An Aptamer-based Sensor for Unchelated Gadolinium(III)

Summary

The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor.

Abstract

A method for determining the presence of unchelated trivalent gadolinium ion (Gd³⁺) in aqueous solution is demonstrated. Gd³⁺ is often present in samples of gadolinium-based contrast agents as a result of incomplete reactions between the ligand and the ion, or as a dissociation product. Since the ion is toxic, its detection is of critical importance. Herein, the design and usage of an aptamer-based sensor (Gd-sensor) for Gd³⁺ are described. The sensor produces a fluorescence change in response to increasing concentrations of the ion, and has a limit of detection in the nanomolar range (~100 nM with a signal-to-noise ratio of 3). The assay may be run in an aqueous buffer at ambient pH (~7 - 7.4) in a 384-well microplate. The sensor is relatively unreactive toward other physiologically relevant metal ions such as sodium, potassium, and calcium ions, although it is not specific for Gd³⁺ over other trivalent lanthanides such as europium(III) and terbium(III). Nevertheless, the lanthanides are not commonly found in contrast agents or the biological systems, and the sensor may therefore be used to selectively determine unchelated Gd³⁺ in aqueous conditions.

Introduction

The increasing importance of magnetic resonance imaging (MRI) in clinical diagnosis, which is limited by the inherent sensitivity of the technique, has resulted in the rapid growth of research into the development of novel gadolinium-based contrast agents (GBCAs)¹. GBCAs are molecules that are administered to improve the image quality, and they typically have the chemical structure of a trivalent gadolinium ion (Gd³⁺) coordinated to a polydentate ligand. This complexation is of critical importance as unchelated Gd³⁺ is toxic; it has been implicated in the development of nephrogenic systemic fibrosis in some patients with renal disease ....

Protocol

NOTE: Molecular biology grade water is used in all buffer and solution preparations. All disposable tubes (microcentrifuge and PCR) and pipet tips are DNase- and RNase-free. Please consult the material safety data sheet (MSDS) for all chemicals prior to use. Use of appropriate personal protective equipment (PPE) is strongly recommended.

1. Preparation of the Aptamer Stock Solutions

1. Purchase 2 strands of polydeoxynucleotide commercially. Order both strands with purification via high performance liquid chromatography (HPLC).
   Strand 1 (Gd-aptamer):
   5'-/56-FAM/AGGCTCTCGGGACGACCAGTTGGTCCCGCTTTATGTGTCCCGAG-3'

Results

A typical fluorescence change of the Gd-sensor solution in the presence of unchelated Gd³⁺ is shown in Figure 2. The emission may be plotted as the fluorescence fold change (Figure 2A) or the raw fluorescence reading (Figure 2B) in arbitrary units (AFU). Both plots yield very similar calibration curves with a linear range for concentrations of Gd³⁺ below 1 μM and saturation of the signal at > 3 μM. The limit of de.......

Discussion

Using the aptamer-based Gd-sensor, an increase in fluorescence emission that is proportional to the concentration of unchelated Gd³⁺ is observed. To minimize the amount of sample used, the assay may be run in a 384-well microplate with a total sample volume of 45 μL per well. In this design, the choice of fluorescein (FAM) and dabcyl (Dab) was primarily based on the cost of the reagents; to modify the emission wavelength, a different pairing of fluorophore and quencher may be used¹¹.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gd-aptamer	IDTDNA	Input sequence and fluorophore modification in the order form	A fluorophore with a different emission wavelength may be used. The aptamer may also be ordered from another company.
Quenching strand	IDTDNA	Input sequence and quencher modification in the order form	A different quencher for optimal energy transfer from the fluorophore may be used. The aptamer may also be ordered from another company.
Molecular biology grade water			No specific manufacturer, both DEPC or non-DEPC treated work equally well
Gadolinium(III) chloride anhydrous	Strem	936416	Toxic
HEPES	Fisher Scientific	BP310-500
Magnesium chloride anhydrous	MP Biomedicals	0520984480 - 100 g
Sodium Chloride	Acros Organics	327300025
Potassium chloride	Fisher Scientific	P333-500
Sodium hydroxide, pellets	Fisher Scientific	BP359	Corrosive
Hydrochloric acid	Fisher Scientific	SA49	Toxic and corrosive
384-well low flange black flat bottom polystyrene NBS plates	Corning	3575	Plates which are suitable for fluorescence reading are required.
Nalgene Rapid-Flow sterile disposable bottle top filter	Thermo Scientific	5680020	The bottle top is fitted with 0.2 micron PES membrane
Disposable sterile bottles 250 mL	Corning	430281	A larger or smaller bottle may be used
1.5 mL microcentrifuge tubes			No specific manufacturer, as long as they are DNAse and RNAse-free
0.2 mL PCR tubes			No specific manufacturer, as long as they are DNAse and RNAse-free
Micropipets			No specific manufacturer
Pipet tips (non filter) of appropriate sizes			No specific manufacturer, as long as they are DNAse and RNAse-free
Equipment
Plate reader	Biotek Synergy H1		Plate readers from other manufacturers would work equally well