Name: an aptamer-based sensor for unchelated gadolinium(iii)
Uploaded: 2017-01-09T15:00:00
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...

<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>

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.
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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 ...

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.

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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 ...