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Tomagra G, Re A, Varzi V, Aprà P, Britel A, Franchino C, Sturari S, Amine NH, Westerink RHS, Carabelli V, Picollo F. Enhancing the Study of Quantal Exocytotic Events: Combining Diamond Multi-Electrode Arrays with Amperometric PEak Analysis (APE) an Automated Analysis Code. BIOSENSORS 2023; 13:1033. [PMID: 38131793 PMCID: PMC10741388 DOI: 10.3390/bios13121033] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/13/2023] [Revised: 12/11/2023] [Accepted: 12/14/2023] [Indexed: 12/23/2023]
Abstract
MicroGraphited-Diamond-Multi Electrode Arrays (μG-D-MEAs) can be successfully used to reveal, in real time, quantal exocytotic events occurring from many individual neurosecretory cells and/or from many neurons within a network. As μG-D-MEAs arrays are patterned with up to 16 sensing microelectrodes, each of them recording large amounts of data revealing the exocytotic activity, the aim of this work was to support an adequate analysis code to speed up the signal detection. The cutting-edge technology of microGraphited-Diamond-Multi Electrode Arrays (μG-D-MEAs) has been implemented with an automated analysis code (APE, Amperometric Peak Analysis) developed using Matlab R2022a software to provide easy and accurate detection of amperometric spike parameters, including the analysis of the pre-spike foot that sometimes precedes the complete fusion pore dilatation. Data have been acquired from cultured PC12 cells, either collecting events during spontaneous exocytosis or after L-DOPA incubation. Validation of the APE code was performed by comparing the acquired spike parameters with those obtained using Quanta Analysis (Igor macro) by Mosharov et al.
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Affiliation(s)
- Giulia Tomagra
- Department of Drug and Science Technology, NIS Interdepartmental Centre, University of Torino, Corso Raffaello 30, 10125 Torino, Italy; (G.T.); (C.F.); (V.C.)
| | - Alice Re
- Department of Physics, NIS Interdepartmental Centre, University of Torino and Italian Institute of Nuclear Physics, Via Giuria 1, 10125 Torino, Italy (P.A.); (A.B.); (S.S.); (N.-H.A.); (F.P.)
| | - Veronica Varzi
- Department of Physics, NIS Interdepartmental Centre, University of Torino and Italian Institute of Nuclear Physics, Via Giuria 1, 10125 Torino, Italy (P.A.); (A.B.); (S.S.); (N.-H.A.); (F.P.)
| | - Pietro Aprà
- Department of Physics, NIS Interdepartmental Centre, University of Torino and Italian Institute of Nuclear Physics, Via Giuria 1, 10125 Torino, Italy (P.A.); (A.B.); (S.S.); (N.-H.A.); (F.P.)
| | - Adam Britel
- Department of Physics, NIS Interdepartmental Centre, University of Torino and Italian Institute of Nuclear Physics, Via Giuria 1, 10125 Torino, Italy (P.A.); (A.B.); (S.S.); (N.-H.A.); (F.P.)
| | - Claudio Franchino
- Department of Drug and Science Technology, NIS Interdepartmental Centre, University of Torino, Corso Raffaello 30, 10125 Torino, Italy; (G.T.); (C.F.); (V.C.)
| | - Sofia Sturari
- Department of Physics, NIS Interdepartmental Centre, University of Torino and Italian Institute of Nuclear Physics, Via Giuria 1, 10125 Torino, Italy (P.A.); (A.B.); (S.S.); (N.-H.A.); (F.P.)
| | - Nour-Hanne Amine
- Department of Physics, NIS Interdepartmental Centre, University of Torino and Italian Institute of Nuclear Physics, Via Giuria 1, 10125 Torino, Italy (P.A.); (A.B.); (S.S.); (N.-H.A.); (F.P.)
| | - Remco H. S. Westerink
- Neurotoxicology Research Group, Division of Toxicology, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.177, NL-3508 TD Utrecht, The Netherlands;
| | - Valentina Carabelli
- Department of Drug and Science Technology, NIS Interdepartmental Centre, University of Torino, Corso Raffaello 30, 10125 Torino, Italy; (G.T.); (C.F.); (V.C.)
| | - Federico Picollo
- Department of Physics, NIS Interdepartmental Centre, University of Torino and Italian Institute of Nuclear Physics, Via Giuria 1, 10125 Torino, Italy (P.A.); (A.B.); (S.S.); (N.-H.A.); (F.P.)
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2
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Ackermann J, Stegemann J, Smola T, Reger E, Jung S, Schmitz A, Herbertz S, Erpenbeck L, Seidl K, Kruss S. High Sensitivity Near-Infrared Imaging of Fluorescent Nanosensors. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2023; 19:e2206856. [PMID: 36610045 DOI: 10.1002/smll.202206856] [Citation(s) in RCA: 17] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/06/2022] [Revised: 11/27/2022] [Indexed: 06/17/2023]
Abstract
Biochemical processes are fast and occur on small-length scales, which makes them difficult to measure. Optical nanosensors based on single-wall carbon nanotubes (SWCNTs) are able to capture such dynamics. They fluoresce in the near-infrared (NIR, 850-1700 nm) tissue transparency window and the emission wavelength depends on their chirality. However, NIR imaging requires specialized indium gallium arsenide (InGaAs) cameras with a typically low resolution because the quantum yield of normal Si-based cameras rapidly decreases in the NIR. Here, an efficient one-step phase separation approach to isolate monochiral (6,4)-SWCNTs (880 nm emission) from mixed SWCNT samples is developed. It enables imaging them in the NIR with high-resolution standard Si-based cameras (>50× more pixels). (6,4)-SWCNTs modified with (GT)10 -ssDNA become highly sensitive to the important neurotransmitter dopamine. These sensors are 1.7× brighter and 7.5× more sensitive and allow fast imaging (<50 ms). They enable high-resolution imaging of dopamine release from cells. Thus, the assembly of biosensors from (6,4)-SWCNTs combines the advantages of nanosensors working in the NIR with the sensitivity of (Si-based) cameras and enables broad usage of these nanomaterials.
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Affiliation(s)
- Julia Ackermann
- Fraunhofer Institute for Microelectronic Circuits and Systems, Finkenstrasse 61, 47057, Duisburg, Germany
- Department EBS, University Duisburg-Essen, Bismarkstrasse 81, 47057, Duisburg, Germany
| | - Jan Stegemann
- Fraunhofer Institute for Microelectronic Circuits and Systems, Finkenstrasse 61, 47057, Duisburg, Germany
- Department of Chemistry, Ruhr-University Bochum, Universitätsstrasse 150, 44801, Bochum, Germany
| | - Tim Smola
- Fraunhofer Institute for Microelectronic Circuits and Systems, Finkenstrasse 61, 47057, Duisburg, Germany
- Department EBS, University Duisburg-Essen, Bismarkstrasse 81, 47057, Duisburg, Germany
| | - Eline Reger
- Fraunhofer Institute for Microelectronic Circuits and Systems, Finkenstrasse 61, 47057, Duisburg, Germany
- Department EBS, University Duisburg-Essen, Bismarkstrasse 81, 47057, Duisburg, Germany
| | - Sebastian Jung
- ZEMOS Center for Solvation Science, Ruhr-University Bochum, Universitätsstrasse 150, 44801, Bochum, Germany
| | - Anne Schmitz
- Department of Dermatology, University Hospital Münster, Von-Esmarch-Strasse 58, 48149, Münster, Germany
| | - Svenja Herbertz
- Fraunhofer Institute for Microelectronic Circuits and Systems, Finkenstrasse 61, 47057, Duisburg, Germany
| | - Luise Erpenbeck
- Department of Dermatology, University Hospital Münster, Von-Esmarch-Strasse 58, 48149, Münster, Germany
| | - Karsten Seidl
- Fraunhofer Institute for Microelectronic Circuits and Systems, Finkenstrasse 61, 47057, Duisburg, Germany
- Department EBS, University Duisburg-Essen, Bismarkstrasse 81, 47057, Duisburg, Germany
- Center for Nanointegration Duisburg-Essen (CENIDE), Carl-Benz-Strasse 199, 47057, Duisburg, Germany
| | - Sebastian Kruss
- Fraunhofer Institute for Microelectronic Circuits and Systems, Finkenstrasse 61, 47057, Duisburg, Germany
- Department of Chemistry, Ruhr-University Bochum, Universitätsstrasse 150, 44801, Bochum, Germany
- Center for Nanointegration Duisburg-Essen (CENIDE), Carl-Benz-Strasse 199, 47057, Duisburg, Germany
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3
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Kim S, Kang S, Choe J, Moon C, Choi H, Kim JY, Choi JW. A Microfluidic System for Investigating Anticipatory Medication Effects on Dopamine Homeostasis in Dopaminergic Cells. Anal Chem 2023; 95:3153-3159. [PMID: 36656793 DOI: 10.1021/acs.analchem.2c04923] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
Dopamine (DA) homeostasis influences emotions, neural circuit development, cognition, and the reward system. Dysfunctions in DA regulation can lead to neurological disorders, including depression, developmental disorders, and addiction. DA homeostasis disruption is a primary cause of Parkinson's Disease (PD). Therefore, understanding the relationship between DA homeostasis and PD progression may clarify the mechanisms for pharmacologically treating PD. This study developed a novel in vitro DA homeostasis platform which consists of three main parts: (1) a microfluidic device for culturing DAergic neurons, (2) an optical detection system for reading DA levels, and (3) an automatic closed-loop control system that establishes when and how much medication to infuse; this uses a microfluidic device that can cultivate DAergic neurons, perfuse solutions, perform in vitro PD modeling, and continuously monitor DA concentrations. The automatically controlled closed-loop control system simultaneously monitors pharmacological PD treatment to support long-term monitoring of DA homeostasis. SH-SY5Y neuroblastoma cells were chosen as DAergic neurons. They were cultivated in the microfluidic device, and real-time cellular DA level measurements successfully achieved long-term monitoring and modulation of DA homeostasis. When applied in combination with multiday cell culture, this advanced system can be used for drug screening and fundamental biological studies.
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Affiliation(s)
- Samhwan Kim
- Department of Electrical Engineering and Computer Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Techno jungang-daero 333, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea.,Brain Engineering Convergence Research Center, DGIST, Techno jungang-daero 333, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea.,Research Advanced Centre for Olfaction, DGIST, Techno jungang-daero 333, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea
| | - Seongtak Kang
- Department of Electrical Engineering and Computer Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Techno jungang-daero 333, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea
| | - Jiyun Choe
- Department of Brain Sciences, DGIST, Techno jungang-daero 333, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea
| | - Cheil Moon
- Department of Brain Sciences, DGIST, Techno jungang-daero 333, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea.,Research Advanced Centre for Olfaction, DGIST, Techno jungang-daero 333, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea
| | - Hongsoo Choi
- Department of Robotics and Mechatronics Engineering, DGIST, Techno jungang-daero 333, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea.,DGIST-ETH Microrobotic Research Center, DGIST, Techno jungang-daero 333, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea
| | - Jin-Young Kim
- Brain Engineering Convergence Research Center, DGIST, Techno jungang-daero 333, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea.,Department of Robotics and Mechatronics Engineering, DGIST, Techno jungang-daero 333, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea.,DGIST-ETH Microrobotic Research Center, DGIST, Techno jungang-daero 333, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea.,Division of Biotechnology, DGIST, Techno jungang-daero 333, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea
| | - Ji-Woong Choi
- Department of Electrical Engineering and Computer Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Techno jungang-daero 333, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea.,Brain Engineering Convergence Research Center, DGIST, Techno jungang-daero 333, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea
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PC-12 Cell Line as a Neuronal Cell Model for Biosensing Applications. BIOSENSORS 2022; 12:bios12070500. [PMID: 35884303 PMCID: PMC9313070 DOI: 10.3390/bios12070500] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/15/2022] [Revised: 07/04/2022] [Accepted: 07/07/2022] [Indexed: 12/02/2022]
Abstract
PC-12 cells have been widely used as a neuronal line study model in many biosensing devices, mainly due to the neurogenic characteristics acquired after differentiation, such as high level of secreted neurotransmitter, neuron morphology characterized by neurite outgrowth, and expression of ion and neurotransmitter receptors. For understanding the pathophysiology processes involved in brain disorders, PC-12 cell line is extensively assessed in neuroscience research, including studies on neurotoxicity, neuroprotection, or neurosecretion. Various analytical technologies have been developed to investigate physicochemical processes and the biosensors based on optical and electrochemical techniques, among others, have been at the forefront of this development. This article summarizes the application of different biosensors in PC-12 cell cultures and presents the modern approaches employed in neuronal networks biosensing.
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A fluorescent nanosensor paint detects dopamine release at axonal varicosities with high spatiotemporal resolution. Proc Natl Acad Sci U S A 2022; 119:e2202842119. [PMID: 35613050 DOI: 10.1073/pnas.2202842119] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
SignificanceThe neurotransmitter dopamine controls normal behavior and dopaminergic dysfunction is prevalent in multiple brain diseases. To reach a detailed understanding of how dopamine release and signaling are regulated at the subcellular level, we developed a near infrared fluorescent dopamine nanosensor 'paint' (AndromeDA) to directly image dopamine release and its spatiotemporal characteristics. With AndromeDA, we can ascribe discrete DA release events to defined axonal varicosities, directly assess the heterogeneity of DA release events across such release sites, and determine the molecular components of the DA release machinery. AndromeDA thus provides a new method for gaining fundamental insights into the core mechanisms of dopamine release, which with greatly benefit our knowledge of dopamine biology and pathobiology.
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Kong W, Zhu D, Luo R, Yu S, Ju H. Framework-promoted charge transfer for highly selective photoelectrochemical biosensing of dopamine. Biosens Bioelectron 2022; 211:114369. [PMID: 35594626 DOI: 10.1016/j.bios.2022.114369] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2021] [Revised: 05/04/2022] [Accepted: 05/10/2022] [Indexed: 11/16/2022]
Abstract
Traditional photoelectrochemical (PEC) systems with inorganic semiconductors as photoactive materials generally involve effortless recombination of electron-hole pairs, which greatly limit the detection sensitivity. The arrangement of multiple components with tunable bandgaps provides an effective way to accelerate charge transfer. In this work, a framework material with adjustable structure was used to promote the charge transfer in the PEC process. The framework was constructed with 9,10-di(p-carboxyphenyl)anthracene (DPA) ligands as the light collector to coordinate with Zn2+ nodes, which formed an electronegative metal-organic framework (ZnMOF), and showed good conductivity and PEC performance due to the π-π stacking of DPA and the intrareticular charge transfer. Based on the band and charge matching of dopamine (DA) with ZnMOF, the ZnMOF modified electrode as a biosensor showed excellent PEC response to DA with good selectivity, thus realized sensitive detection of DA ranging from 0.03 to 10 μM with a detection limit of 17.7 nM. The biosensor could be used to monitor the release of DA from PC12 cells and evaluate the stimulation of K+ to DA release. The conductive framework material provided an approach to develop highly selective sensing platform for trace bioanalysis.
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Affiliation(s)
- Weisu Kong
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, PR China
| | - Da Zhu
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, PR China
| | - Rengan Luo
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, PR China
| | - Siqi Yu
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, PR China
| | - Huangxian Ju
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, PR China.
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Hu K, Le Vo KL, Hatamie A, Ewing AG. Quantifying Intracellular Single Vesicular Catecholamine Concentration with Open Carbon Nanopipettes to Unveil the Effect of L‐DOPA on Vesicular Structure. Angew Chem Int Ed Engl 2022. [DOI: 10.1002/ange.202113406] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Affiliation(s)
- Keke Hu
- Department of Chemistry and Molecular Biology University of Gothenburg Kemivägen 10 41296 Gothenburg Sweden
| | - Kim Long Le Vo
- Department of Chemistry and Molecular Biology University of Gothenburg Kemivägen 10 41296 Gothenburg Sweden
| | - Amir Hatamie
- Department of Chemistry and Molecular Biology University of Gothenburg Kemivägen 10 41296 Gothenburg Sweden
| | - Andrew G. Ewing
- Department of Chemistry and Molecular Biology University of Gothenburg Kemivägen 10 41296 Gothenburg Sweden
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8
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Hu K, Le Vo KL, Hatamie A, Ewing AG. Quantifying Intracellular Single Vesicular Catecholamine Concentration with Open Carbon Nanopipettes to Unveil the Effect of L-DOPA on Vesicular Structure. Angew Chem Int Ed Engl 2021; 61:e202113406. [PMID: 34734466 PMCID: PMC9299131 DOI: 10.1002/anie.202113406] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2021] [Indexed: 12/03/2022]
Abstract
Understanding the regulatory mechanisms of exocytosis is essential for uncovering the pathologies of neuronal disorders and developing related pharmaceuticals. In this work intracellular vesicle impact electrochemical cytometry (IVIEC) measurements with different‐sized (50–500 nm radius) open carbon nanopipettes (CNPs) were performed to quantify the vesicular content and release kinetics of specific vesicle populations grouped by orifice sizes. Intracellular vesicles with radius below 100 nm were captured and narrowed between 50 and 100 nm. On the basis of this, single vesicular catecholamine concentrations in the intracellular environment were quantified as 0.23–1.1 M. Our results with L‐3,4‐dihydroxyphenylalanine (L‐DOPA)‐exposure indicate that L‐DOPA regulates exocytosis by increasing the dense core size and vesicular content while catecholamine concentrations did not show obvious alterations. These were all achieved simultaneously and relatively noninvasively with open CNPs.
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Affiliation(s)
- Keke Hu
- Department of Chemistry and Molecular Biology, University of Gothenburg, Kemivägen 10, 41296, Gothenburg, Sweden
| | - Kim Long Le Vo
- Department of Chemistry and Molecular Biology, University of Gothenburg, Kemivägen 10, 41296, Gothenburg, Sweden
| | - Amir Hatamie
- Department of Chemistry and Molecular Biology, University of Gothenburg, Kemivägen 10, 41296, Gothenburg, Sweden
| | - Andrew G Ewing
- Department of Chemistry and Molecular Biology, University of Gothenburg, Kemivägen 10, 41296, Gothenburg, Sweden
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Murti BT, Putri AD, Huang YJ, Wei SM, Peng CW, Yang PK. Clinically oriented Alzheimer's biosensors: expanding the horizons towards point-of-care diagnostics and beyond. RSC Adv 2021; 11:20403-20422. [PMID: 35479927 PMCID: PMC9033966 DOI: 10.1039/d1ra01553b] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2021] [Accepted: 05/28/2021] [Indexed: 12/30/2022] Open
Abstract
The development of minimally invasive and easy-to-use sensor devices is of current interest for ultrasensitive detection and signal recognition of Alzheimer's disease (AD) biomarkers. Over the years, tremendous effort has been made on diagnostic platforms specifically targeting neurological markers for AD in order to replace the conventional, laborious, and invasive sampling-based approaches. However, the sophistication of analytical outcomes, marker inaccessibility, and material validity strongly limit the current strategies towards effectively predicting AD. Recently, with the promising progress in biosensor technology, the realization of a clinically applicable sensing platform has become a potential option to enable early diagnosis of AD and other neurodegenerative diseases. In this review, various types of biosensors, which include electrochemical, fluorescent, plasmonic, photoelectrochemical, and field-effect transistor (FET)-based sensor configurations, with better clinical applicability and analytical performance towards AD are highlighted. Moreover, the feasibility of these sensors to achieve point-of-care (POC) diagnosis is also discussed. Furthermore, by grafting nanoscale materials into biosensor architecture, the remarkable enhancement in durability, functionality, and analytical outcome of sensor devices is presented. Finally, future perspectives on further translational and commercialization pathways of clinically driven biosensor devices for AD are discussed and summarized.
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Affiliation(s)
- Bayu Tri Murti
- Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University Taipei Taiwan
- Semarang College of Pharmaceutical Sciences (STIFAR) Semarang City Indonesia
| | - Athika Darumas Putri
- Semarang College of Pharmaceutical Sciences (STIFAR) Semarang City Indonesia
- Department of Pharmaceutical Sciences, School of Pharmacy, College of Pharmacy, Taipei Medical University Taipei Taiwan
| | - Yi-June Huang
- Graduate Institute of Nanomedicine and Medical Engineering, College of Biomedical Engineering, Taipei Medical University Taipei Taiwan
- International Ph.D. Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University Taipei Taiwan
| | - Shih-Min Wei
- Graduate Institute of Nanomedicine and Medical Engineering, College of Biomedical Engineering, Taipei Medical University Taipei Taiwan
- International Ph.D. Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University Taipei Taiwan
| | - Chih-Wei Peng
- International Ph.D. Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University Taipei Taiwan
- School of Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University Taipei Taiwan
| | - Po-Kang Yang
- Graduate Institute of Nanomedicine and Medical Engineering, College of Biomedical Engineering, Taipei Medical University Taipei Taiwan
- International Ph.D. Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University Taipei Taiwan
- Department of Biomedical Sciences and Engineering, National Central University Chung-li Taiwan
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10
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Gu C, Ewing AG. Simultaneous detection of vesicular content and exocytotic release with two electrodes in and at a single cell. Chem Sci 2021; 12:7393-7400. [PMID: 34163829 PMCID: PMC8171312 DOI: 10.1039/d1sc01190a] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
We developed a technique employing two electrodes to simultaneously and dynamically monitor vesicular neurotransmitter storage and vesicular transmitter release in and at the same cell. To do this, two electrochemical techniques, single-cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC), were applied using two nanotip electrodes. With one electrode being placed on top of a cell measuring exocytotic release and the other electrode being inserted into the cytoplasm measuring vesicular transmitter storage, upon chemical stimulation, exocytosis is triggered and the amount of release and storage can be quantified simultaneously and compared. By using this technique, we made direct comparison between exocytotic release and vesicular storage, and investigated the dynamic changes of vesicular transmitter content before, during, and after chemical stimulation of PC12 cells, a neuroendocrine cell line. While confirming that exocytosis is partial, we suggest that chemical stimulation either induces a replenishment of the releasable pool with a subpool of vesicles having higher amount of transmitter storage, or triggers the vesicles within the same subpool to load more transiently at approximately 10–20 s. Thus, a time scale for vesicle reloading is determined. The effect of l-3,4-dihydroxyphenylalanine (l-DOPA), the precursor to dopamine, on the dynamic alteration of vesicular storage upon chemical stimulation for exocytosis was also studied. We found that l-DOPA incubation reduces the observed changes of vesicular storage in regular PC12 cells, which might be due to an increased capacity of vesicular transmitter loading caused by l-DOPA. Our data provide another mechanism for plasticity after stimulation via quantitative and dynamic changes in the exocytotic machinery. Simultaneous measurements of IVIEC and SCA by two nanotip electrodes allows direct and dynamic comparison between vesicular transmitter content and vesicular transmitter release to shed light on stimulation-induced plasticity.![]()
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Affiliation(s)
- Chaoyi Gu
- Department of Chemistry and Molecular Biology, University of Gothenburg Kemivägen 10 412 96 Gothenburg Sweden
| | - Andrew G Ewing
- Department of Chemistry and Molecular Biology, University of Gothenburg Kemivägen 10 412 96 Gothenburg Sweden
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11
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White KA, Kim BN. Quantifying neurotransmitter secretion at single-vesicle resolution using high-density complementary metal-oxide-semiconductor electrode array. Nat Commun 2021; 12:431. [PMID: 33462204 PMCID: PMC7813837 DOI: 10.1038/s41467-020-20267-0] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2020] [Accepted: 11/17/2020] [Indexed: 11/21/2022] Open
Abstract
Neuronal exocytosis facilitates the propagation of information through the nervous system pertaining to bodily function, memory, and emotions. Using amperometry, the sub-millisecond dynamics of exocytosis can be monitored and the modulation of exocytosis due to drug treatment or neurodegenerative diseases can be studied. Traditional single-cell amperometry is a powerful technique for studying the molecular mechanisms of exocytosis, but it is both costly and labor-intensive to accumulate statistically significant data. To surmount these limitations, we have developed a silicon-based electrode array with 1024 on-chip electrodes that measures oxidative signal in 0.1 millisecond intervals. Using the developed device, we are able to capture the modulation of exocytosis due to Parkinson's disease treatment (L-Dopa), with statistical significance, within 30 total minutes of recording. The validation study proves our device's capability to accelerate the study of many pharmaceutical treatments for various neurodegenerative disorders that affect neurotransmitter secretion to a matter of minutes.
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Affiliation(s)
- Kevin A White
- Department of Electrical and Computer Engineering, University of Central Florida, Orlando, FL, 32827, USA
| | - Brian N Kim
- Department of Electrical and Computer Engineering, University of Central Florida, Orlando, FL, 32827, USA.
- Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, 32827, USA.
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12
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McCormick HK, Dick JE. Nanoelectrochemical quantification of single-cell metabolism. Anal Bioanal Chem 2020; 413:17-24. [PMID: 32915282 DOI: 10.1007/s00216-020-02899-9] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Revised: 08/10/2020] [Accepted: 08/18/2020] [Indexed: 12/12/2022]
Abstract
At the most fundamental level, the behavior of tissue is governed by the activity of its single cells. A detailed examination of single-cell biology is necessary in order to gain a deeper understanding of disease progression. While single-cell genomics and transcriptomics are mature due to robust amplification strategies, the metabolome is difficult to quantify. Nanoelectrochemical techniques stand poised to quantify single-cell metabolism as a result of the fabrication of nanoelectrodes, which allow one to make intracellular electrochemical measurements. This article is concerned with intracellular nanoelectrochemistry, focusing on the sensitive and selective quantification of various metabolites within a single, living cell. We will review the strong literature behind this field, discuss the potential deleterious effects of passing charge inside cells, and provide future outlooks for this promising avenue of inquiry. We also present a mathematical relationship based on Faraday's Law and bulk electrolysis theory to examine the consumption of analyte within a cell due to passing charge at the nanotip.Graphical abstract.
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Affiliation(s)
- Hadley K McCormick
- Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA
| | - Jeffrey E Dick
- Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA. .,Lineberger Comprehensive Cancer Center, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
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13
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Keighron JD, Wang Y, Cans AS. Electrochemistry of Single-Vesicle Events. ANNUAL REVIEW OF ANALYTICAL CHEMISTRY (PALO ALTO, CALIF.) 2020; 13:159-181. [PMID: 32151142 DOI: 10.1146/annurev-anchem-061417-010032] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/10/2023]
Abstract
Neuronal transmission relies on electrical signals and the transfer of chemical signals from one neuron to another. Chemical messages are transmitted from presynaptic neurons to neighboring neurons through the triggered fusion of neurotransmitter-filled vesicles with the cell plasma membrane. This process, known as exocytosis, involves the rapid release of neurotransmitter solutions that are detected with high affinity by the postsynaptic neuron. The type and number of neurotransmitters released and the frequency of vesicular events govern brain functions such as cognition, decision making, learning, and memory. Therefore, to understand neurotransmitters and neuronal function, analytical tools capable of quantitative and chemically selective detection of neurotransmitters with high spatiotemporal resolution are needed. Electrochemistry offers powerful techniques that are sufficiently rapid to allow for the detection of exocytosis activity and provides quantitative measurements of vesicle neurotransmitter content and neurotransmitter release from individual vesicle events. In this review, we provide an overview of the most commonly used electrochemical methods for monitoring single-vesicle events, including recent developments and what is needed for future research.
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Affiliation(s)
- Jacqueline D Keighron
- Department of Chemical and Biological Sciences, New York Institute of Technology, Old Westbury, New York 11568, USA
| | - Yuanmo Wang
- Department of Chemistry and Chemical Engineering, Chalmers University of Technology, 412 96 Gothenburg, Sweden;
| | - Ann-Sofie Cans
- Department of Chemistry and Chemical Engineering, Chalmers University of Technology, 412 96 Gothenburg, Sweden;
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14
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Chen R, Alanis K, Welle TM, Shen M. Nanoelectrochemistry in the study of single-cell signaling. Anal Bioanal Chem 2020; 412:6121-6132. [PMID: 32424795 DOI: 10.1007/s00216-020-02655-z] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2020] [Revised: 04/02/2020] [Accepted: 04/08/2020] [Indexed: 12/28/2022]
Abstract
Label-free biosensing has been the dream of scientists and biotechnologists as reported by Vollmer and Arnold (Nat Methods 5:591-596, 2008). The ability of examining living cells is crucial to cell biology as noted by Fang (Int J Electrochem 2011:460850, 2011). Chemical measurement with electrodes is label-free and has demonstrated capability of studying living cells. In recent years, nanoelectrodes of different functionality have been developed. These nanometer-sized electrodes, coupled with scanning electrochemical microscopy (SECM), have further enabled nanometer spatial resolution study in aqueous environments. Developments in the field of nanoelectrochemistry have allowed measurement of signaling species at single cells, contributing to better understanding of cell biology. Leading studies using nanoelectrochemistry of a variety of cellular signaling molecules, including redox-active neurotransmitter (e.g., dopamine), non-redox-active neurotransmitter (e.g., acetylcholine), reactive oxygen species (ROS), and reactive nitrogen species (RNS), are reviewed here.
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Affiliation(s)
- Ran Chen
- Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL, 61801, USA
| | - Kristen Alanis
- Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL, 61801, USA
| | - Theresa M Welle
- Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL, 61801, USA
| | - Mei Shen
- Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL, 61801, USA.
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15
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Thomen A, Najafinobar N, Penen F, Kay E, Upadhyay PP, Li X, Phan NTN, Malmberg P, Klarqvist M, Andersson S, Kurczy ME, Ewing AG. Subcellular Mass Spectrometry Imaging and Absolute Quantitative Analysis across Organelles. ACS NANO 2020; 14:4316-4325. [PMID: 32239916 PMCID: PMC7199216 DOI: 10.1021/acsnano.9b09804] [Citation(s) in RCA: 46] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/13/2019] [Accepted: 04/02/2020] [Indexed: 05/22/2023]
Abstract
Mass spectrometry imaging is a field that promises to become a mainstream bioanalysis technology by allowing the combination of single-cell imaging and subcellular quantitative analysis. The frontier of single-cell imaging has advanced to the point where it is now possible to compare the chemical contents of individual organelles in terms of raw or normalized ion signal. However, to realize the full potential of this technology, it is necessary to move beyond this concept of relative quantification. Here we present a nanoSIMS imaging method that directly measures the absolute concentration of an organelle-associated, isotopically labeled, pro-drug directly from a mass spectrometry image. This is validated with a recently developed nanoelectrochemistry method for single organelles. We establish a limit of detection based on the number of isotopic labels used and the volume of the organelle of interest, also offering this calculation as a web application. This approach allows subcellular quantification of drugs and metabolites, an overarching and previously unmet goal in cell science and pharmaceutical development.
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Affiliation(s)
- Aurélien Thomen
- Department
of Chemistry and Molecular Biology, University
of Gothenburg, Gothenburg, 412 96, Sweden
| | - Neda Najafinobar
- Medicinal
Chemistry, Research and Early Development, Respiratory, Inflammation,
and Autoimmune, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, 430 51, Sweden
| | - Florent Penen
- Department
of Chemistry and Chemical Engineering, Chalmers
University of Technology, Gothenburg, 412 96, Sweden
| | - Emma Kay
- Bioscience,
Research and Early Development, Cardiovascular, Renal and Metabolism,
BioPharmaceuticals R&D, AstraZeneca, Gothenburg, 430 51, Sweden
| | - Pratik P. Upadhyay
- Pharmaceutical
Technolgy and Development, AstraZeneca R&D, Gothenburg, 430 52, Sweden
| | - Xianchan Li
- Center
for Imaging and Systems Biology, College of Life and Environmental
Sciences, Minzu University of China, Beijing, 100081, China
| | - Nhu T. N. Phan
- Department
of Chemistry and Molecular Biology, University
of Gothenburg, Gothenburg, 412 96, Sweden
| | - Per Malmberg
- Department
of Chemistry and Chemical Engineering, Chalmers
University of Technology, Gothenburg, 412 96, Sweden
| | - Magnus Klarqvist
- Early
Product Development, Pharmaceutical Science, R&D, AstraZeneca, Gothenburg, 431 50, Sweden
| | - Shalini Andersson
- New Modalities,
Discovery Sciences, R&D, AstraZeneca, Gothenburg, 430 51, Sweden
| | - Michael E. Kurczy
- DMPK,
Research and Early Development, Cardiovascular, Renal and Metabolism,
BioPharmaceuticals R&D, AstraZeneca, Gothenburg, 430 51, Sweden
| | - Andrew G. Ewing
- Department
of Chemistry and Molecular Biology, University
of Gothenburg, Gothenburg, 412 96, Sweden
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16
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Yang K, Jiang X, Cheng S, Bai L, Xia Y, Chen C, Meng P, Wang J, Li C, Tang Q, Cao X, Tu B. Synaptic dopamine release is positively regulated by SNAP-25 that involves in benzo[a]pyrene-induced neurotoxicity. CHEMOSPHERE 2019; 237:124378. [PMID: 31376700 DOI: 10.1016/j.chemosphere.2019.124378] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/05/2019] [Revised: 07/13/2019] [Accepted: 07/15/2019] [Indexed: 06/10/2023]
Abstract
Benzo[a]pyrene (B[a]P) is a ubiquitous neurotoxic pollutant that widely distributes in the natural environment. However, the exact mechanism of B[a]P-induced neurotoxicity has not been well established. As one key synaptic protein, SNAP-25 plays an important role in the regulation of neurotransmitter release, including synaptic dopamine release. In this study, we demonstrated that, after intragastric administration of B[a]P in rats aged postnatal day 5 for 7 weeks, B[a]P significantly increased the level of dopamine and the expression of SNAP-25, dopamine receptor 1 (DRD1) and DRD 3. Moreover, treatment of B[a]P also caused the ultra-structural pathological changes in the cerebral cortex of rats. To further reveal the potential role of SNAP-25 in the regulation of DRDs, we treated the dopaminergic PC-12 cells with 20 μM B[a]P for 24 h. A significant cytotoxicity and apoptosis were observed, and more importantly, we found that SNAP-25, DRD 1 and DRD 3 co-localized in the cells, and down-regulation of SNAP-25 by CRISPR-Cas9 plasmid remarkably reduced the expression of DRD1 and DRD3. Together, our findings suggest that, synaptic dopamine release may be positively regulated by SNAP-25 via its receptors, and thus affecting the neurotoxicity induced by B[a]P.
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Affiliation(s)
- Kai Yang
- Emergency and Business Management Office, Chengdu Center for Disease Control and Prevention, Chengdu, Sichuan, People's Republic of China; Department of Occupational and Environmental Health, School of Public Health and Management, Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, People's Republic of China
| | - Xuejun Jiang
- Center of Experimental Teaching for Public Health, Experimental Teaching and Management Center, Chongqing Medical University, Chongqing, People's Republic of China; Laboratory of Tissue and Cell Biology, Experimental Teaching and Management Center, Chongqing Medical University, Chongqing, People's Republic of China
| | - Shuqun Cheng
- Department of Occupational and Environmental Health, School of Public Health and Management, Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, People's Republic of China
| | - LuLu Bai
- Department of Occupational and Environmental Health, School of Public Health and Management, Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, People's Republic of China
| | - Yinyin Xia
- Department of Occupational and Environmental Health, School of Public Health and Management, Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, People's Republic of China
| | - Chengzhi Chen
- Department of Occupational and Environmental Health, School of Public Health and Management, Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, People's Republic of China
| | - Pan Meng
- Department of Occupational and Environmental Health, School of Public Health and Management, Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, People's Republic of China
| | - Jing Wang
- Department of Occupational and Environmental Health, School of Public Health and Management, Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, People's Republic of China
| | - Chunlin Li
- Department of Occupational and Environmental Health, School of Public Health and Management, Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, People's Republic of China
| | - Qianghu Tang
- Department of Occupational and Environmental Health, School of Public Health and Management, Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, People's Republic of China
| | - Xianqing Cao
- Department of Occupational and Environmental Health, School of Public Health and Management, Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, People's Republic of China
| | - Baijie Tu
- Department of Occupational and Environmental Health, School of Public Health and Management, Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, People's Republic of China.
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17
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Li X, Dunevall J, Ewing AG. Electrochemical quantification of transmitter concentration in single nanoscale vesicles isolated from PC12 cells. Faraday Discuss 2019; 210:353-364. [PMID: 29989629 DOI: 10.1039/c8fd00020d] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
We use an electrochemical platform, nanoparticle tracking analysis, and differential centrifugation of single catecholamine vesicles to study the properties of nanometer transmitter vesicles, including the number of molecules, size, and catecholamine concentration inside. Vesicle impact electrochemical cytometry (VIEC) was used to quantify the catecholamine content of single vesicles in different batches isolated from pheochromocytoma (PC12) cells with different ultracentrifugation speeds. We show that, vesicles containing less catecholamine are obtained at subsequent centrifugation steps with higher speed (force). Important to quantification, the cumulative content after subsequent centrifugation steps is equivalent to that of one-step centrifugation at the highest speed, 70 000g. Moreover, as we count molecules in the vesicles, we compared molecular numbers from VIEC, flow VIEC, and intracellular VIEC to corresponding vesicle size measured by nanoparticle tracking analysis to evaluate catecholamine concentration in vesicles. The data suggest that vesicular catecholamine concentration is relatively constant and independent of the vesicular size, indicating vesicular transmitter content as a main factor regulating the vesicle size.
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Affiliation(s)
- Xianchan Li
- Department of Chemistry and Molecular Biology, University of Gothenburg, SE-405 30 Gothenburg, Sweden.
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18
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Tomagra G, Franchino C, Pasquarelli A, Carbone E, Olivero P, Carabelli V, Picollo F. Simultaneous multisite detection of quantal release from PC12 cells using micro graphitic-diamond multi electrode arrays. Biophys Chem 2019; 253:106241. [PMID: 31398633 DOI: 10.1016/j.bpc.2019.106241] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2019] [Revised: 07/29/2019] [Accepted: 07/29/2019] [Indexed: 12/28/2022]
Abstract
Micro graphitic - diamond - multi electrode arrays (μG-D-MEAs) are suitable for measuring multisite quantal dopamine (DA) release from PC12 cells. Following cell stimulation with high extracellular KCl and electrode polarization at +650 mV, amperometric spikes are detected with a mean frequency of 0.60 ± 0.16 Hz. In each recording, simultaneous detection of secretory events is occurred in approximately 50% of the electrodes. Kinetic spike parameters and background noise are preserved among the different electrodes. Comparing the amperometric spikes recorder under control conditions with those recorders from PC12 cells previously incubated for 30 min with the dopamine precursor Levodopa (L-DOPA, 20 μM) it appears that the quantal size of amperometric spikes is increased by 250% and the half-time width (t1/2) by over 120%. On the contrary, L-DOPA has no effect on the frequency of secretory events. Overall, these data demonstrate that the μG-D-MEAs represent a reliable bio-sensor to simultaneously monitor quantal exocytotic events from different cells and in perspective can be exploited as a drug-screening tool.
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Affiliation(s)
- Giulia Tomagra
- Department of Drug and Science Technology, NIS Inter-departmental Centre, University of Torino, Corso Raffaello 30, 10125 Torino, Italy.
| | - Claudio Franchino
- Department of Drug and Science Technology, NIS Inter-departmental Centre, University of Torino, Corso Raffaello 30, 10125 Torino, Italy
| | - Alberto Pasquarelli
- Institute of Electron Devices and Circuits, University of Ulm, 89069 Ulm, Germany
| | - Emilio Carbone
- Department of Drug and Science Technology, NIS Inter-departmental Centre, University of Torino, Corso Raffaello 30, 10125 Torino, Italy
| | - Paolo Olivero
- Department of Physics, NIS Inter-departmental Centre, University of Torino, Italian Institute of Nuclear Physics, via Giuria 1, 10125 Torino, Italy
| | - Valentina Carabelli
- Department of Drug and Science Technology, NIS Inter-departmental Centre, University of Torino, Corso Raffaello 30, 10125 Torino, Italy
| | - Federico Picollo
- Department of Physics, NIS Inter-departmental Centre, University of Torino, Italian Institute of Nuclear Physics, via Giuria 1, 10125 Torino, Italy
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19
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Zhang HW, Hu XB, Qin Y, Jin ZH, Zhang XW, Liu YL, Huang WH. Conductive Polymer Coated Scaffold to Integrate 3D Cell Culture with Electrochemical Sensing. Anal Chem 2019; 91:4838-4844. [PMID: 30864440 DOI: 10.1021/acs.analchem.9b00478] [Citation(s) in RCA: 31] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Remarkable progresses have been made in electrochemical monitoring of living cells based on one-dimensional (1D) or two-dimensional (2D) sensors, but the cells cultured on 2D substrate under these circumstances are departed from their three-dimensional (3D) microenvironments in vivo. Significant advances have been made in developing 3D culture scaffolds to simulate the 3D microenvironment yet most of them are insulated, which greatly restricts their application in electrochemical sensing. Herein, we propose a versatile strategy to endow 3D insulated culture scaffolds with electrochemical performance while granting their biocompatibility through conductive polymer coating. More specifically, 3D polydimethylsiloxane scaffold is uniformly coated by poly(3,4-ethylenedioxythiophene) and further modified by platinum nanoparticles. The integrated 3D device demonstrates desirable biocompatibility for long-term 3D cell culture and excellent electrocatalytic ability for electrochemical sensing. This allows real-time monitoring of reactive oxygen species release from cancer cells induced by a novel potential anticancer drug and reveals its promising application in cancer treatment. This work provides a new idea to construct 3D multifunctional electrochemical sensors, which will be of great significance for physiological and pathological research.
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Affiliation(s)
- Hai-Wei Zhang
- Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences , Wuhan University , Wuhan 430072 , China
| | - Xue-Bo Hu
- Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences , Wuhan University , Wuhan 430072 , China
| | - Yu Qin
- Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences , Wuhan University , Wuhan 430072 , China
| | - Zi-He Jin
- Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences , Wuhan University , Wuhan 430072 , China
| | - Xin-Wei Zhang
- Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences , Wuhan University , Wuhan 430072 , China
| | - Yan-Ling Liu
- Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences , Wuhan University , Wuhan 430072 , China
| | - Wei-Hua Huang
- Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences , Wuhan University , Wuhan 430072 , China
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20
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Mehl BT, Martin RS. Integrating 3D Cell Culture of PC12 Cells with Microchip-Based Electrochemical Detection. ANALYTICAL METHODS : ADVANCING METHODS AND APPLICATIONS 2019; 11:1064-1072. [PMID: 31244918 PMCID: PMC6594695 DOI: 10.1039/c8ay02672f] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/11/2023]
Abstract
Developing in vitro cell culture models that accurately mimic in vivo processes in a manner that also enables near real-time analysis of neurotransmitters is an important research area. New technologies being developed such as 3D scaffolds for cell culture and 3D printed microfluidics provide an opportunity for such advancements. In this work, PC12 cells were used as a model system and they were immobilized onto a 3D scaffold of polystyrene (PS) fibers. These fibers were created by electrospinning onto PS sheets, which were laser cut and, after cell seeding, inserted into a 3D printed microfluidic device. The 3D printed device was designed with threads for connecting commercial fittings (to integrate automated pumps and a 4-port injection system) and a steel pin for simple coupling with PDMS/polystyrene analytical devices. A straight PDMS channel was used for simple (and continuous) flow-based detection by sealing onto a PS base containing an embedded gold array working electrode and a platinum pseudo-reference. Electrochemical detection of stimulated catecholamine release was demonstrated. The insert-based system was then integrated with a bilayer valving PDMS device (for microchip electrophoresis) sealed onto a PS base (with electrodes for electrochemical detection). This base was embedded with a Pd decoupler (for grounding the separation voltage and adsorbing hydrogen) and a 33 µm carbon fiber working electrode for in-channel detection. PC12 cells were stimulated in the 3D cell culture device, and the valving/electrophoresis microchip was able to separate and detect dopamine and norepinephrine release. This work demonstrates the ability to integrate 3D cell scaffolds with microchip-based analysis for detection of multiple analytes released from cells.
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21
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An electrochemical biosensor for sensitive detection of nicotine-induced dopamine secreted by PC12 cells. J Electroanal Chem (Lausanne) 2019. [DOI: 10.1016/j.jelechem.2018.10.018] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
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22
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Mehl BT, Martin RS. Enhanced Microchip Electrophoresis Separations Combined with Electrochemical Detection Utilizing a Capillary Embedded in Polystyrene. ANALYTICAL METHODS : ADVANCING METHODS AND APPLICATIONS 2018; 10:37-45. [PMID: 29707044 PMCID: PMC5915312 DOI: 10.1039/c7ay02505j] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/14/2023]
Abstract
The ability to use microchip-based electrophoresis for fast, high-throughput separations provides researchers with a tool for close-to real time analysis of biological systems. While PDMS-based electrophoresis devices are popular, the separation efficiency is often an issue due to the hydrophobic nature of PDMS. In this study, a hybrid microfluidic capillary device was fabricated to utilize the positive features of PDMS along with the electrophoretic performance of fused silica. A capillary loop was embedded in a polystyrene base that can be coupled with PDMS microchannels at minimal dead volume interconnects. A method for cleaning out the capillaries after a wet-polishing step was devised through the use of 3D printed syringe attachment. By comparing the separation efficiency of fluorescein and CBI-glycine with both a PDMS-based serpentine device and the embedded capillary loop device, it was shown that the embedded capillary loop device maintained higher theoretical plates for both analytes. A Pd decoupler with a carbon or Pt detection electrode were embedded along with the loop allowing integration of the electrophoretic separation with electrochemical detection. A series of catecholamines were separated to show the ability to resolve similar analytes and detect redox active species. The release of dopamine and norepinephrine from PC 12 cells was also analyzed showing the compatibility of these improved microchip separations with high ionic cell buffers associated with cell culture.
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23
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Ferapontova EE. Electrochemical Analysis of Dopamine: Perspectives of Specific In Vivo Detection. Electrochim Acta 2017. [DOI: 10.1016/j.electacta.2017.05.183] [Citation(s) in RCA: 52] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
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24
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Lovrić J, Dunevall J, Larsson A, Ren L, Andersson S, Meibom A, Malmberg P, Kurczy ME, Ewing AG. Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles. ACS NANO 2017; 11:3446-3455. [PMID: 27997789 DOI: 10.1021/acsnano.6b07233] [Citation(s) in RCA: 77] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/23/2023]
Abstract
We report an approach to spatially resolve the content across nanometer neuroendocrine vesicles in nerve-like cells by correlating super high-resolution mass spectrometry imaging, NanoSIMS, with transmission electron microscopy (TEM). Furthermore, intracellular electrochemical cytometry at nanotip electrodes is used to count the number of molecules in individual vesicles to compare to imaged amounts in vesicles. Correlation between the NanoSIMS and TEM provides nanometer resolution of the inner structure of these organelles. Moreover, correlation with electrochemical methods provides a means to quantify and relate vesicle neurotransmitter content and release, which is used to explain the slow transfer of dopamine between vesicular compartments. These nanoanalytical tools reveal that dopamine loading/unloading between vesicular compartments, dense core and halo solution, is a kinetically limited process. The combination of NanoSIMS and TEM has been used to show the distribution profile of newly synthesized dopamine across individual vesicles. Our findings suggest that the vesicle inner morphology might regulate the neurotransmitter release event during open and closed exocytosis from dense core vesicles with hours of equilibrium needed to move significant amounts of catecholamine from the protein dense core despite its nanometer size.
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Affiliation(s)
- Jelena Lovrić
- Department of Chemistry and Chemical Engineering, Chalmers University of Technology , Gothenburg SE-412 96, Sweden
- National Centre for Imaging Mass Spectrometry, Chalmers University of Technology and University of Gothenburg , Gothenburg SE-412 96, Sweden
| | - Johan Dunevall
- Department of Chemistry and Chemical Engineering, Chalmers University of Technology , Gothenburg SE-412 96, Sweden
| | - Anna Larsson
- Department of Chemistry and Molecular Biology, University of Gothenburg , Gothenburg SE-412 96, Sweden
| | - Lin Ren
- Department of Chemistry and Chemical Engineering, Chalmers University of Technology , Gothenburg SE-412 96, Sweden
| | - Shalini Andersson
- Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, AstraZeneca , Mölndal SE-431 50, Sweden
| | - Anders Meibom
- Laboratory for Biological Geochemistry, École Polytechnique Fédérale de Lausanne and Center for Advanced Surface Analysis, Institute of Earth Sciences, University of Lausanne , Lausanne CH-1015, Switzerland
| | - Per Malmberg
- Department of Chemistry and Chemical Engineering, Chalmers University of Technology , Gothenburg SE-412 96, Sweden
- National Centre for Imaging Mass Spectrometry, Chalmers University of Technology and University of Gothenburg , Gothenburg SE-412 96, Sweden
| | - Michael E Kurczy
- Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, AstraZeneca , Mölndal SE-431 50, Sweden
| | - Andrew G Ewing
- Department of Chemistry and Chemical Engineering, Chalmers University of Technology , Gothenburg SE-412 96, Sweden
- National Centre for Imaging Mass Spectrometry, Chalmers University of Technology and University of Gothenburg , Gothenburg SE-412 96, Sweden
- Department of Chemistry and Molecular Biology, University of Gothenburg , Gothenburg SE-412 96, Sweden
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25
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Moreira B, Tuoriniemi J, Kouchak Pour N, Mihalčíková L, Safina G. Surface Plasmon Resonance for Measuring Exocytosis from Populations of PC12 Cells: Mechanisms of Signal Formation and Assessment of Analytical Capabilities. Anal Chem 2017; 89:3069-3077. [DOI: 10.1021/acs.analchem.6b04811] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Affiliation(s)
- Beatriz Moreira
- Department
of Chemistry and Molecular Biology, University of Gothenburg, Kemigården
4, 412 96 Gothenburg, Sweden
| | - Jani Tuoriniemi
- Department
of Chemistry and Molecular Biology, University of Gothenburg, Kemigården
4, 412 96 Gothenburg, Sweden
| | - Naghmeh Kouchak Pour
- Department
of Chemistry and Molecular Biology, University of Gothenburg, Kemigården
4, 412 96 Gothenburg, Sweden
| | - Lýdia Mihalčíková
- Department
of Chemistry and Molecular Biology, University of Gothenburg, Kemigården
4, 412 96 Gothenburg, Sweden
| | - Gulnara Safina
- Department
of Chemistry and Molecular Biology, University of Gothenburg, Kemigården
4, 412 96 Gothenburg, Sweden
- Division
of Biological Physics, Department of Physics, Chalmers University of Technology, Kemigården 1, 412 96 Gothenburg, Sweden
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26
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Tian H, Yang FF, Liu CY, Liu XM, Pan RL, Chang Q, Zhang ZS, Liao YH. Effects of phenolic constituents of daylily flowers on corticosterone- and glutamate-treated PC12 cells. BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE 2017; 17:69. [PMID: 28109297 PMCID: PMC5251317 DOI: 10.1186/s12906-017-1582-x] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/16/2016] [Accepted: 01/13/2017] [Indexed: 01/30/2023]
Abstract
Background Daylily flowers, the flower and bud parts of Hemerocallis citrina or H. fulva, are well known as Wang-You-Cao in Chinese, meaning forget-one’s sadness plant. However, the major types of active constituents responsible for the neurological effects remain unclear. This study was to examine the protective effects of hydroalcoholic extract and fractions and to identify the active fractions. Methods The extract of daylily flowers was separated with AB-8 resin into different fractions containing non-phenolic compounds, phenolic acid derivatives and flavonoids as determined using UPLC-DAD chromatograms. The neuroprotective activity was measured by evaluating the cell viability and lactate dehydrogenase release using PC12 cell damage models induced by corticosterone and glutamate. The neurological mechanisms were explored by determining their effect on the levels of dopamine (DA), 5-hydroxy tryptamine (5-HT), γ-aminobutyric acid (GABA), noradrenaline (NE) and acetylcholine (ACh) in the cell culture medium measured using an LC-MS/MS method. Results Pretreatment of PC12 cells with the extract and phenolic fractions of daylily flowers at concentrations ranging from 0.63 to 5 mg raw material/mL significantly reversed corticosterone- and glutamate-induced neurotoxicity in a dose-dependent manner. The fractions containing phenolic acid derivatives (0.59% w/w in the flowers) and/or flavonoids (0.60% w/w) exerted similar dose-dependent neuroprotective effect whereas the fractions with non-phenolic compounds exhibited no activity. The presence of phenolic acid derivatives in the corticosterone- and glutamate-treated PC12 cells elevated the DA level in the cell culture medium whereas flavonoids resulted in increased ACH and 5-HT levels. Conclusion Phenolic acid derivatives and flavonoids were likely the active constituents of daylily flowers and they conferred a similar extent of neuroprotection, but affected the release of neurotransmitters in a different manner.
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Oleinick A, Svir I, Amatore C. 'Full fusion' is not ineluctable during vesicular exocytosis of neurotransmitters by endocrine cells. Proc Math Phys Eng Sci 2017; 473:20160684. [PMID: 28265193 PMCID: PMC5312129 DOI: 10.1098/rspa.2016.0684] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2016] [Accepted: 11/17/2016] [Indexed: 11/12/2022] Open
Abstract
Vesicular exocytosis is an essential and ubiquitous process in neurons and endocrine cells by which neurotransmitters are released in synaptic clefts or extracellular fluids. It involves the fusion of a vesicle loaded with chemical messengers with the cell membrane through a nanometric fusion pore. In endocrine cells, unless it closes after some flickering ('Kiss-and-Run' events), this initial pore is supposed to expand exponentially, leading to a full integration of the vesicle membrane into the cell membrane-a stage called 'full fusion'. We report here a compact analytical formulation that allows precise measurements of the fusion pore expansion extent and rate to be extracted from individual amperometric spike time courses. These data definitively establish that, during release of catecholamines, fusion pores enlarge at most to approximately one-fifth of the radius of their parent vesicle, hence ruling out the ineluctability of 'full fusion'.
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Affiliation(s)
| | | | - Christian Amatore
- Ecole Normale Supérieure-PSL Research University, Département de Chimie, Sorbonne Universités-UPMC Paris 06, CNRS UMR 8640 PASTEUR, 24 rue Lhomond, 75005 Paris, France
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28
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Najafinobar N, Mellander LJ, Kurczy ME, Dunevall J, Angerer TB, Fletcher JS, Cans AS. Cholesterol Alters the Dynamics of Release in Protein Independent Cell Models for Exocytosis. Sci Rep 2016; 6:33702. [PMID: 27650365 PMCID: PMC5030643 DOI: 10.1038/srep33702] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2015] [Accepted: 09/01/2016] [Indexed: 11/25/2022] Open
Abstract
Neurons communicate via an essential process called exocytosis. Cholesterol, an abundant lipid in both secretory vesicles and cell plasma membrane can affect this process. In this study, amperometric recordings of vesicular dopamine release from two different artificial cell models created from a giant unilamellar liposome and a bleb cell plasma membrane, show that with higher membrane cholesterol the kinetics for vesicular release are decelerated in a concentration dependent manner. This reduction in exocytotic speed was consistent for two observed modes of exocytosis, full and partial release. Partial release events, which only occurred in the bleb cell model due to the higher tension in the system, exhibited amperometric spikes with three distinct shapes. In addition to the classic transient, some spikes displayed a current ramp or plateau following the maximum peak current. These post spike features represent neurotransmitter release from a dilated pore before constriction and show that enhancing membrane rigidity via cholesterol adds resistance to a dilated pore to re-close. This implies that the cholesterol dependent biophysical properties of the membrane directly affect the exocytosis kinetics and that membrane tension along with membrane rigidity can influence the fusion pore dynamics and stabilization which is central to regulation of neurochemical release.
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Affiliation(s)
- Neda Najafinobar
- Department of Chemistry and Chemical Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
| | - Lisa J. Mellander
- Department of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, Sweden
| | - Michael E. Kurczy
- Department of Chemistry and Chemical Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
| | - Johan Dunevall
- Department of Chemistry and Chemical Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
| | - Tina B. Angerer
- Department of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, Sweden
| | - John S. Fletcher
- Department of Chemistry and Chemical Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
| | - Ann-Sofie Cans
- Department of Chemistry and Chemical Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
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29
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Sulzer D, Cragg SJ, Rice ME. Striatal dopamine neurotransmission: regulation of release and uptake. ACTA ACUST UNITED AC 2016; 6:123-148. [PMID: 27141430 DOI: 10.1016/j.baga.2016.02.001] [Citation(s) in RCA: 262] [Impact Index Per Article: 29.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Dopamine (DA) transmission is governed by processes that regulate release from axonal boutons in the forebrain and the somatodendritic compartment in midbrain, and by clearance by the DA transporter, diffusion, and extracellular metabolism. We review how axonal DA release is regulated by neuronal activity and by autoreceptors and heteroreceptors, and address how quantal release events are regulated in size and frequency. In brain regions densely innervated by DA axons, DA clearance is due predominantly to uptake by the DA transporter, whereas in cortex, midbrain, and other regions with relatively sparse DA inputs, the norepinephrine transporter and diffusion are involved. We discuss the role of DA uptake in restricting the sphere of influence of DA and in temporal accumulation of extracellular DA levels upon successive action potentials. The tonic discharge activity of DA neurons may be translated into a tonic extracellular DA level, whereas their bursting activity can generate discrete extracellular DA transients.
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Affiliation(s)
- David Sulzer
- Depts of Psychiatry, Neurology, & Pharmacology, NY State Psychiatric Institute, Columbia University, New York, NY, USA
| | - Stephanie J Cragg
- Dept Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
| | - Margaret E Rice
- Depts of Neurosurgery & Neuroscience and Physiology, New York University School of Medicine, New York, NY, USA
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30
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Lin HJ, Lu HH, Liu KM, Chau CM, Hsieh YZ, Li YK, Liau I. Toward live-cell imaging of dopamine neurotransmission with fluorescent neurotransmitter analogues. Chem Commun (Camb) 2016; 51:14080-3. [PMID: 26251847 DOI: 10.1039/c5cc03050a] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
We report a novel 'fluorescent dopamine' that possesses essential features of natural dopamine. Our method is simple and is readily extended to monoamine neurotransmitters such as L-norepinephrine, serotonin and GABA, providing a more practical approach. Because of its compatibility with sensitive fluorescent measurements, we envisage that our approach will have a broad range of applications in neural research.
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Affiliation(s)
- Hui-Jen Lin
- Department of Applied Chemistry and Institute of Molecular Science, National Chiao Tung University, Hsinchu 30010, Taiwan.
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31
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Li X, Hu H, Zhao S, Liu YM. Microfluidic Platform with In-Chip Electrophoresis Coupled to Mass Spectrometry for Monitoring Neurochemical Release from Nerve Cells. Anal Chem 2016; 88:5338-44. [PMID: 27111409 DOI: 10.1021/acs.analchem.6b00638] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
Chemical stimulus-induced neurotransmitter release from neuronal cells is well-documented. However, the dynamic changes in neurochemical release remain to be fully explored. In this work, a three-layered microfluidic chip was fabricated and evaluated for studying the dynamics of neurotransmitter release from PC-12 cells. The chip features integration of a nanoliter sized chamber for cell perfusion, pneumatic pressure valves for fluidic control, a microfluidic channel for electrophoretic separation, and a nanoelectrospray emitter for ionization in MS detection. Deploying this platform, a microchip electrophoresis-mass spectrometric method (MCE-MS) was developed to simultaneously quantify important neurotransmitters, including dopamine (DA), serotonin (5-HT), aspartic acid (Asp), and glutamic acid (Glu) without need for labeling or enrichment. Monitoring neurotransmitter release from PC-12 cells exposed to KCl (or alcohol) revealed that all four neurotransmitters investigated were released. Two release patterns were observed, one for the two monoamine neurotransmitters (i.e., DA and 5-HT) and another for the two amino acid neurotransmitters. Release dynamics for the two monoamine neurotransmitters was significantly different. The cells released DA most quickly and heavily in response to the stimulation. After exposure to the chemical stimulus for 4 min, the DA level in the perfusate from the cells was 86% lower than that at the beginning. Very interestingly, the cells started to release 5-HT in large quantities when they stopped releasing DA. These results suggest that DA and 5-HT are packaged into different vesicle pools and they are mobilized differently in response to chemical stimuli. The microfluidic platform proposed is proven useful for monitoring cellular release in biological studies.
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Affiliation(s)
- Xiangtang Li
- Department of Chemistry and Biochemistry, Jackson State University , 1400 Lynch Street, Jackson, Mississippi 39217, United States.,Wuhan Yaogu Bio-tech Company, Ltd. , Wuhan 430075, China
| | - Hankun Hu
- Wuhan Yaogu Bio-tech Company, Ltd. , Wuhan 430075, China.,Zhongnan Hospital, Wuhan University , Wuhan 430071, China
| | - Shulin Zhao
- College of Chemistry and Chemical Engineering, Guangxi Normal University , Guilin 51004, China
| | - Yi-Ming Liu
- Department of Chemistry and Biochemistry, Jackson State University , 1400 Lynch Street, Jackson, Mississippi 39217, United States.,Wuhan Yaogu Bio-tech Company, Ltd. , Wuhan 430075, China
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32
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Jonsson M, Brackmann C, Puchades M, Brattås K, Ewing A, Gatenholm P, Enejder A. Neuronal Networks on Nanocellulose Scaffolds. Tissue Eng Part C Methods 2015; 21:1162-70. [DOI: 10.1089/ten.tec.2014.0602] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Affiliation(s)
- Malin Jonsson
- Department of Biology and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden
| | - Christian Brackmann
- Department of Biology and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden
| | - Maja Puchades
- Department of Biology and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden
| | - Karoline Brattås
- Department of Biology and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden
| | - Andrew Ewing
- Department of Biology and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden
| | - Paul Gatenholm
- Department of Biology and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden
| | - Annika Enejder
- Department of Biology and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden
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33
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Wang J, Ewing AG. Simultaneous study of subcellular exocytosis with individually addressable multiple microelectrodes. Analyst 2015; 139:3290-5. [PMID: 24740449 DOI: 10.1039/c4an00058g] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
We report the application of individually addressable microelectrode arrays (MEAs) to study the heterogeneity of cell exocytosis at the subcellular level. Multiple subcellular-size electrodes are covered by a single PC12 cell for the investigation of subcellular exocytosis. PC12 cells have been seeded and cultured on top of three kinds of MEAs containing 16, 25, or 36 square microelectrodes (4 μm width in a 4 by 4 MEA, 3 μm width in a 5 by 5 MEA, 2 μm width in a 6 by 6 MEA). After collagen coating, single cells were found to cover several electrodes and these were selected for the study of subcellular exocytosis. Amperometric results show that single cell and subcellular heterogeneity in single cell exocytosis can be electrochemically detected with these MEAs. The results also show that these MEAs are suitable for detecting fast chemical events at single cells, as well as for developing multifunctional electrochemical sensors.
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Affiliation(s)
- Jun Wang
- Department of Chemistry and Molecular Biology, University of Gothenburg, Sweden
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34
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Abe H, Ino K, Li CZ, Kanno Y, Inoue KY, Suda A, Kunikata R, Matsudaira M, Takahashi Y, Shiku H, Matsue T. Electrochemical Imaging of Dopamine Release from Three-Dimensional-Cultured PC12 Cells Using Large-Scale Integration-Based Amperometric Sensors. Anal Chem 2015; 87:6364-70. [DOI: 10.1021/acs.analchem.5b01307] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Affiliation(s)
- Hiroya Abe
- Graduate School
of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8579, Japan
| | - Kosuke Ino
- Graduate School
of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8579, Japan
| | - Chen-Zhong Li
- Nanobioengineering/Nanobioelectronics Laboratory, Department
of Biomedical Engineering, Florida International University, 10555 West
Flagler Street, Miami, Florida 33174, United States
- WPI-Advanced Institute for Materials Research, Tohoku University, 2-1-1
Katahira, Aoba, Sendai 980-8577, Japan
| | - Yusuke Kanno
- Graduate School
of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8579, Japan
| | - Kumi Y. Inoue
- Graduate School
of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8579, Japan
| | - Atsushi Suda
- Japan Aviation Electronics Industry, Ltd. 1-1, Musashino 3-chome, Akishima-shi, Tokyo 196-8555, Japan
| | - Ryota Kunikata
- Japan Aviation Electronics Industry, Ltd. 1-1, Musashino 3-chome, Akishima-shi, Tokyo 196-8555, Japan
| | - Masahki Matsudaira
- Micro
System Integration Center, Tohoku University, 519-1176 Aramaki-aza Aoba, Aoba-ku, Sendai 980-0845, Japan
| | - Yasufumi Takahashi
- Graduate School
of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8579, Japan
- WPI-Advanced Institute for Materials Research, Tohoku University, 2-1-1
Katahira, Aoba, Sendai 980-8577, Japan
- PRESTO, JST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
| | - Hitoshi Shiku
- Graduate School
of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8579, Japan
| | - Tomokazu Matsue
- Graduate School
of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8579, Japan
- WPI-Advanced Institute for Materials Research, Tohoku University, 2-1-1
Katahira, Aoba, Sendai 980-8577, Japan
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35
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Liu Y, Li M, Zhang F, Zhu A, Shi G. Development of Au Disk Nanoelectrode Down to 3 nm in Radius for Detection of Dopamine Release from a Single Cell. Anal Chem 2015; 87:5531-8. [PMID: 25940227 DOI: 10.1021/ac5042999] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
A Au disk nanoelectrode down to 3 nm in radius was developed by a facile and reliable method and successfully applied for monitoring dopamine release from single living vesicles. A fine etched Au wire was coated with cathodic electrophoretic paint followed by polyimide, which retracted from the tip end during curing to expose the Au nanotip. By cyclic voltammetric scanning the above tip in 0.5 M KCl, the transformation of a core-shaped apex into a geometrically well-defined Au disk nanoelectrode with different dimensions can be controllably and reproducibly achieved. Scanning electron microscopy, transmission electron microscopy, and steady-state voltammetry were used to determine the size of nanoelectrodes. The results showed that the specific etching and insulation method not only avoids the use of toxic etching solution and the uncontrollable treatment to expose the tip but also makes possible the controllable and reproducible fabrication of Au disk nanoelectrode down to 3 nm in radius. The nanoelectrodes with well-demonstrated analytical performance were further applied for amperometrically monitoring dopamine release from single rat pheochromacytoma cells with high spatial resolution.
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Affiliation(s)
- Yingzi Liu
- School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai 200241, P.R. China
| | - Meina Li
- School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai 200241, P.R. China
| | - Fan Zhang
- School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai 200241, P.R. China
| | - Anwei Zhu
- School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai 200241, P.R. China
| | - Guoyue Shi
- School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai 200241, P.R. China
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36
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Modification of carbon paste electrode with cucurbit[8]uril and its recognition to phenols. J INCL PHENOM MACRO 2015. [DOI: 10.1007/s10847-015-0480-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
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37
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Johnson AS, Mehl BT, Martin RS. Integrated hybrid polystyrene-polydimethylsiloxane device for monitoring cellular release with microchip electrophoresis and electrochemical detection. ANALYTICAL METHODS : ADVANCING METHODS AND APPLICATIONS 2015; 7:884-893. [PMID: 25663849 PMCID: PMC4318258 DOI: 10.1039/c4ay02569e] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/06/2023]
Abstract
In this work, a polystyrene (PS)-polydimethylsiloxane (PDMS) hybrid device was developed to enable the integration of cell culture with analysis by microchip electrophoresis and electrochemical detection. It is shown that this approach combines the fundamental advantages of PDMS devices (the ability to integrate pumps and valves) and PS devices (the ability to permanently embed fluidic tubing and electrodes). The embedded fused-silica capillary enables high temporal resolution measurements from off-chip cell culture dishes and the embedded electrodes provide close to real-time analysis of small molecule neurotransmitters. A novel surface treatment for improved (reversible) adhesion between PS and PDMS is described using a chlorotrimethylsilane stamping method. It is demonstrated that a Pd decoupler is efficient at handling the high current (and cathodic hydrogen production) resulting from use of high ionic strength buffers needed for cellular analysis; thus allowing an electrophoretic separation and in-channel detection. The separation of norepinephrine (NE) and dopamine (DA) in highly conductive biological buffers was optimized using a mixed surfactant system. This PS-PDMS hybrid device integrates multiple processes including continuous sampling from a cell culture dish, on-chip pump and valving technologies, microchip electrophoresis, and electrochemical detection to monitor neurotransmitter release from PC 12 cells.
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Affiliation(s)
- Alicia S Johnson
- Saint Louis University, Department of Chemistry, 3501 Laclede Avenue, St. Louis, MO 63103
| | - Benjamin T Mehl
- Saint Louis University, Department of Chemistry, 3501 Laclede Avenue, St. Louis, MO 63103
| | - R Scott Martin
- Saint Louis University, Department of Chemistry, 3501 Laclede Avenue, St. Louis, MO 63103
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38
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INO K. Microchemistry- and MEMS-based Integrated Electrochemical Devices for Bioassay Applications. ELECTROCHEMISTRY 2015. [DOI: 10.5796/electrochemistry.83.688] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Affiliation(s)
- Kosuke INO
- Graduate School of Environmental Studies, Tohoku University
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39
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Hwang SH, Lee BH, Choi SH, Kim HJ, Jung SW, Kim HS, Shin HC, Park HJ, Park KH, Lee MK, Nah SY. Gintonin, a novel ginseng-derived lysophosphatidic acid receptor ligand, stimulates neurotransmitter release. Neurosci Lett 2014; 584:356-61. [PMID: 25445364 DOI: 10.1016/j.neulet.2014.11.007] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2014] [Revised: 10/30/2014] [Accepted: 11/06/2014] [Indexed: 10/24/2022]
Abstract
Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. Gintonin elicits an intracellular calcium concentration [Ca(2+)]i transient via activation of LPA receptors and regulates calcium-dependent ion channels and receptors. [Ca(2+)]i elevation by neurotransmitters or depolarization is usually coupled to neurotransmitter release in neuronal cells. Little is known about whether gintonin-mediated [Ca(2+)]i transients are also coupled to neurotransmitter release. The PC12 cell line is derived from a pheochromocytoma of the rat adrenal medulla and is widely used as a model for catecholamine release. In the present study, we examined the effects of gintonin on dopamine release in PC12 cells. Application of gintonin to PC12 cells induced [Ca(2+)]i transients in concentration-dependent and reversible manners. However, ginsenoside Rg3, another active ingredient of ginseng, induced a lagged and irreversible [Ca(2+)]i increase. The induction of gintonin-mediated [Ca(2+)]i transients was attenuated or blocked by the LPA1/3 receptor antagonist Ki16425, a phospholipase C inhibitor, an inositol 1,4,5-triphosphate receptor antagonist, and an intracellular Ca(2+) chelator. Repeated treatment with gintonin induced homologous desensitization of [Ca(2+)]i transients. Gintonin treatment in PC12 cells increased the release of dopamine in a concentration-dependent manner. Intraperitoneal administration of gintonin to mice also increased serum dopamine concentrations. The present study shows that gintonin-mediated [Ca(2+)]i transients are coupled to dopamine release via LPA receptor activation. Finally, gintonin-mediated [Ca(2+)]i transients and dopamine release via LPA receptor activation might explain one mechanism of gintonin-mediated inter-neuronal modulation in the nervous system.
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Affiliation(s)
- Sung-Hee Hwang
- Department of Pharmaceutical Engineering, College of Health Sciences, Sangji University, Wonju 220-702, South Korea
| | - Byung-Hwan Lee
- Department of Physiology, College of Veterinary Medicine and Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, South Korea
| | - Sun-Hye Choi
- Department of Physiology, College of Veterinary Medicine and Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, South Korea
| | - Hyeon-Joong Kim
- Department of Physiology, College of Veterinary Medicine and Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, South Korea
| | - Seok-Won Jung
- Department of Physiology, College of Veterinary Medicine and Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, South Korea
| | - Hyun-Sook Kim
- Department of Physiology, College of Veterinary Medicine and Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, South Korea
| | - Ho-Chul Shin
- Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, Seoul 143‑701, South Korea
| | - Hyun Jin Park
- College of Pharmacy and Research Center for Bioresource and Health, Chungbuk National University, Cheongju 361-763, South Korea
| | - Keun Hong Park
- College of Pharmacy and Research Center for Bioresource and Health, Chungbuk National University, Cheongju 361-763, South Korea
| | - Myung Koo Lee
- College of Pharmacy and Research Center for Bioresource and Health, Chungbuk National University, Cheongju 361-763, South Korea
| | - Seung-Yeol Nah
- Department of Physiology, College of Veterinary Medicine and Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, South Korea.
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40
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Ribeiro PRDS, Duarte RM. Development and validation of a simple spectrophotometric method for the determination of methyldopa in both bulk and marketed dosage formulations. BRAZ J PHARM SCI 2014. [DOI: 10.1590/s1984-82502014000300017] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
A simple, precise, sensitive, rapid, specific and economical spectrophotometric method was developed to determine methyldopa (MTD) content in bulk and pharmaceutical dosage formulations. The proposed method was based on the formation of a colored product from the nitrosation reaction of MTD with sodium nitrite in an acid medium. The resultant nitroso derivative species reacts further with sodium hydroxide and is converted it into a more stable compound. This yellow nitrosation product exhibited an absorption maximum at 430 nm. Beer's Law was obeyed in a concentration range of 6.37 to 82.81 μg mL-1 MTD with an excellent coefficient of determination (R2 = 0.9998). No interference was observed from common excipients in formulations. The results showed the method to be simple, accurate and readily applied for the determination of MTD in pure form and in pharmaceutical preparations. The analytical results obtained for these products using the proposed method are in agreement with those of the Brazilian Pharmacopoeia procedure at a 95% confidence level.
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41
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Wang J, Trouillon R, Dunevall J, Ewing AG. Spatial resolution of single-cell exocytosis by microwell-based individually addressable thin film ultramicroelectrode arrays. Anal Chem 2014; 86:4515-20. [PMID: 24712854 PMCID: PMC4014142 DOI: 10.1021/ac500443q] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2014] [Accepted: 04/08/2014] [Indexed: 01/24/2023]
Abstract
We report the fabrication and characterization of microwell-based individually addressable microelectrode arrays (MEAs) and their application to spatially and temporally resolved detection of neurotransmitter release across a single pheochromocytoma (PC12) cell. The microwell-based MEAs consist of 16 4-μm-width square ultramicroelectrodes, 25 3-μm-width square ultramicroelectrodes, or 36 2-μm-width square ultramicroelectrodes, all inside a 40 × 40 μm square SU-8 microwell. MEAs were fabricated on glass substrates by photolithography, thin film deposition, and reactive ion etching. The ultramicroelectrodes in each MEA are tightly defined in a 30 × 30 μm square area, which is further encased inside the SU-8 microwell. With this method, we demonstrate that these microelectrodes are stable, reproducible, and demonstrate good electrochemical properties using cyclic voltammetry. Effective targeting and culture of a single cell is achieved by combining cell-sized microwell trapping and cell-picking micropipet techniques. The surface of the microelectrodes in the MEA was coated with collagen IV to promote cell adhesion and further single-cell culture, as good adhesion between the cell membrane and the electrode surface is critical for the quality of the measurements. Imaging the spatial distribution of exocytosis at the surface of a single PC12 cell has also been demonstrated with this system. Exocytotic signals have been successfully recorded from eight independent 2-μm-wide ultramicroelectrodes from a single PC12 cell showing that the subcellular heterogeneity in single-cell exocytosis can be precisely analyzed with these microwell-based MEAs.
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Affiliation(s)
- Jun Wang
- Department
of Chemistry and Molecular Biology, University
of Gothenburg, Kemivägen
10, 41296 Gothenburg, Sweden
- Department
of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 41296 Gothenburg, Sweden
| | - Raphaël Trouillon
- Department
of Chemistry and Molecular Biology, University
of Gothenburg, Kemivägen
10, 41296 Gothenburg, Sweden
| | - Johan Dunevall
- Department
of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 41296 Gothenburg, Sweden
| | - Andrew G. Ewing
- Department
of Chemistry and Molecular Biology, University
of Gothenburg, Kemivägen
10, 41296 Gothenburg, Sweden
- Department
of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 41296 Gothenburg, Sweden
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42
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Takahashi K, Yokota M, Ohta T. Molecular mechanism of 2-APB-induced Ca2+ influx in external acidification in PC12. Exp Cell Res 2014; 323:337-45. [DOI: 10.1016/j.yexcr.2014.03.001] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2014] [Revised: 02/27/2014] [Accepted: 03/01/2014] [Indexed: 02/02/2023]
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43
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Sun M, Kaplan SV, Gehringer RC, Limbocker RA, Johnson MA. Localized drug application and sub-second voltammetric dopamine release measurements in a brain slice perfusion device. Anal Chem 2014; 86:4151-6. [PMID: 24734992 PMCID: PMC4018083 DOI: 10.1021/ac5008927] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
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The
use of fast scan cyclic voltammetry (FSCV) to measure the release
and uptake of dopamine (DA) as well as other biogenic molecules in
viable brain tissue slices has gained popularity over the last 2 decades.
Brain slices have the advantage of maintaining the functional three-dimensional
architecture of the neuronal network while also allowing researchers
to obtain multiple sets of measurements from a single animal. In this
work, we describe a simple, easy-to-fabricate perfusion device designed
to focally deliver pharmacological agents to brain slices. The device
incorporates a microfluidic channel that runs under the perfusion
bath and a microcapillary that supplies fluid from this channel up
to the slice. We measured electrically evoked DA release in brain
slices before and after the administration of two dopaminergic stimulants,
cocaine and GBR-12909. Measurements were collected at two locations,
one directly over and the other 500 μm away from the capillary
opening. Using this approach, the controlled delivery of drugs to
a confined region of the brain slice and the application of this chamber
to FSCV measurements, were demonstrated. Moreover, the consumption
of drugs was reduced to tens of microliters, which is thousands of
times less than traditional perfusion methods. We expect that this
simply fabricated device will be useful in providing spatially resolved
delivery of drugs with minimum consumption for voltammetric and electrophysiological
studies of a variety of biological tissues both in vitro and ex vivo.
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Affiliation(s)
- Meng Sun
- Department of Chemistry and R. N. Adams Institute for Bioanalytical Chemistry, University of Kansas , Lawrence, Kansas 66045 United States
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Mellander LJ, Kurczy ME, Najafinobar N, Dunevall J, Ewing AG, Cans AS. Two modes of exocytosis in an artificial cell. Sci Rep 2014; 4:3847. [PMID: 24457949 PMCID: PMC3900996 DOI: 10.1038/srep03847] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2013] [Accepted: 12/31/2013] [Indexed: 01/24/2023] Open
Abstract
The details of exocytosis, the vital cell process of neuronal communication, are still under debate with two generally accepted scenarios. The first mode of release involves secretory vesicles distending into the cell membrane to release the complete vesicle contents. The second involves partial release of the vesicle content through an intermittent fusion pore, or an opened or partially distended fusion pore. Here we show that both full and partial release can be mimicked with a single large-scale cell model for exocytosis composed of material from blebbing cell plasma membrane. The apparent switching mechanism for determining the mode of release is demonstrated to be related to membrane tension that can be differentially induced during artificial exocytosis. These results suggest that the partial distension mode might correspond to an extended kiss-and-run mechanism of release from secretory cells, which has been proposed as a major pathway of exocytosis in neurons and neuroendocrine cells.
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Affiliation(s)
- Lisa J Mellander
- University of Gothenburg, Department of Chemistry and Molecular Biology, 412 96 Gothenburg, Sweden
| | - Michael E Kurczy
- Chalmers University of Technology, Department of Chemical and Biological Engineering, 412 96 Gothenburg, Sweden
| | - Neda Najafinobar
- Chalmers University of Technology, Department of Chemical and Biological Engineering, 412 96 Gothenburg, Sweden
| | - Johan Dunevall
- Chalmers University of Technology, Department of Chemical and Biological Engineering, 412 96 Gothenburg, Sweden
| | - Andrew G Ewing
- 1] University of Gothenburg, Department of Chemistry and Molecular Biology, 412 96 Gothenburg, Sweden [2] Chalmers University of Technology, Department of Chemical and Biological Engineering, 412 96 Gothenburg, Sweden
| | - Ann-Sofie Cans
- Chalmers University of Technology, Department of Chemical and Biological Engineering, 412 96 Gothenburg, Sweden
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45
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The real catecholamine content of secretory vesicles in the CNS revealed by electrochemical cytometry. Sci Rep 2013; 3:1447. [PMID: 23486177 PMCID: PMC3596796 DOI: 10.1038/srep01447] [Citation(s) in RCA: 71] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2013] [Accepted: 02/28/2013] [Indexed: 11/12/2022] Open
Abstract
Resolution of synaptic vesicle neurotransmitter content has mostly been limited to the study of stimulated release in cultured cell systems, and it has been controversial as to whether synaptic vesicle transmitter levels are saturated in vivo. We use electrochemical cytometry to count dopamine molecules in individual synaptic vesicles in populations directly sampled from brain tissue. Vesicles from the striatum yield an average of 33,000 dopamine molecules per vesicle, an amount considerably greater than typically measured during quantal release at cultured neurons. Vesicular content was markedly increased by L-DOPA or decreased by reserpine in a time-dependent manner in response to in vivo administration of drugs known to alter dopamine release. We investigated the effects of the psychostimulant amphetamine on vesicle content, finding that vesicular transmitter is rapidly depleted by 50% following in vivo administration, supporting the “weak base hypothesis” that amphetamine reduces synaptic vesicle transmitter and quantal size.
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46
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Trouillon R, Lin Y, Mellander LJ, Keighron JD, Ewing AG. Evaluating the diffusion coefficient of dopamine at the cell surface during amperometric detection: disk vs ring microelectrodes. Anal Chem 2013; 85:6421-8. [PMID: 23706095 PMCID: PMC3737586 DOI: 10.1021/ac400965d] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
During exocytosis, small quantities of neurotransmitters are released by the cell. These neurotransmitters can be detected quantitatively using electrochemical methods, principally with disk carbon fiber microelectrode amperometry. An exocytotic event then results in the recording of a current peak whose characteristic features are directly related to the mechanisms of exocytosis. We have compared two exocytotic peak populations obtained from PC12 cells with a disk carbon fiber microelectrode and with a pyrolyzed carbon ring microelectrode array, with a 500 nm ring thickness. The specific shape of the ring electrode allows for precise analysis of diffusion processes at the vicinity of the cell membrane. Peaks obtained with a ring microelectrode array show a distorted average shape, owing to increased diffusion pathways. This result has been used to evaluate the diffusion coefficient of dopamine at the surface of a cell, which is up to an order of magnitude smaller than that measured in free buffer. The lower rate of diffusion is discussed as resulting from interactions with the glycocalyx.
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Affiliation(s)
- Raphaël Trouillon
- Department of Chemistry and Molecular Biology, University of Gothenburg, S-41296, Gothenburg, Sweden
| | - Yuqing Lin
- Department of Chemistry and Molecular Biology, University of Gothenburg, S-41296, Gothenburg, Sweden
| | - Lisa J. Mellander
- Department of Chemistry and Molecular Biology, University of Gothenburg, S-41296, Gothenburg, Sweden
| | - Jacqueline D. Keighron
- Department of Chemical and Biological Engineering, Chalmers University of Technology, S-41296 Gothenburg, Sweden
| | - Andrew G. Ewing
- Department of Chemistry and Molecular Biology, University of Gothenburg, S-41296, Gothenburg, Sweden
- Department of Chemical and Biological Engineering, Chalmers University of Technology, S-41296 Gothenburg, Sweden
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47
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Tsai HY, Shih ZY, Lin ZH, Chang HT. Te/Pt nanonetwork modified carbon fiber microelectrodes for methanol oxidation. NANOTECHNOLOGY 2013; 24:195402. [PMID: 23579734 DOI: 10.1088/0957-4484/24/19/195402] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/02/2023]
Abstract
Te/Pt nanonetwork-decorated carbon fiber microelectrodes (CFMEs) have been fabricated and employed as anodic catalysts in a direct methanol fuel cell (DMFC). Te nanowires were prepared from tellurite ions (TeO3(2-)) through a seed-mediated growth process and were deposited onto CFMEs to form three-dimensional Te nanonetworks. The Te nanonetworks then acted as a framework and reducing agent to reduce PtCl6(2-) ions to form Te/Pt through a galvanic replacement reaction, leading to the formation of Te/PtCFMEs. By controlling the reaction time, the amount of Pt and morphology of Te/Pt nanonetworks were controlled, leading to various degrees of electrocatalytic activity. The Te/PtCFMEs provide a high electrochemical active surface area (129.2 m(2) g(-1)), good catalytic activity (1.2 A mg(-1)), high current density (20.0 mA cm(-2)), long durability, and tolerance toward the poisoning species for methanol oxidation in 0.5 M sulfuric acid containing 1 M methanol. We have further demonstrated an enhanced current density by separately using 3 and 5 Te/PtCFMEs. Our results show that the low-cost, stable, and effective Te/PtCFMEs have great potential in the fabrication of cost-effective fuel cells.
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Affiliation(s)
- Hsiang-Yu Tsai
- Department of Chemistry, National Taiwan University, 1 Section 4 Roosevelt Road, Taipei 106, Taiwan
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48
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Wang J, Trouillon R, Lin Y, Svensson MI, Ewing AG. Individually addressable thin-film ultramicroelectrode array for spatial measurements of single vesicle release. Anal Chem 2013; 85:5600-8. [PMID: 23627439 DOI: 10.1021/ac4009385] [Citation(s) in RCA: 73] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Thin-film platinum ultramicroelectrode arrays (MEAs) with subcellular microelectrodes were developed for the spatial measurement of neurotransmitter release across single cells or clusters of single cells. MEAs consisting of 16, 25, and 36 square ultramicroelectrodes with respective widths of 4, 3, and 2 μm were fabricated on glass substrates by photolithography, thin-film deposition, and reactive ion etching. The electrodes in each MEA are tightly defined in a 30 μm × 30 μm square, which is potentially useful to measure exocytosis across a single cell or clusters of single cells. These MEAs have been characterized with scanning electron microscopy and cyclic voltammetry and show excellent stability and reproducibility. Culturing PC12 cells on top of the MEAs has been achieved by modifying the array with a poly(dimethylsiloxane) chamber and coating a thin layer of collagen IV on top of the electrode surface. The electrochemical response to dopamine has been characterized after coating the surface with the cell-adhering molecules and then with cells attached. Amperometric detection demonstrates that individual exocytotic events can be recorded at these arrays with spatial resolution for dynamic electrochemical measurements near 2 μm. In contrast to previous single-cell experiments, the effect of dopaminergic drugs on imaging single vesicle exocytotic release from PC12 cell clusters is presented at cell clusters incubated with the dopamine precursor and Parkinson's therapy agent, L-3,4-dihydroxyphenylalanine, and at cell clusters incubated with the vesicular monoamine transport inhibitor, reserpine. The results of electrochemical imaging demonstrate that the drug effect on PC12 cell clusters is consistent with previous single-cell experiments.
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Affiliation(s)
- Jun Wang
- Department of Chemistry and Molecular Biology, University of Gothenburg, Göteborg, Sweden
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49
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Taskin MB, Sasso L, Dimaki M, Svendsen WE, Castillo-León J. Combined cell culture-biosensing platform using vertically aligned patterned peptide nanofibers for cellular studies. ACS APPLIED MATERIALS & INTERFACES 2013; 5:3323-8. [PMID: 23537161 DOI: 10.1021/am400390g] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/19/2023]
Abstract
This Article presents the development of a combined cell culture-biosensing platform using vertically aligned self-assembled peptide nanofibers. Peptide nanofibers were patterned on a microchip containing gold microelectrodes to provide the cells with a 3D environment enabling them to grow and proliferate. Gold microelectrodes were functionalized with conductive polymers for the electrochemical detection of dopamine released from PC12 cells. The combined cell culture-biosensing platform assured a close proximity of the release site, the cells and the active surface of the sensor, thereby rendering it possible to avoid a loss of sensitivity because of the diffusion of the sample. The obtained results showed that the peptide nanofibers were suitable as a cell culturing substrate for PC12 cells. The peptide nanofibers could be employed as an alternative biological material to increase the adherence properties of PC12 cells. Dopamine was amperometrically detected at a value of 168 fmole.
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Affiliation(s)
- Mehmet B Taskin
- Department of Micro- and Nanotechnology, Technical University of Denmark, Ørsted Plads 345B. 2800 Kgs. Lyngby, Denmark
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50
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Farjami E, Campos R, Nielsen JS, Gothelf KV, Kjems J, Ferapontova EE. RNA aptamer-based electrochemical biosensor for selective and label-free analysis of dopamine. Anal Chem 2012; 85:121-8. [PMID: 23210972 DOI: 10.1021/ac302134s] [Citation(s) in RCA: 139] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
The inherent redox activity of dopamine enables its direct electrochemical in vivo analysis ( Venton , B. J.; Wightman, M. R. Anal. Chem. 2003, 75, 414A). However, dopamine analysis is complicated by the interference from other electrochemically active endogenous compounds present in the brain, including dopamine precursors and metabolites and other neurotransmitters (NT). Here we report an electrochemical RNA aptamer-based biosensor for analysis of dopamine in the presence of other NT. The biosensor exploits a specific binding of dopamine by the RNA aptamer, immobilized at a cysteamine-modified Au electrode, and further electrochemical oxidation of dopamine. Specific recognition of dopamine by the aptamer allowed a selective amperometric detection of dopamine within the physiologically relevant 100 nM to 5 μM range in the presence of competitive concentrations of catechol, epinephrine, norepinephrine, 3,4-dihydroxy-phenylalanine (L-DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), methyldopamine, and tyramine, which gave negligible signals under conditions of experiments (electroanalysis at 0.185 V vs Ag/AgCl). The interference from ascorbic and uric acids was eliminated by application of a Nafion-coated membrane. The aptasensor response time was <1 s, and the sensitivity of analysis was 62 nA μM(-1) cm(-2). The proposed design of the aptasensor, based on electrostatic interactions between the positively charged cysteamine-modified electrode and the negatively charged aptamer, may be used as a general strategy not to restrict the conformational freedom and binding properties of surface-bound aptamers and, thus, be applicable for the development of other aptasensors.
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Affiliation(s)
- Elaheh Farjami
- Interdisciplinary Nanoscience Center, Aarhus University, Denmark
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