed electrodes.

We believe that the biologically assembled conductive nanomesh enzyme platform presents a promising solution for future health-monitoring systems.

About The Author

Dr. Hyunjung Yi received her B.S. and M.S. degrees from Pohang University of Science and Technology (POSTECH), Pohang, Korea, in the department of Material Science and Engineering and Ph. D. degree from Massachusetts Institute of Technology (MIT), Cambridge, USA in the department of Material Science and Engineering in 2001, 2003, and 2011, respectively.

Dr. Hyungjung Yi joined Korea Institute of Science and Technology (KIST), Seoul, Korea, in 2003 as a research scientist and is currently a Principal Investigator and a Senior Research Scientist.

Dr. Hyunjung Yi’s research focuses on developing nano-bio hybrid materials for wearable biosensors, electronics, tactile sensors, and flexible energy devices. Biotechnology to genetically engineer biomaterials and processes to assemble and fabricate nano-bio hybrid are also being developed.

About The Author

Dr. Seung-Woo Lee received B.E. in Fine chemical engineering & Applied chemistry at Chungnam National University in 2006, and Ph.D. in chemical and biomolecular engineering at University of Nebraska-Lincoln in 2013, respectively.

He joined Dr. Hyunjung Yi’s Lab at Korea Institute of Science and Technology (KIST) in 2014.

His research mainly focuses on the electrochemical biosensor, wearable/flexible nano-bio electronics, non-destructively assembly of nano-bio hybrid biosensing materials, and electrochemical bio-energy generation.

Reference

Adv Mater. 2016 Feb;28(8):1577-84.

Lee SW¹, Lee KY¹, Song YW¹, Choi WK², Chang J¹, Yi H¹.

Show Affiliations

¹Post-Silicon Semiconductor Institute, Korea Institute of Science and Technology, Seoul, 136-791, Republic of Korea.

²Materials and Life Science Research Division, Korea Institute of Science and Technology, Seoul, 136-791, Republic of Korea.

Abstract

Nondestructive assembly of a nanostructured enzyme platform is developed in combination of the specific biomolecular attraction and electrostatic coupling for highly efficient direct electron transfer (DET) of enzymes with unprecedented applicability and versatility. The biologically assembled conductive nanomesh enzyme platform enables DET-based flexible integrated biosensors and DET of eight different enzyme with various catalytic activities.

Go To Advanced Materials

Wednesday, June 8, 2016

Global Medical Discovery features paper: Resveratrol induces ordered domains formation in biomembranes: Implication for its pleiotropic action

Significance Statement

Membrane Approach to Understand the Pleiotropic Effects of Resveratrol

Resveratrol is a natural polyphenol compound with great potential in the cancer therapy, cardiovascular protection, and neurodegenerative disorders. The mechanism of action of resveratrol may be associated to its capacity of modulating cell membrane structure and function, thereby influencing the activity of several membrane associating receptors, proteins and enzymes. The present research reveals potential molecular interactions between resveratrol and lipid rafts found in cell membranes, bringing a new membrane approach to understand the pleiotropic effects of this phytochemical.

Förster resonance energy transfer (FRET), DPH fluorescence quenching by TEMPO, and Triton X-100 detergent resistance assay have been employed to monitor lipid phase separation in model membranes. Unilamellar liposomes composed of phosphatidylcholine, cholesterol and sphingomyelin were chosen as membrane mimetic systems. The results indicate that resveratrol is able to incorporate in lipid bilayers, inducing phase separation, stabilizing and promoting the formation of ordered domains (lipid rafts) that can act as organizing centers for the assembly of proteins and receptors, which in turn may be involved in the cell signaling and cellular processes.

In conclusion, the influence of resveratrol on the regulation of lipid domains formation and stability provide a rational approach to better understand the therapeutic effects of this promising compound.

About The Author

Dr. Ana Rute Neves (PhD Pharmaceutical Sciences in 2015) is a Post-doctoral researcher in the Molecular Biophysics and Biotechnology Unit at GABAI/UCIBIO/REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Portugal. Her research focuses on biophysical and biochemical approaches to study the interaction of phytochemicals with biomembranes (using liposomes as biomimetic systems) in order to explain their therapeutic properties and the development of nanopharmaceuticals (lipid nanoparticles) as drug delivery systems to create new and more efficient therapies for a range of diseases.

About The Author

Dr. Cláudia Nunes (PhD Pharmaceutical Sciences in 2011) is a Post-doctoral researcher in the Molecular Biophysics and Biotechnology Unit at GABAI/UCIBIO/REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Portugal. Her research activities include the assessment of drug-membrane interactions, using membrane mimetic models; the development of nanotechnology based drug delivery systems (silica nanotubes, solid lipid nanoparticles, nanostructured lipid particles and liposomes) that can be efficiently carried to the inflamed tissues avoiding the gastric local toxic effects of the classical therapies.

About The Author

Professor Salette Reis (PhD Analytical Chemistry in 1995) is the Director of the Department of Chemical Sciences and the group leader of the Molecular Biophysics and Biotechnology Unit at GABAI/UCIBIO/REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Portugal. Her investigation has been focused in biophysics and pharmaceutical chemistry, namely in the use of biomimetic membrane models to study the effect of drugs on biological membranes trying to establish a relationship between this effect and their activity/mechanism of action; and the development of nanocarrier systems for drug delivery to overcome the disadvantages of the classical therapies.

Journal Reference

Biochim Biophys Acta. 2016;1858(1):12-8.

Neves AR¹, Nunes C¹, Reis S².

Show Affiliations

UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. Electronic address: shreis@ff.up.pt.

Abstract

Resveratrol is a polyphenol compound with great value in cancer therapy, cardiovascular protection, and neurodegenerative disorders. The mechanism by which resveratrol exerts such pleiotropic effects is not yet clear and there is a huge need to understand the influence of this compound on the regulation of lipid domains formation on membrane structure. The aim of the present study was to reveal potential molecular interactions between resveratrol and lipid rafts found in cell membranes by means of Förster resonance energy transfer, DPH fluorescence quenching, and triton X-100 detergent resistance assay. Liposomes composed of egg phosphatidylcholine, cholesterol, and sphingomyelin were used as model membranes. The results revealed that resveratrol induces phase separation and formation of liquid-ordered domains in bilayer structures. The formation of such tightly packed lipid rafts is important for different signal transduction pathways, through the regulation of membrane-associating proteins, that can justify several pharmacological activities of this compound.

Go To Biochim Biophys Acta.

Global Medical Discovery features paper: Functional differentiation of cholinergic and noradrenergic modulation in a biophysical model of olfactory bulb granule cells

Significance Statement

The mammalian olfactory bulb (OB), the first center of synaptic integration in the olfactory systems, receives massive cholinergic inputs from the basal forebrain and dense noradrenergic innervations from the locus coeruleus, both of which have profound effects on odor processing as well as olfactory learning and memory. However, the effects of acetylcholine (ACh) and norepinephrine (NE) have not been clearly distinguished. Given the important roles of cholinergic and noradrenergic modulation in the olfactory bulb, achieving clear functional dissociation among the roles of these two modulators is not trivial and is required for a mechanistic and integrated understanding of olfactory information processing in the brain. Using detailed biophysical simulations of granule cells, the major interneurons in the olfactory bulb, both alone and embedded in a microcircuit with mitral cells (MCs), the principal output neurons of olfactory bulb, we demonstrated computationally for the first time that the effects of ACh and NE on olfactory bulb function are both distinct and functionally complementary to one another. While ACh increases MC spike synchronization and sharpens MC firing rate representation, NE mainly modulates the neuronal signal-to-noise ratio (S/N) and can regulate cholinergic function. Co-application of ACh and NE sharpens MC tuning, improves the S/N ratio and enhances spike synchronization among mitral cells. Our main conclusions are that ACh is particularly important for odor discrimination and sensory information encoding via a spike-timing code, while NE is more important for odor detection. Therefore, this work is significant in understanding the cholinergic and noradrenergic function in the olfactory bulb and offers important specific and testable hypotheses for future work.

Figure Legend: The effects of acetylcholine (ACh) and norepinephrine (NE) in the olfactory bulb are both distinct and complementary to each other. ACh modulation increases mitral cell (MC) spike synchronization and sharpens odor representation by suppressing the weakly activated MCs. By comparison, NE modulation increases the signal-to-noise (S/N) ratio by suppressing MC spontaneous activities. Simultaneous activation of ACh and NE leads to highly synchronized MCs, large S/N ratio and highly tuned MC responses with little overlap between different odors.

About The Author

Dr. Guoshi Li is currently a postdoctoral research associate in the Department of Psychiatry at University of North Carolina at Chapel Hill. His primary focus is to perform cutting edge computational neuroscience research. He is currently working on two research projects: (1) Developing biophysically realistic models of the thalamocortical network to understand the cellular and circuit mechanisms underlying distinct states of oscillatory activities and how brain stimulation impacts the thalamocortical network dynamics; and (2) Extending an existing olfactory bulb network model (Li and Cleland, 2013) he developed previously to examine the dynamical mechanisms of external tufted cells in olfactory information processing. His research in olfaction is funded by a NIH/NIDCD R03 grant.

Prior to joining the Frohlich lab at UNC, Dr. Li was a postdoctoral research associate in the Computational Physiology Lab at Cornell University directed by Dr. Thomas Cleland and Dr. Christiane Linster. His postdoc research at Cornell focused on olfactory information processing in the olfactory bulb with a particular interest in cholinergic and noradrenergic neuromodulation and gamma oscillations.

Dr. Li obtained his PhD in Electrical Engineering from University of Missouri – Columbia in 2009 and MS in Mechanical Engineering from State University of New York at Buffalo in 2003. His PhD research concentrated on understanding the neural mechanisms of fear learning and extinction using a computational modeling approach.

About The Author

Dr. Christiane Linster is a professor in the Department of Neurobiology and Behavior at Cornell University. Her research focuses on the neural basis of sensory information processing, using olfaction as a model system. She is primarily interested in the relationship between perceptual qualities, as measured by behavioral experiments, and neural activity patterns, as observed electrophysiologically. Her present work concerns how the central nervous system neuromodulators acetylcholine and noradrenaline, both of which have been implicated in memory deficits such as those symptomatic of Alzheimer’s disease, influence the representation and storage of olfactory information. This approach necessitates coordinated behavioral and electrophysiological experiments based on predictive theories.

About The Author

Dr. Thomas Cleland is an associate professor in the Department of Psychology at Cornell University. His research concerns how complex cognitive and perceptual phenomena can arise from, and be regulated by, cellular and neural circuit properties. Primarily using the sense of smell (olfaction), Dr. Cleland investigates how learning, memory, expectation, and like processes shape the transformations performed on sensory inputs by relatively peripheral (i.e., experimentally accessible) cortical circuitry, and how these different transformations in turn influence behavior and subsequent learning. He and his colleagues triangulate on these questions using a range of techniques including electrophysiology, pharmacology, behavior and behavior genetics, and biophysically constrained computational modeling. In collaboration with colleagues in the College of Engineering, he also implemented circuit models of olfactory processing in neuromorphic digital chips.

Journal Reference

J Neurophysiol. 2015 Dec;114(6):3177-200.

Li G¹, Linster C², Cleland TA³.

Show Affiliations

Department of Psychology, Cornell University, Ithaca, New York; guoshi_li@med.unc.edu.
Department of Neurobiology and Behavior, Cornell University, Ithaca, New York.
Department of Psychology, Cornell University, Ithaca, New York;

Abstract

Olfactory bulb granule cells are modulated by both acetylcholine (ACh) and norepinephrine (NE), but the effects of these neuromodulators have not been clearly distinguished. We used detailed biophysical simulations of granule cells, both alone and embedded in a microcircuit with mitral cells, to measure and distinguish the effects of ACh and NE on cellular and microcircuit function. Cholinergic and noradrenergic modulatory effects on granule cells were based on data obtained from slice experiments; specifically, ACh reduced the conductance densities of the potassium M current and the calcium-dependent potassium current, whereas NE nonmonotonically regulated the conductance density of an ohmic potassium current. We report that the effects of ACh and NE on granule cell physiology are distinct and functionally complementary to one another. ACh strongly regulates granule cell firing rates and after potentials, whereas NE bidirectionally regulates subthreshold membrane potentials. When combined, NE can regulate the ACh-induced expression of after depolarizing potentials and persistent firing. In a microcircuit simulation developed to investigate the effects of granule cell neuromodulation on mitral cell firing properties, ACh increased spike synchronization among mitral cells, whereas NE modulated the signal-to-noise ratio. Coapplication of ACh and NE both functionally improved the signal-to-noise ratio and enhanced spike synchronization among mitral cells. In summary, our computational results support distinct and complementary roles for ACh and NE in modulating olfactory bulb circuitry and suggest that NE may play a role in the regulation of cholinergic function.

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Global Medical Discovery features paper: Surface nitridation improves bone cell response to melt-derived bioactive silicate/borosilicate glass composite scaffolds

Significance Statement

Biomaterials have long been used in the orthopaedic surgery to guide and assist bone repair. Nowadays, they also have the potential of being used as substrates for bone tissue engineering. Bioglasses like 45S5 present optimal bioactivity, but when sinterized into 3D monoliths they crystallize and partly loose their properties, mainly in vivo absorb ability. We have developed a novel bioglass, called ICIE16/BSG-NITRI, that not only overcomes this limitation but also displays improved reactivity and biocompatibility due to surface nitridation. ICIE16/SBG-NITRI was synthesized from a mixture of two melt-derived glasses through combined gel casting and foam replication techniques, followed by nitridation. It is highly porous but mechanically stable and mimics the architecture of bone tissue. Nitridation confers it improved reactivity and bioactivity facilitating its resorption and deposition of apatite (bone-like mineral) at its surface. The nitrided surface also improved its interaction with bone cells, which were found to attach better to ICIE16/SBG-NITRI and to differentiate earlier on its surface.

Figure legend: bone on the left and our ICIE16/BSG-Nitru bioglass on the right (in both cases bar is 500 µm).

Figure legend: merge of visible and fluorescent-blue channels to show cell distribution on the bioglass surface. The hoechst-stained nuclei can be seen as blue dots.

About The Author

Felipe Orgaz, Ph. D.

Felipe Orgaz is a tenured researcher at the Glass and Ceramic Research Institute (Madrid, Spain). He obtained his Ph. D. from the University of Madrid on strengthening glasses by ion exchange, and then conducted his post-doctoral studies at Sheffield University (UK) on sol-gel process. He joined the Spanish company Explosivos Río Tinto, and then Encros as Head of the Department of Ceramic Technology, conducting projects on structural ceramics for aerospace applications. He has also worked for the Spanish Ministry of Science and Technology evaluating technology transfer activities. He has conducted research on glasses and coatings from sol-gel process, nano-structured materials, dynamic fracture of advanced materials, transparent ceramics for armors, mechanical behavior of silicon nitride structural ceramics, TiO2 catalysts, and ion-densified ceramic foams and materials. His current research is focused on surface nitridation and its effects on the interface of the materials with living tissues, including protein adsorption and cell response. He has authored 10 international patents and more than 55 articles, in national and international journals and conference proceedings. He has also been part of several technical committees of the International Commission on Glass (ICG). He has been member of the steering committee of Fundación Círculo para la Defensa y la Seguridad, he has been member of the editorial board of the Bulletin of the Spanish Society of Ceramics and Glass, and of the Journal of Sol-Gel Science and Technology. He is currently the president of the Education and Innovation Area of the Spanish Society of Glass and Ceramic.

About The Author

Leonor Santos-Ruiz, Ph. D.

Leonor Santos-Ruiz is a Senior Researcher at the Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN, Spain), and part-time associate professor at the Faculty of Sciences of the University of Málaga (Spain). She earned her PhD degree in Regenerative Biology in 2001, from the University of Málaga, and completed her post-doctoral fellowship at the Advanced Biotechnology Centre (Genoa, Italy) and the Institute for Child Health (ICH-UCL; London, UK). Her research is focused on bone repair and seeks the application of regenerative biology principles to develop tissue engineering products that stimulate bone regeneration, particularly in individuals where bone endogenous natural repair is hampered due to age or disease. She is interested in conditions like osteoporosis, osteonecrosis and skeletal dysplasias, particularly craniosynostosis, and her work includes the use of adult and perinatal stem cells for stem cell therapy, and the development and evaluation of novel biomaterials to be used as cell carriers and/or drug deliverers. She is the author of 2 international patents and more than thirty articles in international journals and conference proceedings.

Journal Reference

Acta Biomater. 2016;29:424-34.

Orgaz F¹, Dzika A¹, Szycht O¹, Amat D^2,3, Barba F¹, Becerra J^3,4,5, Santos-Ruiz L^3,4,5.

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¹ Instituto de Cerámica y Vidrio, Consejo Superior de Investigaciones Científicas (ICV-CSIC), c/ Kelsen n° 5, 28049 Madrid, Spain

² Universidad de Málaga, Departamento de Anatomía y Medicina Legal, Facultad de Medicina, Campus de Teatinos, 29071 Málaga, Spain

³ Centro de Investigación Biomédica en Red. Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto de Salud Carlos III, c/ Monforte de Lemos 3-5, Pabellón 11, Planta 0, 28029 Madrid, Spain

⁴ Universidad de Málaga & IBIMA, Departamento de Biología Celular, Genética y Fisiología, Facultad de Ciencias, Campus de Teatinos, 29071 Málaga, Spain

⁵ BIONAND-Universidad de Málaga, c/ Severo Ochoa 35, Campanillas, 29590 Málaga, Spain

Abstract

Novel bioactive amorphous glass-glass composite scaffolds (ICIE16/BSG) with interconnected porosity have been developed. Hierarchically interconnected porous glass scaffolds were prepared from a mixture of two melt-derived glasses: a ICIE16 bioactive glass that was previously developed by Wu et al. (2011) to prevent crystallization, and a borosilicate glass of composition 73.48 SiO2-11.35 B2O3-15.15 Na2O (wt%). The resulting melt derived glass-glass composite scaffolds (ICIE16/BSG) were subject to surface functionalization to further improve its interaction with biological systems. Surface functionalization was performed by a nitridation process with hot gas N2/ammonia at 550°C for 2h, obtaining the ICIE16/BSG-NITRI. Evaluation of the degradation rate and the conversion to hydroxyapatite after immersion in simulated body fluid predicted a good biological activity of all the scaffolds, but particularly of the nitrided ones. In vitro evaluation of osteoblastic cells cultured onto the nitrided and non-nitrided scaffolds showed cell attachment, proliferation and differentiation on all scaffolds, but both proliferation and differentiation were improved in the nitrided ICIE16/BSG-NITRI.

STATEMENT OF SIGNIFICANCE:

Biomaterials are often required in the clinic to stimulate bone repair. We have developed a novel bioglass (ICIE16/SBG-NITRI) that can be sintered into highly porous 3D scaffolds, and we have further improved its bioactivity by nitridation. ICIE16/SBG-NITRI was synthesized from a mixture of two melt-derived glasses through combined gel casting and foam replication techniques, followed by nitridation. To mimic bone, it presents high-interconnected porosity while being mechanically stable. Nitridation improved its reactivity and bioactivity facilitating its resorption and the deposition of apatite (bone-like mineral) on its surface and increasing its degradation rate. The nitrided surface also improved the bioglass’ interaction with bone cells, which were found to attach better to ICIE16/SBG-NITRI and to differentiate earlier on its surface.

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Global Medical Discovery features paper: Controlling the mechanical behavior of dual-material 3D printed meta-materials for patient-specific tissue-mimicking phantoms

Significance Statement

Mimicking the dynamic mechanical properties of the human aorta in 3D printed models is challenging because of the inherent difference between mechanical behaviors of polymeric materials and human tissues (Fig. A). We sought to print the aortic root using materials which achieved the strain-stiffening behavior of the human aortic tissues using commercial polymer printing materials. The mechanical behavior of aortic tissue is mimicked by a 3D printed meta-material, in which sinusoidal wave-shaped stiff fibers were embedded in a soft polymeric matrix (Fig. B). The wavelength, amplitude, and fiber diameter of the embedded sinusoidal fiber were tuned to study the meta-material’s stress-strain relationship. Then, fibers of ideal configurations were embedded in a 3D printed aortic root phantom (Fig. D). The designed meta-material demonstrated strain-stiffening behavior similar to the human aortic tissues. The stress-strain curve of the meta-materials was controlled by the design of the embedded fibers (Fig. C). As a follow-up to the study in this paper, a CoreValve prosthesis was deployed to simulate TAVR. The model was connected to a flow loop, and CMR images were acquired to visualize the in-vitro anatomy, and characterize and quantify the flow velocity field (Fig. E). 3D printed tissue-mimicking aortic root may enable predictions of post-TAVR root strain and distribution and aortic flow pattern, for pre-TAVR planning.

About The Author

Kan Wang received the B.S. degree in Theoretical and Applied Mechanics from Peking University, Beijing, China, in 2005, the M.S. degree in Aircraft Design from Beihang University, Beijing, China, in 2007, and the Ph.D. degree in Industrial and Manufacturing Engineering from Florida State University, Tallahassee, USA in 2013. Currently, he is a post-doctoral fellow at the H. Milton Stewart School of Industrial and Systems Engineering and Georgia Tech Manufacturing Institute, Georgia Institute of Technology, Atlanta, USA. His research interest include nanomanufacturing, additive manufacturing, printed electronics technologies and their applications in smart materials and biomedical devices.

Journal Reference

Materials & Design, Volume 90, 15 January 2016, Pages 704–712.

Kan Wang^1,2, Yuanshuo Zhao¹ , Yung-Hang Chang^1,2^, ,Zhen Qian⁴, Chuck Zhang^1,2, Ben Wang^1,2,3, Mani A. Vannan⁴, Mao-Jiun Wang⁵

Show Affiliations

Milton Stewart School of Industrial and Systems Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA
Georgia Tech Manufacturing Institute, Georgia Institute of Technology, Atlanta, GA 30332, USA
School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA
Department of Cardiovascular Imaging, Piedmont Heart Institute, 95 Collier Road, Atlanta, GA 30309, USA
Department of Industrial Engineering and Engineering Management, National Tsing-Hua University, Hsinchu, Taiwan

Abstract

Patient-specific tissue-mimicking phantoms are becoming available with the advent of additive manufacturing. Phantoms currently in use are focused on the geometrical accuracy and mechanical properties under small deformation. Mimicking the mechanical properties at large deformation is challenging because of the inherent difference between the mechanical behaviors of polymeric materials and that of human tissues. In this study, the mechanical behavior of soft tissues under a uniaxial tension is mimicked by dual-material 3D printed meta-materials with stiff micro-structured fibers embedded in a soft polymeric matrix. Although the two base materials are strain-softening polymers, some of the designed meta-materials demonstrate certain degree of strain-stiffening behavior. Further investigation shows how the stress–strain curve of the meta-materials can be controlled by the design parameters. Sensitivity analysis is used to study the effects of each parameter. General design guidelines are proposed based on the results of the experiments. Dual-material 3D printed meta-materials have great potential in fabricating patient-specific phantoms with accurate mechanical properties that are associated with the gender, age, ethnicity, and other physiological/pathological characteristics. Mechanically accurate phantoms can play an important role in a variety of biomedical applications, including validation of computational models, testing of medical devices, surgery planning, medical education and training, and doctor-patient interaction.

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Global Medical Discovery features paper: Development of neuraminidase detection using gold nanoparticles boron-doped diamond electrodes

Significance Statement

Neuraminidase detection is substantial in controlling the disease caused by related virus and microbes since Neuraminidase is an important enzyme in the pathogenic viruses and microbes spreading. Common detection methods of neuraminidase, such as RT-PCR, ELISA, and enzymatic reaction require several types of chemical reagents, specific instruments, and highly skilled operators. Therefore, a more simple and practical method is urgently required. In this work, a sensor for Neuraminidase has been developed using gold nanoparticles modified boron-doped diamond (AuNPs-BDD) electrodes. The detection method was performed based on the difference of electrochemical responses of Zanamivir at gold surface before and after the reaction with neuraminidase in phosphate buffer solution (PBS) pH 5.5.

In order to have a stable response, the modified electrodes was prepared through a self-assembly deposition of AuNPs at an amine-terminated boron-doped diamond. Cyclic voltammetry was used for the method. A linear calibration curve for Zanamivir in 0.1 M PBS in the absence of neuraminidase was achieved in the concentration range of 1 x 10^-6 – 1 x 10^-5 M (R² = 0.99), with the estimated limit of detection (LOD) of 2.29 x 10^-6 M.

Furthermore, using its reaction with 1.00 x 10^-5 M Zanamivir a linear calibration curve of neuraminidase can be obtained in the concentration range of 0 – 12 mU (R² = 0.99) with an estimated LOD of 0.12 mU. High reproducibility was shown with an RSD of 1.14 % (n=30). These performances could be maintained when the detection was performed in mucin matrix. Comparison made using gold-modified BDD (Au-BDD) electrodes suggested that the good performance of the detection method is due to the stability of the gold particles position at the BDD surface.

About The Author

Dr. Tribidasari A. Ivandini is a member of the teaching and research staff at Department of Chemistry, Faculty of Mathematics and Science, Universitas Indonesia, since 1997. She was born in Jakarta, Jakarta, on January 29th, 1970. She received her magister degrees in 1996 from Universitas Indonesia. Then, in 2003 she accomplished her doctoral degree at The University of Tokyo, Tokyo, Japan. Between 2003 and 2007 she performed post-doctoral research at the Department of Chemistry, Keio University, Tokyo, Japan with JSPS fellowship during 2004-2006. Up to the present time she is still performing research collaboration with Keio University.

Her research interests are mostly concerning electrochemistry of diamond, including its development for sensors and biosensors, electrocatalytics, and waste-water treatments. She has published about 50 scientific papers in highly respectable journals and conference proceedings, such as Analytical Chemistry, Physical Chemistry Chemical Physics, Sensors Actuators B: Chemicals, and Diamond Related Materials. She also holds some patents under European and Japan Patents. Ivandini can be reached at the Department of Chemistry, Faculty of Mathematics and Science, Universitas Indonesia, Indonesia.E-mail : ivandini.tri@sci.ui.ac.id

About The Author

Dr. Yasuaki Einaga is a professor in the Department of Chemistry at Keio University, Japan. He received his BS (1994) , MS (1996) , and PhD (1999) degree from The University of Tokyo. After 2 years as a research associate at The University of Tokyo, he started a faculty career as an assistant professor in Keio University in 2001. He was promoted to associate professor in 2003, and to professor in 2011. He was also a research director of JST-CREST (2011-2014), and JST-ACCEL (2014-present). His research interests include functional materials science, photochemistry, electrochemistry, and diamond electrodes. Einaga can be reached at the Department of Chemistry, Keio University, Yokohama, Japan. E-mail einaga@chem.keio.ac.jp

Journal Reference

Anal Biochem. 2016 Mar 15;497:68-75.

Wahyuni WT¹, Ivandini TA², Saepudin E¹, Einaga Y³.

Show Affiliations

Department of Chemistry, Faculty of Mathematics and Sciences, University of Indonesia, Kampus UI Depok, Jakarta 16424, Indonesia.
Department of Chemistry, Faculty of Mathematics and Sciences, University of Indonesia, Kampus UI Depok, Jakarta 16424, Indonesia. Electronic address: ivandini.tri@ui.ac.id.
Department of Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan; JST ACCEL, Yokohama 223-8522, Japan. Electronic address: einaga@chem.keio.ac.jp.

Abstract

Gold nanoparticles-modified boron-doped diamond (AuNPs-BDD) electrodes, which were prepared with a self-assembly deposition of AuNPs at amine-terminated boron-doped diamond, were examined for voltammetric detection of neuraminidase (NA). The detection method was performed based on the difference of electrochemical responses of zanamivir at gold surface before and after the reaction with neuraminidase in phosphate buffer solution (PBS, pH 5.5). A linear calibration curve for zanamivir in 0.1 M PBS in the absence of NA was achieved in the concentration range of 1 × 10(-6) to 1 × 10(-5) M (R(2) = 0.99) with an estimated limit of detection (LOD) of 2.29 × 10(-6) M. Furthermore, using its reaction with 1.00 × 10(-5) M zanamivir, a linear calibration curve of neuraminidase can be obtained in the concentration range of 0-12mU (R(2) = 0.99) with an estimated LOD of 0.12mU. High reproducibility was shown with a relative standard deviation (RSD) of 1.14% (n = 30). These performances could be maintained when the detection was performed in mucin matrix. Comparison performed using gold-modified BDD (Au-BDD) electrodes suggested that the good performance of the detection method is due to the stability of the gold particles position at the BDD surface.

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