The overall goal of this fluorescence-based assay is to detect the presence of toxic unchelated gadolinium ion in aqueous solutions containing magnetic resonance imaging contrast agents. This method can help facilitate the gadolinium-based MRI contrast agents by providing a means to ensure high parity of synthesized agents. The main advantage of this technique is that it is able to detect submicromolar concentration of unchelated toxic gadolinium with relatively high selectivity over other biological metal cations.
To begin this procedure, prepare the assay buffer as outlined in the text protocol. Using sodium hydroxide and hydrochloric acid, adjust the pH to 7.4. Then, filter the buffer through a sterile disposable bottle-top filter with a 0.2 micron PES membrane.
Next, transfer 497 microliters of the assay buffer to a fresh micro centrifuge tube. Add one microliter of prepared gadolinium aptamer stock solution and two microliters of prepared QS stock solution. Transfer 50 microliters of this 2X Gadolinium sensor solution into each of nine PCR tubes.
After this, place the tubes into a thermal cycler. Set the thermal cycler to heat the samples to 95 degrees celsius for five minutes, then slowly cool the solutions to 25 degrees celsius over 15 minutes. It's important to remember to heat the 2X gadolinium sensor solutions to 95 degrees, followed by slow cooling to room temperature before adding the gadolinium ion solution.
To begin, dissolve solid gadolinium trichloride in assay buffer to create a gadolinium three stock solution of the desired concentration. Using serial dilution, prepare six 100 microliter gadolinium three solutions as outlined in the text protocol. After this, dissolve the contrasting agent to be tested in assay buffer.
Use serial dilution to prepare three different concentrations. Then, remove the PCR tubes containing the 2X gadolinium sensor solution from the thermal cycler. Transfer 50 microliters of each gadolinium three solution to separate PCR tubes.
Pipette each solution up and down several times to mix. Next, transfer 50 microliters of each contrasting agent solution separately to the remaining PCR tubes. Mix by pipetting up and down several times.
Incubate all nine solutions for five minutes at room temperature. After this, transfer 45 microliters of each solution to a 384 well plate in duplicates. Then, record and analyze fluorescence data as outlined in the text protocol.
In this study, unchelated trivalent gadolinium ion is detected in aqueous solution using a fluorescence-based method. The fluorescent sensor is developed by attaching a fluorophore to the aptamer. The sensor is then hybridized with a quenching strand, which is tagged with a dark quencher molecule.
The addition of gadolinium three displaces the quenching strand from the aptamer, causing an increase in fluorescence emission. Calibration curves are obtained using 100 nanomolar gadolinium aptamer and 200 nanomolar QS and plotted either raw fluorescence or fluorescence fold change. Both curves show a linear range for gadolinium three concentrations below one micromolar and saturation of the signal at concentrations over three micromolar.
Two different batches of gadoteric acid are then analyzed for concentrations ranging from zero millimolar to 20 millimolar. While the fluorescence emission of the high purity sample does not increase noticeably over this range, a significant change is observed in the sample containing unchelated gadolinium three at concentrations as low as five millimolar. While attempting this procedure, it is important to make sure there are no air bubbles in the wells of the microplate, as this will alter the fluorescent reading.
Using this procedure, we may be able to detect even small amounts of calculated gadolinium ions in solutions containing contrast agents. This will be a good method for determining the purity of the agents. Working with chemical can be hazardous and taking precautions such as wearing personal protective equipment is important while performing this procedure.
