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>

<ol>
	<li>Shen, C., New, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promising+strategies+for+Gd-based+responsive+magnetic+resonance+imaging+contrast+agents.">Promising strategies for Gd-based responsive magnetic resonance imaging contrast agents.</a> Curr. Opin. Chem. Biol. 17 (2), 158-166 (2013).</li><li>Cheong, B. Y. C., Muthupillai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephrogenic+systemic+fibrosis:+a+concise+review+for+cardiologists.">Nephrogenic systemic fibrosis: a concise review for cardiologists.</a> Tex. Heart Inst. J. 37 (5), 508-515 (2010).</li><li>Hao, D., Ai, T., Goerner, F., Hu, X., Runge, V. 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. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+gadolinium-based+magnetic+resonance+imaging+contrast+agents+in+human+serum+at+37+degrees+C.">Stability of gadolinium-based magnetic resonance imaging contrast agents in human serum at 37 degrees C.</a> Invest. Radiol. 43 (12), 817-828 (2008).</li><li>Loreti, V., Bettmer, J. <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+the+MRI+contrast+agent+Gd-DTPA+by+SEC-ICP-MS.">Determination of the MRI contrast agent Gd-DTPA by SEC-ICP-MS.</a> Anal. Bioanal. 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.
<p cla...

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="http://cloudflare.jove.com/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 border="0" cellpadding="0" cellspacing="0" width="2018">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Gd-aptamer</td>
			<td style="width:187px;">IDTDNA</td>
			<td style="width:439px;">Input sequence and fluorophore modification in the order form</td>
			<td style="width:943px;">A fluorophore with a different emission wavelength may be used. The aptamer may also be ordered from another company.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Gadolinium(III) chloride anhydrous</td>
			<td>Strem</td>
			<td>936416</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride anhydrous</td>
			<td>MP Biomedicals</td>
			<td>0520984480 - 100 g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Acros Organics</td>
			<td>327300025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride</td>
			<td>Fisher Scientific</td>
			<td>P333-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide, pellets</td>
			<td>Fisher Scientific</td>
			<td>BP359</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>SA49</td>
			<td>Toxic and corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalgene Rapid-Flow sterile disposable bottle top filter</td>
			<td>Thermo Scientific&#160;</td>
			<td>5680020</td>
			<td>The bottle top is fitted with&#160; 0.2 micron PES membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable sterile bottles 250 mL</td>
			<td>Corning</td>
			<td>430281</td>
			<td>A larger or smaller bottle may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Micropipets</td>
			<td></td>
			<td></td>
			<td>No specific manufacturer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Name of Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 ...

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

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by &#960;-&#960; Stacking Interactions

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE). Characterization of the copolymer revealed a narrow molecular distribution (PDI &#60; 1.03) and confirmed the degree of polymerization of mPEG122-b-(PheGE)15. The critical micelle concentration of the copolymer was evaluated using an established fluorescence method with the aggregation behavior evaluated by dynamic light scattering and transmission electronic microscopy. The potential of the copolymer for use in drug delivery applications was evaluated in a preliminary manner including in vitro biocompatibility, loading and release of the hydrophobic anti-cancer drug doxorubicin (DOX). A stable micelle formulation of DOX was prepared with drug loading levels up to 14% (wt%), drug loading efficiencies &#62; 60% (w/w) and sustained release of drug over 4 days under physiologically relevant conditions (acidic and neutral pH, presence of albumin). The high drug loading level and sustained release is attributed to stabilizing &#960;-&#960; interactions between DOX and the core-forming block of the micelles.

The key steps of living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE) are described. The resulting block copolymer micelles (BCMs) were loaded with doxorubicin 14% (wt%) and sustained release of drug over 4 days under physiologically relevant conditions was obtained.

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables the acquisition of photoluminescence spectra covering the time regime from nanoseconds up to 0.1 s. This enables us to follow the changes in the intensity (decay) and emission of the spectra over time. Using this method, it is possible to study diverse photophysical phenomena, such as the emission of phosphorescence, and the contributions of prompt and delayed fluorescence in molecules showing thermally activated delayed fluorescence (TADF). Remarkably, all spectra and decays are obtained in a single experiment. This can be done for solids (thin film, powder, crystal) and liquid samples, where the only limitations are the spectral sensitivity of the camera and the excitation wavelength (532 nm, 355 nm, 337 nm, and 266 nm). This technique is, thus, very important when investigating the excited state dynamics in organic emitters for their application in organic light-emitting diodes and other areas where triplet harvesting is of paramount importance. Since triplet states are strongly quenched by oxygen, emitters with efficient TADF luminescence, or those showing room temperature phosphorescence (RTP), must be correctly prepared in order to remove any dissolved oxygen from solutions and films. Otherwise, no long-lived emission will be observed. The method of degassing solid samples as presented in this work is basic and simple, but the degassing of liquid samples creates additional difficulties and is particularly interesting. A method of minimizing solvent loss and changing the sample concentration, while still enabling to remove oxygen in a very efficient and a repeatable manner, is presented in this work.

Here, we present a method of the spectroscopic characterization of organic molecules by means of time-resolved photoluminescence spectroscopy on the nanosecond-to-millisecond timescale in oxygen-free conditions. Methods to efficiently remove oxygen from the samples and, thus, limit luminescence quenching are also described.

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables ...

An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control cellular functions. The reversal of DNA methylation, or DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family of dioxygenases. Although it has been widely reported that aberrant DNA methylation and demethylation are associated with developmental defects and cancer, how these epigenetic changes directly contribute to the subsequent alteration in gene expression or disease progression remains unclear, largely owing to the lack of reliable tools to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution. To overcome this hurdle, we designed a split-TET2 enzyme to enable temporal control of 5-methylcytosine (5mC) oxidation and subsequent remodeling of epigenetic states in mammalian cells by simply adding chemicals. Here, we describe methods for introducing a chemical-inducible epigenome remodeling tool (CiDER), based on an engineered split-TET2 enzyme, into mammalian cells and quantifying the chemical inducible production of 5-hydroxymethylcytosine (5hmC) with immunostaining, flow cytometry or a dot-blot assay. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype&#8722;phenotype relations in various biological systems.

Detailed protocols for introducing an engineered split-TET2 enzyme (CiDER) into mammalian cells for chemical inducible DNA hydroxymethylation and epigenetic remodeling are presented.

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control ...

An Experimental Protocol for Femtosecond NIR/UV - XUV Pump-Probe Experiments with Free-Electron Lasers

This protocol describes key steps in performing and analyzing femtosecond pump-probe experiments that combine a femtosecond optical laser with a free-electron laser. This includes methods to establish the spatial and temporal overlap between the optical and free-electron laser pulses during the experiment, as well as important aspects of the data analysis, such as corrections for arrival time jitter, which are necessary to obtain high-quality pump-probe data sets with the best possible temporal resolution. These methods are demonstrated for an exemplary experiment performed at the FLASH (Free-electron LASer Hamburg) free-electron laser in order to study ultrafast photochemistry in gas-phase molecules by means of velocity map ion imaging. However, most of the strategies are also applicable to similar pump-probe experiments using other targets or other experimental techniques.

This protocol describes the key steps for performing and analyzing pump-probe experiments combining a femtosecond optical laser with a free-electron laser in order to study ultrafast photochemical reactions in gas-phase molecules.

This protocol describes key steps in performing and analyzing femtosecond pump-probe experiments that combine a femtosecond optical laser with a ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

Key Factors Affecting the Performance of Sb2S3-sensitized Solar Cells During an Sb2S3 Deposition via SbCl3-thiourea Complex Solution-processing

Sb2S3 is considered as one of the emerging light absorbers that can be applied to next-generation solar cells because of its unique optical and electrical properties. Recently, we demonstrated its potential as next-generation solar cells by achieving a high photovoltaic efficiency of &#62; 6% in Sb2S3-sensitized solar cells using a simple thiourea (TU)-based complex solution method. Here, we describe the key experimental procedures for the deposition of Sb2S3 on a mesoporous TiO2 (mp-TiO2) layer using a SbCl3-TU complex solution in the fabrication of solar cells. First, the SbCl3-TU solution is synthesized by dissolving SbCl3 and TU in N,N-dimethylformamide at different molar ratios of SbCl3:TU. Then, the solution is deposited on as-prepared substrates consisting of mp-TiO2/TiO2-blocking layer/F-doped SnO2 glass by spin coating. Finally, to form crystalline Sb2S3, the samples are annealed in an N2-filled glove box at 300 &#176;C. The effects of the experimental parameters on the photovoltaic device performance are also discussed.

Key Factors Affecting the Performance of Sb2S3-sensitized Solar Cells During an Sb2S3 Deposition via SbCl3-thiourea Complex Solution-processing

This work provides a detailed experimental procedure for the deposition of Sb2S3 on a mesoporous TiO2 layer using a SbCl3-thiourea complex solution for applications in Sb2S3-sensitized solar cells. This article also determines key factors governing the deposition process.

Sb2S3 is considered as one of the emerging light absorbers that can be applied to next-generation solar cells because of its unique optical and electrical ...

An Electrochemical Cholesteric Liquid Crystalline Device for Quick and Low-Voltage Color Modulation

We demonstrate a method for fabricating a prototype reflective display device that contains cholesteric liquid crystal (LC) as an active component. The cholesteric LC is composed of a nematic LC 4&#39;-pentyloxy-4-cyanobiphenyl (5OCB), redox-responsive chiral dopant (FcD), and a supporting electrolyte 1-ethyl-3-methylimidazolium trifluoromethanesulfonate (EMIm-OTf). The most important component is FcD. This molecule changes its helical twisting power (HTP) value in response to redox reactions. Therefore, in situ electrochemical redox reactions in the LC mixture allow for the device to change its reflection color in response to electrical stimuli. The LC mixture was introduced, by a capillary action, into a sandwich-type ITO glass cell comprising two glass slides with patterned indium tin oxide (ITO) electrodes, one of which was coated with poly(3,4-ethylenedioxythiophene)-co-poly(ethylene glycol) doped with perchlorate (PEDOT+). Upon application of +1.5 V, the reflection color of the device changed from blue (467 nm) to green (485 nm) in 0.4 s. Subsequent application of 0 V made the device recover the original blue color in 2.7 s. This device is characterized by its fastest electrical response and lowest operating voltage among any previously reported cholesteric LC device. This device could pave the way for the development of next generation reflective displays with low energy consumption rates.

A protocol for the fabrication of a reflective cholesteric liquid crystalline display device containing a redox-responsive chiral dopant allowing quick and low-voltage operation is presented.

We demonstrate a method for fabricating a prototype reflective display device that contains cholesteric liquid crystal (LC) as an active component. The ...