Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Summary

Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H₂O₂ in the kidney.

Abstract

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made  possible the measuring of cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H₂O₂ signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor are coated with a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H₂O₂. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H₂O₂ on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the in vivo studies are instead based on the reduction of H₂O₂ on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H₂O₂ and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H₂O₂ in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs.

Introduction

Enzymatic microelectrode biosensors (also referenced as sensors in the present manuscript) have been a valuable tool for studying dynamic signaling processes in living cells and tissues. The sensors provide increased temporal and spatial resolution of cell signaling molecules in biologically relevant concentrations. Instead of sampling and analyzing extracellular fluids taken at intervals over long periods of time, these sensors respond as fast as their enzymes react to the analyte, thereby producing real-time measurements^1,2. Fast detection of interstitial concentrations of autocrine and paracrine factors, like purines or hydrogen peroxide, and the dynamic....

Protocol

The following animal procedures  adhered to the NIH Guide for the Care and Use of Laboratory Animals. Prior approval was obtained from the Institutional Animal Care and Use Committee (IACUC).
NOTE: Review of the sensor manufacturer instructions should be done during the experiment design and prior to their use. Following these instructions will produce optimal results when using the sensors.

1. Sensor Calibration

1. Prepare fresh stock solutions prior the start of the experiment.
2. Create Buffer A containing 10 mM NaPi buffer, 100 mM NaCl, 1 mM MgCl₂, and 2 mM glycerol. Adjust the pH to ....

Results

The design of the enzymatic microelectrode biosensor allows the real-time detection of analytes in whole kidneys. The general experiment design for either ex vivo or in vivo studies is illustrated in Figure 1.The sensors used and the surgical procedures differ depending on whether the study is ex vivo or in vivo.

To obtain reproducible results, accurate pre- and post- calibrations are critical. Figure 6A shows a rep.......

Discussion

The present protocols were developed to provide enhanced temporal and spatial resolution of ATP and H₂O₂ signaling for ex vivo isolated, perfused and in vivo blood-perfused kidneys. The differences between the protocols and the sensors used here provide optimal data acquisition for either pharmacological agents or physiological studies. The protocols consist of 1) sensor calibration, 2) surgical procedure, 3) data acquisition setup, and 4) data analysis. They enable the real-time m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sensor Kit	Sarissa Biomedical	SBK-ATP-05-125	The kit includes storage bottle, rehydration chamber, electrode leads, and reference electrodes.  Also included with the kit is the user's choice of sensors.
Sarissaprobe ATP Biosensor 125 μm	Sarissa Biomedical	SBS-ATP-05-125	store at 2-8 oC before use
Sarissagold ATP Biosensor 50 μm	Sarissa Biomedical	SGS-ATP-10-50	store at 2-8 oC before use
Sarissaprobe null sensor 125 μm	Sarissa Biomedical	SBS-NUL-20-125	store at 2-8 oC before use
Sarissagold null sensor 50 μm	Sarissa Biomedical	SGS-NUL-10-50	store at 2-8 oC before use
Sarissaprobe ATP Manual	Sarissa Biomedical		http://www.sarissa-biomedical.com/media/31563/instructions-atp.pdf
Faraday cage	TMC
Dual channel potentiostat	Digi-Ivy	DY2021	Type II Faraday cage
Data acquisition program	Digi-Ivy	DY2000
Perfusion pump	Razel Scientific Instruments	Model R99E
Fiber optic illuminator	Schott	ACE 1
micromanipulator	Narishige	MM-3
micromanipulator magnetic stand	Narishige	GJ-8
air table	TMC	63-500
isoflurane ventilator	LEI Medical	M2000
3 ml petri dish	Fisher Scientific	S3358OA
needle	Santa Cruz		26-30 G
pins			Standard dissection pins
catheter			Polyethylene tubing (PE50)
catheter tissue glue	Vetbond	1469SB
suture	Look	SP117
rubber bands			any 2-4 mm wide rubber bands
silicone	Momentive	RTV-615 Clear 1#
clamp	Fine Science Tools	18052-03
standard dissection kit			Kit should include scalpel and dissection sissors
Kidney Cup			Of own design
standard chemicals	Sigma-Aldrich
ATP	Sigma-Aldrich	A6559-25UMO	100 mM ATP solution
hydrogen peroxide	Sigma-Aldrich	216763
glycerol	Sigma-Aldrich	G9012
Apyrase	Sigma-Aldrich	A7646
Catalase	Sigma-Aldrich	C40
isoflurane	Clipper	10250
inactin	Sigma-Aldrich	T133
ketamine	Clipper	2010012
Hanks Balanced Salt Solution	Gibco	14025092