CMOS-Based Redox-Type Label-Free ATP Image Sensor for In Vitro Sensitive Imaging of Extracellular ATP.

Hideo Doi,Kazuhiro Takahashi,Yong-Joon Choi,Toshihiko Noda,Kazuaki Sawada,Tomoko Horio

doi:10.3390/s22010075

Hideo Doi, Kazuhiro Takahashi + Show 4 more

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https://doi.org/10.3390/s22010075

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Abstract

Adenosine 5′-triphosphate (ATP) plays a crucial role as an extracellular signaling molecule in the central nervous system and is closely related to various nerve diseases. Therefore, label-free imaging of extracellular ATP dynamics and spatiotemporal analysis is crucial for understanding brain function. To decrease the limit of detection (LOD) of imaging extracellular ATP, we fabricated a redox-type label-free ATP image sensor by immobilizing glycerol-kinase (GK), L-α-glycerophosphate oxidase (LGOx), and horseradish peroxidase (HRP) enzymes in a polymer film on a gold electrode-modified potentiometric sensor array with a 37.3 µm-pitch. Hydrogen peroxide (H2O2) is generated through the enzymatic reactions from GK to LGOx in the presence of ATP and glycerol, and ATP can be detected as changes in its concentration using an electron mediator. Using this approach, the LOD for ATP was 2.8 µM with a sensitivity of 77 ± 3.8 mV/dec., under 10 mM working buffers at physiological pH, such as in in vitro experiments, and the LOD was great superior 100 times than that of the hydrogen ion detection-based image sensor. This redox-type ATP image sensor may be successfully applied for in vitro sensitive imaging of extracellular ATP dynamics in brain nerve tissue or cells.

Highlights

Adenosine 50 -triphosphate (ATP) is a well-known intracellular energy currency in all living cells and is necessary for many biological processes
Adenosine-50 -triphosphate (98%), adenosine diphosphate (ADP) (97%), adenosine monophosphate (AMP) (99.3%), adenosine (Ado) (98%), L-glutamic acid sodium (99%), sodium chloride (99.5%), potassium chloride (99.5%), calcium chloride (95%), magnesium chloride, D-glucose (98%), potassium hexacyanoferrate (III) [K4 Fe(CN)6 ] (99%), potassium hexacyanoferrate (II) [K3 Fe(CN)6 ] (99.5%), glycerol
LGO, and horseradish peroxidase (HRP) at the surface of the gold electrode deposited on the potentiometric sensor and at the surface of the gold electrode deposited on the potentiometric sensor arrays having a spatial resolution of 37.3 μm, and the detection performance for Adenosine -triphosphate (ATP) sensing having a spatial resolution μm, and the detection performance for ATP sensing was investigated

Summary

Introduction

Adenosine 50 -triphosphate (ATP) is a well-known intracellular energy currency in all living cells and is necessary for many biological processes. ATP plays a key role in the regulation of synaptic transmission as an extracellular signaling molecule from neurons and glial cells (non-neuronal types in the brain) in the central nervous system [1,2,3]. ATP levels in living tissues, including the brain, have usually been measured by luminescence [5] or high-performance liquid chromatography (HPLC) [6] using microdialysis [7] samples. These analysis systems are very sensitive to ATP, they have a low temporal resolution, ranging from a few tens of seconds to several minutes, due to analytical samples being collected using the dialysis probe and no spatial resolution. Various electrochemical ATP biosensors with enzymes immobilized on Pt disk [8], glassy carbon [9], Clark-type oxygen [10], and interdigitated gold [11]

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