Name: an aptamer-based sensor for unchelated gadolinium(iii)
Uploaded: 2017-01-09T15:00:00.000+00:00
Duration: 5 min 15 s
Description: Watch this Scientific Journal Video about An Aptamer-based Sensor for Unchelated GadoliniumIII at JoVE.com

We would like to gratefully acknowledge Dr. Milan N Stojanovic from Columbia University, New York, NY for valuable scientific input. This work is supported by funding from the California State University East Bay (CSUEB) and the CSUEB Faculty Support Grant-Individual Researcher. O.E., T.C., and A.L. were supported by the CSUEB Center for Student Research (CSR) Fellowship.

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
<ol>
	<li>Purchase 2 strands of polydeoxynucleotide commercially. Order both strands with purification via high performance liquid chromatography (HPLC). 
	Strand 1 (Gd-aptamer): 
	5&#39;-/56-FAM/AGGCTCTCGGGACGACCAGTTGGTCCCGCTTTATGTGTCCCGAG-3&#39; 
	Strand 2 (QS): 
	5&#39;-GTCCCGAGAGCCT/3Dab/-3&#39;</li>
	<li>Dissolve each strand in water to make 100 &#956;M individual stock solutions of the Gd-aptamer and the QS.</li>
	<li>Store these solutions at -20 &#176;C. The solutions are stable thus far, for 3 years.</li>
	<li>To minimize freeze-thaw cycles, store the stock solutions in 10 &#956;L aliquots.</li>
</ol>
2. Preparation of the 2x Gd-sensor Solution
<ol>
	<li>Prepare the assay buffer (20 mM HEPES, 2 mM MgCl2, 150 mM NaCl, 5 mM KCl). Adjust the pH to ~7.4 with NaOH and HCl, and filter through sterile disposable bottle top filters with 0.2 &#956;m PES membrane. Store in sterile bottles. If filtered and stored properly (at room temperature), the buffer is stable thus far, for 2 years.</li>
	<li>Dilute 1 &#956;L of the Gd-aptamer stock solution (from step 1.2) and 2 &#956;L of the QS stock solution (from step 1.2) in 497 &#956;L of the assay buffer. Mix well using a vortex. Their concentrations in the 2x Gd-sensor solution are 200 nM and 400 nM, respectively. 
	NOTE: The volume of the 2x Gd-sensor solution prepared in this step is 500 &#956;L, which is sufficient to test 6 - 7 varying concentrations of Gd3+ for the calibration curve and the contrast agent solutions (step 3). Each sample will give duplicate wells in a 384-well plate.
	<ol>
		<li>Adjust the volume of the 2x Gd-sensor solution according to the number of Gd3+ solutions that needs to be tested.</li>
	</ol>
	</li>
	<li>Transfer the 2x Gd-sensor solution into 9 PCR tubes, with 50 &#956;L into each tube. Place the tubes in a thermal cycler.</li>
	<li>Set the program in the thermal cycler to heat the solution in the tubes to 95 &#176;C, hold for 5 min, and then slowly cool the solutions to 25 &#176;C over ~15 min (at the rate of ~0.05 - 0.1 &#176;C/s). The heating and cooling cycle is to ensure optimal hybridization between the Gd-aptamer and the QS. Partial hybridization results in incomplete quenching and a higher background fluorescence of the sensor. If a thermal cycler is not available, carry out this process using a hot water bath instead.</li>
	<li>Once cooled to 25 &#176;C, immediately use the solution, or keep in the thermal cycler (up to about 2 h) until ready to be used. When a water bath is used for the heating, leave the tubes in the bath as the water slowly cools to room temperature.</li>
</ol>
3. Constructing the Fluorescence Calibration Curve and Detecting the Presence of Unchelated Gd3+ in a Solution of Gd Contrast Agent
<ol>
	<li>Dissolve GdCl3 solid in the assay buffer (the same buffer as in step 2.1).</li>
	<li>Through serial dilution, prepare 100 &#956;L each of 6 different Gd3+ solutions in microcentrifuge tubes at double of the final desired concentrations for the calibration curve (2x solutions).
	<ol>
		<li>For example, to construct a calibration for 0 (buffer only with no GdCl3), 50, 100, 200, 400, and 800 nM of Gd3+, prepare solutions containing 0, 100, 200, 400, 800, and 1,600 nM of the ion. Make sure to always include the &#39;blank&#39; with 0 nM Gd3+ as the negative control.</li>
	</ol>
	</li>
	<li>Dissolve the contrast agent to be tested in the assay buffer. Prepare 2 or 3 different concentrations of the contrast agent solutions via serial dilution. 
	NOTE: Testing 3 different concentrations of the contrast agent solution is recommended. This is to ensure that these concentrations are within the linear range. If the samples tested do not display a linear relationship, reduce the concentrations of the contrast agent used.</li>
	<li>Take the PCR tubes containing the 2x Gd-sensor solution from step 2.5 out of the thermal cycler.</li>
	<li>Add 50 &#956;L of each Gd3+ solution from step 3.2 into 6 of the 9 PCR tubes containing the 2x Gd-sensor solution. Mix by pipetting up and down. Each PCR tube now contains the desired concentration of Gd3+ to be tested, 100 nM Gd-aptamer, and 200 nM QS.</li>
	<li>To the remaining PCR tubes containing the 2x Gd-sensor solution, add 50 &#956;L of the contrast agent solutions from step 3.3. Mix well by pipetting up and down a few times.</li>
	<li>Incubate the solutions in the PCR tubes for around 5 min at room temperature. They may be left to stand for up to 30 min.</li>
	<li>Transfer 45 &#956;L of each tube into a 384-well plate. Each PCR tube will give duplicate wells.</li>
	<li>Record the fluorescence of each well on a plate-reader. The fluorophore (FAM) used in the Gd-sensor design has excitation and emission maxima of 495 and 520 nm respectively, as listed on the supplier&#39;s website. Choose appropriate excitation and emission wavelengths or filters depending on whether the plate-reader is monochromator- or filter-based.</li>
	<li>Plot the graph of fluorescence in arbitrary fluorescence units (AFU) against concentration of Gd3+.</li>
	<li>Plot the graph as fluorescence fold change against concentration of Gd3+. Calculate the fluorescence fold change by dividing the AFU of each concentration by the AFU of the &#39;blank&#39; solution (with 0 nM of Gd3+). The fluorescence fold change will allow for the normalization of the results, should the AFU display some periodical (different days, etc.) variations.</li>
	<li>Compare the fluorescence emission of the solution containing the contrast agent and the &#39;blank&#39;, which is the solution containing 0 nM GdCl3 (buffer only). 
	NOTE: A higher fluorescence of the GBCA solution implies the presence of unchelated Gd3+, which may necessitate further purification of the contrast agent. The amount of unchelated Gd3+ present may be estimated using the calibration curve constructed in step 3.10 or 3.11.</li>
</ol>

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M., Tweedle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+contrast+agents:+basic+chemistry+and+safety.">MRI contrast agents: basic chemistry and safety.</a> J Magn. Reson. Imaging. 36 (5), 1060-1071 (2012).</li><li>Telgmann, L., Sperling, M., Karst, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+gadolinium-based+MRI+contrast+agents+in+biological+and+environmental+samples:+a+review.">Determination of gadolinium-based MRI contrast agents in biological and environmental samples: a review.</a> Anal. Chim. Acta. 764, 1-16 (2013).</li><li>Frenzel, T., Lengsfeld, P., Schirmer, H., H&#252;tter, J., Weinmann, H. -. 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Chem. 379 (7), 1050-1054 (2004).</li><li>Telgmann, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Speciation+and+isotope+dilution+analysis+of+gadolinium-based+contrast+agents+in+wastewater.">Speciation and isotope dilution analysis of gadolinium-based contrast agents in wastewater.</a> Environ. Sci. Technol. 46 (21), 11929-11936 (2012).</li><li>Cleveland, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatographic+methods+for+the+quantification+of+free+and+chelated+gadolinium+species+in+MRI+contrast+agent+formulations.">Chromatographic methods for the quantification of free and chelated gadolinium species in MRI contrast agent formulations.</a> Anal. Bioanal. Chem. 398 (7), 2987-2995 (2010).</li><li>Edogun, O., Nguyen, N. H., Halim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+single-stranded+DNA-based+assay+for+detecting+unchelated+gadolinium(III)+ions+in+aqueous+solution.">Fluorescent single-stranded DNA-based assay for detecting unchelated gadolinium(III) ions in aqueous solution.</a> Anal. Bioanal. Chem. 408 (15), 4121-4131 (2016).</li><li>Barge, A., Cravotto, G., Gianolio, E., Fedeli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+determine+free+Gd+and+free+ligand+in+solution+of+Gd+chelates.+A+technical+note.">How to determine free Gd and free ligand in solution of Gd chelates. A technical note.</a> Contrast Med. Mol. Imaging. 1 (5), 184-188 (2006).</li><li>Johansson, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+reporter-quencher+pairs+for+efficient+quenching+through+formation+of+intramolecular+dimers.">Choosing reporter-quencher pairs for efficient quenching through formation of intramolecular dimers.</a> Methods Mol. Biol. 335, 17-29 (2006).</li><li>Sherry, A. D., Caravan, P., Lenkinski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+on+gadolinium+chemistry.">A primer on gadolinium chemistry.</a> J. Magn. Reson. Imaging. 30 (6), 1240-1248 (2009).</li><li>Shakhverdov, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cross-relaxation+mechanism+of+fluorescence+quenching+in+complexes+of+lanthanide+ions+with+organic+ligands.">A cross-relaxation mechanism of fluorescence quenching in complexes of lanthanide ions with organic ligands.</a> Opt. Spectrosc. 95 (4), 571-580 (2003).</li><li>Brittain, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submicrogram+determination+of+lanthanides+through+quenching+of+calcein+blue+fluorescence.">Submicrogram determination of lanthanides through quenching of calcein blue fluorescence.</a> Anal. Chem. 59 (8), 1122-1125 (1987).</li></ol>

The authors declare that they have no competing financial interests.

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 &#956;L 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.
It is important to note that to obtain the best result with the sensor, one of the critical steps is the heating to 95 &#176;C and slow cooling (step 2.4) in the assay buffer to achieve optimal hybridization between the Gd-aptamer and the QS strands. As previously mentioned in the protocol, if a thermal cycler is not available, the incubation at 95 &#176;C may be carried out in a water bath. Another key parameter to control is the compositions of the buffer solutions; the use of the assay buffer listed in step 2.1 to dissolve the contrast agent is recommended, or deionized water may also be used. However, solutions that contain potential interferents should be avoided. An example of such a buffer is one that contains phosphate anions, which can coordinate to unchelated Gd3+ to form insoluble gadolinium phosphate12. The precipitate will not react with the sensor, resulting in a false negative result.
A few steps in the experiments may be modified without affecting the outcome. First, to simplify the assay and calculation, prepare both the Gd-sensor solution and the aqueous GdCl3 for the calibration curve at 2x concentrations. If desired, other dilution factors may be used (for example, 10x solutions), provided that the final assay concentrations of the Gd-aptamer and the QS are maintained at 100 nM and 200 nM, respectively. Second, the assay buffer does not have to be exactly pH 7.4. Any value between 7 - 7.4 will produce the desired fluorescence increase, as long as the same buffer is used throughout the experiment. Third, once the fluorescence emission reading is obtained, the data points may be plotted either as raw fluorescence in arbitrary unit (AFU) or as fluorescence fold change. To calculate the fluorescence fold change, the raw fluorescence reading of each concentration is normalized to (divided by) the reading of the negative control (0 nM Gd3+). As shown in Figures 2A and B, the fluorescence emission trends in both plots are almost identical. The fold change may be a more convenient way to analyze the data if the plate reader displays some variations in the raw readings recorded at different times. Finally, if the laboratory is equipped with a fluorometer, but not a plate reader, each data point may be measured using a cuvette, instead of a microplate. Depending on the size of the available cuvettes, the volumes of the solutions prepared in the assay may need to be adjusted.
The method reported herein provides an alternative to chromatographic- and/or spectrometric-based techniques for detecting aqueous Gd3+. Compared to the latter, the Gd-sensor assay is more limited in terms of sensitivity, accuracy, and ability for simultaneous detection of multiple species. On the other hand, the spectroscopic-based sensor requires an instrumentation that may possibly be more readily available, may be performed within a shorter time period, and the sample preparation is minimal. The contrast agent may be simply dissolved in the buffer solution, mixed with the Gd-sensor solution, and the fluorescence emission directly measured. Furthermore, the sensor is able to detect a much lower concentration of unchelated Gd3+ than the xylenol orange indicator (about 2 orders of magnitude difference between the two methods) and has a higher selectivity for Gd3+ over several other biologically important and transition metal ions9.
There are two drawbacks of this assay that may restrict its use under some experimental conditions. One limitation is that the sensor is not specific for Gd3+; it displays a response to other lanthanide ions (such as Eu3+ and Tb3+)9. However, these are not ions commonly found in contrast agents or the biological systems and therefore, their interference are minimal. The second point to note is that at higher concentrations (above ~10 &#956;M) of Gd3+, a gradual decrease in the Gd-sensor fluorescence emission is observed. The effect of quenching by lanthanide ions is a well-documented phenomenon13 that has also been used as a technique for detecting and quantifying them14. While this limits the utility of the sensor for measuring high concentrations of Gd3+, the goal of this design is to detect small amounts of the free ion in the solution to ensure a higher purity of the contrast agent.
In this work, the use of a convenient fluorescence-based technique for detecting toxic unchelated Gd3+ in aqueous solution has been described. This assay is meant for the early-stage evaluation of gadolinium-based contrast agent purity, specifically during the synthesis and formulation for in vitro experiments. With the current growth of magnetic resonance imaging in diagnosis, an increasing number of novel contrast agents are continually being designed and tested. The aptamer-based Gd-sensor will facilitate this development by providing a means for rapidly detecting the presence of sub-micromolar concentrations of unreacted or dissociated Gd3+ in aqueous solution at ambient pH. Furthermore, since the sensor displays cross-reactivity with other trivalent lanthanide ions, its application may be extended to these areas of research.

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 or failure2. Consequently, detecting the aqueous free ion is instrumental in ensuring the safety of GBCAs. The presence of unchelated Gd3+ in GBCA solutions often is the result of an incomplete reaction between the ligand and the ion, dissociation of the complex, or displacement by other biological metal cations3.
Among the several techniques currently used for determining the presence of Gd3+, those relying on chromatography and/or spectrometry rank highest in terms of versatility and applicability4. Among their strengths are high sensitivity and accuracy, the ability to analyze various sample matrices (including human serum5, urine and hair6, wastewater7, and contrast agent formulations8), and the simultaneous quantification of multiple Gd3+ complexes (a listing of studies prior to 2013 is described in a comprehensive review by Telgmann et al.)4. The only drawback is that several of these methods require instrumentations (such as inductively coupled plasma-mass spectrometry)4 that some laboratories may not have access to. Within the context of novel GBCA discovery at the research and proof-of-concept levels, a relatively more convenient, rapid, and cost-effective spectroscopic-based method (such as UV-Vis absorption or fluorescence) may serve as a valuable alternative. With these applications in mind, a fluorescent aptamer-based sensor for aqueous Gd3+ was developed9.
The aptamer (Gd-aptamer) is a 44-base long single-stranded DNA molecule with a specific sequence of bases that was isolated through the process of systematic evolution of ligands by exponential enrichment (SELEX)9. To adapt the aptamer into a fluorescent sensor, a fluorophore is attached to the 5&#39; terminus of the strand, which is then hybridized with a quenching strand (QS) via 13 complementary bases (Figure 1). The QS is tagged with a dark quencher molecule at the 3&#39; terminus. In the absence of Gd3+, the sensor (Gd-sensor), comprised of a 1:2 mole ratio of Gd-aptamer and QS respectively, will have minimal fluorescence emission due to energy transfer from the fluorophore to the quencher. The addition of aqueous Gd3+ will displace the QS from the Gd-aptamer, resulting in an increase in fluorescence emission.
<img alt="Figure 1" src="/files/ftp_upload/55216/55216fig1.jpg" /> 
Figure 1. The sensor (Gd-sensor) that consists of the 44-base long aptamer (Gd-aptamer) tagged with fluorescein (a fluorophore) and the 13-base long quenching strand (QS) tagged with dabcyl (a dark quencher). In the absence of unchelated Gd3+, the fluorescence of the sensor is minimal. With addition of Gd3+, displacement of the QS occurs and an increase in fluorescence emission is observed. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55216/55216fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
There is at present, one commonly used spectroscopic-based method for detecting aqueous Gd3+. This assay uses the molecule xylenol orange, which undergoes a shift in the maximum absorption wavelength from 433 to 573 nm upon chelation to the ion10. The ratio of these two absorbance maxima can be used to quantify the amount of unchelated Gd3+. The aptamer sensor is an alternative (may also be complementary) to the xylenol orange assay, as the two methods have different reaction conditions (such as pH and composition of the buffer solutions used), target selectivities, linear ranges of quantification, and detection modalities9.

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

an aptamer-based sensor for unchelated gadolinium(iii)

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.

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 &#956;M and saturation of the signal at &#62; 3 &#956;M. The limit of detection is ~100 nM with a signal-to-noise ratio of 3.
In solutions containing the GBCA of interest, the presence of unchelated Gd3+ will be translated into a fluorescence increase of the sensor when compared to the &#39;blank&#39; solution. The fluorescence changes in solutions of 2 different batches of Gd-DOTA complex, one of higher purity than the other, are shown as examples of representative results (Figure 3). Gd-DOTA (gadoteric acid) is a gadolinium complex of Gd3+ surrounded by an organic ligand DOTA that is found in a commercial contrast agent. The batch of higher purity does not display a significant increase in emission up to 20 mM of Gd-DOTA. When unchelated Gd3+ is present, a change that is noticeable even at Gd-DOTA concentrations below 5 mM is observed. In this example where the data points are plotted as fluorescence fold change of the sensor, quantification of the amount of unchelated Gd3+ may be estimated using the calibration curve in Figure 2A.
<img alt="Figure 2" src="/files/ftp_upload/55216/55216fig2.jpg" /> 
Figure 2. Representative Gd-sensor fluorescence calibration curve plots. All data points were performed at least in duplicates and the average values plotted with standard deviation as the error bars. (A) A calibration curve obtained using 100 nM Gd-aptamer and 200 nM QS. The graph is plotted with fluorescence fold change as the y axis. (B) The same calibration curve as in (A) with raw fluorescence in arbitrary units (AFU) as the y axis. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55216/55216fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55216/55216fig3.jpg" /> 
Figure 3. Representative Gd-sensor fluorescence change when testing the presence of unchelated Gd3+ in samples of Gd-DOTA molecule. Two different batches of Gd-DOTA complex solutions are shown in this plot, one of higher purity (circle marker) and the other containing unchelated Gd3+ (blue triangle marker). Each data point is an average of two readings with standard deviation as the error bars. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55216/55216fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An Aptamer-based Sensor for Unchelated GadoliniumIII at JoVE.com

An Aptamer-based Sensor for Unchelated GadoliniumIII

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.

An Aptamer-based Sensor for Unchelated Gadolinium(III)

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples ...

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7.2K Views.  California State University, East Bay. 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. 

Video: An Aptamer-based Sensor for Unchelated GadoliniumIII

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

Medicine

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Bioengineering

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e.,&#160;gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

AN MRI-compatible&#160;custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

Fluorescent Assay for Detection of Novel Iron Chelators

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

Author Spotlight: Assessing the Impact of Novel Iron Chelators on Cancer Cell Metabolism

We describe a fluorescent assay that can quickly and inexpensively confirm the ability of novel compounds to chelate iron. The assay measures the ability of compounds to outcompete the iron binding activity of the weak iron chelating fluorescent probe Calcein, resulting in a quantifiable increase in fluorescence when chelation occurs.

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target ...

Cancer Research

Automated Preparation of [68Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe targeting the fibroblast activation protein for positron emission tomography imaging of the tumor microenvironment. The reaction conditions involved acetate buffer (final concentration: 0.1 M), methionine as an anti-radiolysis agent (final concentration: 5.4 mg/mL), and 30 &#181;g of 3BP-3940, with heating for 8 min at 98 &#176;C. A final purification step on a C18 cartridge was necessary to obtain a radiolabeled product of high purity. In contrast, the generator-produced 68Ga was used directly without a concentration step on a cation exchange cartridge. The production of three validation batches confirmed the method&#39;s reliability, allowing the synthesis of [68Ga]Ga-3BP-3940 in 22.3 &#177; 0.6 min with high radiochemical purity (RCP), as determined by both radio-HPLC (99.1% &#177; 0.1%) and radio-TLC (99.2% &#177; 0.1%). The average radiochemical yield, based on RCP values measured by radio-HPLC, was 74.4% &#177; 3.3%. The stability of the radiolabeled product was demonstrated for up to 4 h after preparation. This protocol provides a reliable, rapid, and efficient methodology for the preparation of [68Ga]Ga-3BP-3940, which can easily be transposed to a clinical setting.

This research describes the automated process for [68Ga]Ga-3BP-3940 production with the GAIA V2 synthesizer, for PET imaging of fibroblast activation protein. The results of quality control tests performed on three test batches are also presented.

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe ...

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Biology

CRISPR-Cas Synthetic Urine Biomarker Test for Cancer Diagnostics

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

Author Spotlight: Engineering Molecular Tools for Disease Detection and Imaging

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

An Aptamer-based Sensor for Unchelated Gadolinium(III)

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