Name: an optimized protocol for the efficient radiolabeling of gold nanoparticles by using a 125i-labeled azide prosthetic group
Uploaded: 2016-10-10T15:00:00
Description: Watch this Scientific Journal Video about An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group at JoVE.com

This work was supported by grants from the National Research Foundation of Korea, funded by the government of the Republic of Korea, (Grant nos. 2012M2B2B1055245 and 2012M2A2A6011335) and by the RI-Biomics Center of Korea Atomic Energy Research Institute.

Caution: The oxidized form of radioactive iodine is quite volatile and must be handled with adequate lead shields and lead vials. All radiochemical steps should be carried out in a well-ventilated charcoal-filtered hood, and the experimental procedures need to be monitored by radioactivity detection devices.
1. Preparation of Chemicals and the Reverse Phase Cartridge for the Synthesis of the 125I-labeled Azide
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	<li>Preparation of reagents in solution
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		<li>Dissolve 1 mg of ...

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In general, the observed radiochemical yield of the purified 125I-labeled azide (1) was 75 &#177; 10% (n = 8). The radiolabeling was accomplished with 50-150 MBq of radioactivity, and the radiochemical results are quite consistent. If [125I]NaI (t1/2 = 59.4 d) that underwent radioactive decay for more than a month was used in the radioiodination reaction, the radiochemical yield of 1 was observed to be slightly decreased (53-65%). Therefore, it i...

The strain-promoted copper-free click reaction between azides and cyclooctynes has been extensively applied to the efficient bioorthogonal labeling of a wide range of biomolecules, nanomaterials, and living subjects1-7. Due to the excellent site-specificity and rapid reaction rate of this conjugation reaction, it has also been used to synthesize radiolabeled tracers. A few 18F-labeled azide or DBCO prosthetic groups have been prepared for in vitro labeling of various cancers targeting peptides and antibodies, as well as for in vivo pre-targeted imaging of tumors8-13. In addition to these examples, the same conjugation reaction was applied to the metal-radioisotope-labeling of nanomaterials for positron emission tomography (PET) imaging studies14-16.
For several decades, radioactive iodines have been used for biomedical research and clinical trials through PET imaging (124I), single-photon emission computed tomography (SPECT) imaging (123I, 125I), and thyroid cancer treatment (131I)17-21. Therefore, an efficient method for radioactive iodine labeling is fundamentally important for various investigations, including molecular imaging studies, analysis of organ distribution of biomolecules, biomarker identification, and drug development. A copper-free click reaction strategy could be used in radioactive iodine labeling. However, this application has not been investigated as extensively as 18F-labeled biomolecules22-23. Here, we will provide a step-by-step protocol for the synthesis of an 125I-labeled azide for radiolabeling of DBCO-group-derived molecules. The procedures in the present report will include radioiodination of the stannylated precursor, purification steps with HPLC, and solid phase extraction. We also demonstrate efficient radiolabeling of DBCO-group-modified 13-nm-sized gold nanoparticles using the 125I-labeled azide. The detailed protocol in this report will help synthetic chemists understand a new radiolabeling methodology for the synthesis of radiolabeled products.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Chloramine T trihydrate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">402869</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:255px;">[125I]NaI in aq. NaOH</td>
			<td style="width:101px;">Perkin-Elmer</td>
			<td style="width:160px;">NEZ033A010MC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Sodium metabisulfite&#160;</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">S9000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Formic acid</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">251364</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sep-Pak tC18 plus cartridge</td>
			<td style="width:101px;">Waters</td>
			<td>WAT036800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Dimethyl sulfoxide&#160;</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Acetone</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">650501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ethanol</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">459844</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Gold(III) chloride trihydrate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">520918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tween 20&#160;</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DBCO PEG SH (MW 5000)</td>
			<td style="width:101px;">NANOCS</td>
			<td style="width:160px;">PG2-DBTH-5k</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:255px;">TLC silica gel 60 F254</td>
			<td style="width:101px;">Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Analytical HPLC</td>
			<td style="width:101px;">Agilent</td>
			<td style="width:160px;">1290 Infinity</td>
			<td>Model number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Preparative HPLC</td>
			<td style="width:101px;">Agilent</td>
			<td style="width:160px;">1260 Infinity</td>
			<td>Model number</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Analytical C18 reverse-phase column</td>
			<td style="width:101px;">Agilent</td>
			<td style="width:160px;">Zorbax Eclipse XDB-C18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Preparative C18 reverse-phase column</td>
			<td style="width:101px;">Agilent</td>
			<td style="width:160px;">PrepHT XDB-C18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Radio TLC scanner</td>
			<td style="width:101px;">Bioscan</td>
			<td style="width:160px;">AR-2000</td>
			<td>Model number</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Radioisotope dose calibrator</td>
			<td style="width:101px;">Capintec, Inc</td>
			<td style="width:160px;">CRC -25R dose calibrator</td>
			<td>Model number</td>
		</tr>
	</tbody>
</table>

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.

The radioiodination reaction of the stannylated precursor (2) was carried out using 150 MBq of [125I]NaI, acetic acid, and chloramine T at room temperature for 15 min to provide the radiolabeled product (1). After preparative HPLC purification of the crude mixture, the desired product was obtained with 75 &#177; 10% (n = 8) of radiochemical yield. Analytical HPLC revealed that the radiochemical purity of the 125I-labeled product was ...

Watch this Scientific Journal Video about An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group at JoVE.com

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.

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 ...

The overall goal of this protocol is to provide an efficient and rapid synthetic method for radioactive iodine labeled gold nano particles using the strain promoted copper free click reaction. This method can help answer key questions in the radiochemistry field such as the synthesis of radio isotope labeled about three years as well as regular imaging probes for pad or spec imaging studies. The main advantage of this technique is that one to five iodine labeled with golden particles can be efficiently synthesized the using of azide prosthetic group with excellent radiochemical yield and radiochemical purity.Visual demonstration of this method is critical as the radio evaluation steps are difficult to learn. Since such procedures require a suitable facility and handle radio labeling experience. Along with the study, radio student Ha Eun Shim will be demonstrating the procedure.To perform the radio iodination reaction, add a precursor solution to a 1.5 mL tube. Add 10 microliters of acetic acid to the precursor solution at room temperature. Then add 150 mega bekarel of iodine 125 in 50 microliters, 0.1 molar sodium hydroxide to the reaction mixture.Add a chloramine T solution and close the microcentrifuge tube containing the reaction mixture. Incubate the reaction mixture at room temperature for fifteen minutes until the radio iodination reaction is complete. Then add a sodium metabisulfite solution to the reaction mixture to quench the radio iodination reaction.Withdraw 0.2 microliters of the crude product and then dilute it with 100 microliters of 50%aceto nitryl in water for high performance liquid chromatography, or HPLC analysis. Analyze the diluted crude product by using a reversed phase analytical radio HPLC. Transfer the entire reaction mixture to an HPLC vial.Rinse the reaction tube with 0.5 mL of aceto nitryl and add the rinse into the same injection vial. Dilute the collected solution with one mL of water. To purify the crude product with reparative HPLC, inject the crude product onto a preparative radio HPLC.Collect the radioactive peak representing the iodine 125 labeled azide in a glass test tube. Measure the radio chemical yield of the fraction using a radioactivity dose calibrator according to the manufacturer's protocol. Then inject the purified product onto an analytical radio HPLC using the same HPLC conditions for determining the radio chemical purity of the product.To perform solid phase extraction of the product, dilute the fraction containing the desired product with 40 mL of pure water. Add the diluted solution into a preconditioned TC18 cartridge. Wash the cartridge with an additional 15 mL of water.Elute the product trapped in the cartridge with 2 mL of acetone into a 10 mL glass vial that is protected by a lead shield. Measure the radioactivity of the eluted product using radioactivity dose calibrator according to the manufacturer's protocol. After evaporating the acetone dissolve the residue with 100 to 200 microliters of DMSO for the next radio labeling step.Perform the synthesis of DBCO group modified gold nanoparticles as described in the text protocol. Prepare a concentrated solution of the DBCO group modified gold nanoparticles by using centrifugation. And adjust the concentration of the gold nano particles to two micro molar.Add 4.1 mega bekarel of iodine 125 labeled azide in 5 microliters of DMSO to a suspension of 50 microliters of 2 micro molar gold nanoparticles. Incubate the resulting reaction mixture at 40 degrees Celsius for 60 minutes. Withdraw a 0.2 microliter alloquot from the crude product and apply it onto a silica coded thin layer chromatography or TLC plate.Develop the TLC plate using ethyl acetate as a mobile phase. Place the TLC plate on a radio TLC scanner and run the scanner to monitor the radio labeling reaction according to the manufacturer's protocol. Purify the reaction mixture containing the iodine 125 labeled gold nanoparticles by centrifugation.Decant the supernatant and add pure water for resuspension of the gold nanoparticle pellets. Withdraw a 0.2 microliter alloquot from the purified product and apply it onto a silica coated TLC plate. Develop the TLC plate using ethyl acetate as the mobile phase.Place the TLC plate on a radio TLC scanner and run the scanner to determine the radiochemical yield and radiochemical purity of the iodine 125 labeled gold nanoparticles, according to the manufacturer's protocol. Representative results of the iodine 125 labeled azide prosthetic group are shown here. A 75%radiochemical yield was determined by using a radioactivity dose calibrator.The analytical radio HPLC result shows excellent radiochemical purity of the product. Representative results of the iodine 125 labeled gold nanoparticle are shown here. Radio TLC analysis presents that both radiochemical yield and purity of the purified gold nano particle were more than 95%Once mastered this technique can be done in three hours if it is performed properly.The implications of this technique extended toward preparation of various molecular probes for nuclear imaging therapeutic purposes because the labeling chemistry in the present method is highly efficient and straightforward. Don't forget that working with radioactive iodine can be extremely hazardous and the precautions such as lighter bricks or lighter shoes should always be taken while performing this procedure.

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Chemistry

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing ...

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated &#62;300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.

We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats.

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like ...

Preparation of a Corannulene-functionalized Hexahelicene by Copper(I)-catalyzed Alkyne-azide Cycloaddition of Nonplanar Polyaromatic Units

The main purpose of this video is to show 6 reaction steps of a convergent synthesis and prepare a complex molecule containing up to three nonplanar polyaromatic units, which are two corannulene moieties and a racemic hexahelicene linking them. The compound described in this work is a good host for fullerenes. Several common organic reactions, such as free-radical reactions, C-C coupling or click chemistry, are employed demonstrating the versatility of functionalization that this compound can accept. All of these reactions work for planar aromatic molecules. With subtle modifications, it is possible to achieve similar results for nonplanar polyaromatic compounds.

Preparation of a Corannulene-functionalized Hexahelicene by CopperI-catalyzed Alkyne-azide Cycloaddition of Nonplanar Polyaromatic Units

Here, we present a protocol to synthesize a complex organic compound comprised of three nonplanar polyaromatic units, assembled easily with reasonable yields.

The main purpose of this video is to show 6 reaction steps of a convergent synthesis and prepare a complex molecule containing up to three nonplanar ...

A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray coating. These nanoparticles are produced by the polymerization reaction of alkyl cyanoacrylate on the surface of cereal protein (gliadin) molecules. Alkyl cyanoacrylate is a monomer that instantly polymerizes at RT when it is applied to the surface of materials. Its polymerization reaction is initiated by the trace amounts of weakly basic or nucleophilic species on the surface, including moisture. Once polymerized, the polymerized alkyl cyanoacrylates show a strong affinity with the object materials because nitrile groups are in the backbone of poly (alkyl cyanoacrylate). Proteins also work as initiator for this polymerization because they contain amine groups that can initiate the polymerization of cyanoacrylate. If aggregated protein is used as an initiator, protein aggregate is surrounded by the hydrophobic poly(alkyl cyanoacrylate) chains after the polymerization reaction of alkyl cyanoacrylate. By controlling the experimental condition, particles in the nanometer range are produced. The produced nanoparticles readily adsorb to the surface of most materials including glass, metals, plastics, wood, leather, and fabrics. When the surface of a material is sprayed with the produced nanoparticle suspension and rinsed with water, the micellar structure of nanoparticle changes its conformation, and the hydrophilic proteins are exposed to the air. As a result, the nanoparticle-coated surface changes to hydrophilic.

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic. The produced nanoparticle is an assembly of gliadin-cyanoacrylate diblock copolymers. Spray coating with the produced nanoparticle changes the surface of target material to a hydrophilic surface.

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray ...

A Simple and Efficient Protocol for the Catalytic Insertion Polymerization of Functional Norbornenes

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic nature of the monomer is conserved. The resulting polymer, polynorbornene, has a very high glass transition temperature, Tg, and interesting optical and electrical properties. However, the polymerization of functional norbornenes by this mechanism is complicated by the fact that the endo substituted norbornene monomer has, in general, a very low reactivity. Furthermore, the separation of the endo substituted monomer from the exo monomer is a tedious task. Here, we present a simple protocol for the polymerization of substituted norbornenes (endo:exo ca. 80:20) bearing either a carboxylic acid or a pendant double bond. The process does not require that both isomers be separated, and proceeds with low catalyst loadings (0.01 to 0.02 mol%). The polymer bearing pendant double bonds can be further transformed in high yield, to afford a polymer bearing pendant epoxy groups. These simple procedures can be applied to prepare polynorbornenes with a variety of functional groups, such as esters, alcohols, imides, double bonds, carboxylic acids, bromo-alkyls, aldehydes and anhydrides.

We describe the catalytic insertion polymerization of 5-norbornene-2-carboxylic acid and 5-vinyl-2-norbornene to form functional polymers with a very high glass transition temperature.

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em = 640 nm) of BSA-Au(III) complexes. The method adjusts the pH to show that the emergence of the red fluorescence is correlated with the pH-induced equilibrium transitions of the BSA conformations. Red fluorescent BSA-Au(III) complexes can only be formed with an adjustment of pH at or above 9.7, which corresponds to the &#34;A-form&#34; conformation of BSA. The protocol to adjust the BSA to Au molar ratio and to monitor the time-course of the process of Au(III) binding is described. The minimum number of Au(III) per BSA, to produce the red fluorescence, is less than seven. We describe the protocol in steps to illustrate the presence of multiple Au(III) binding sites in BSA. First, by adding copper (Cu(II)) or nickel (Ni(II)) cations followed by Au(III), this method reveals a binding site for Au(III) that is not the red fluorophore. Second, by modifying BSA by thiol capping agents, another nonfluorophore-forming Au(III) binding site is revealed. Third, changing the BSA conformation by cleaving and capping of the disulfide bonds, the possible Au(III) binding site(s) are illustrated. The protocol described, to control the BSA conformations and Au(III) binding, can be generally applied to study the interactions of other proteins and metal cations.

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

The protocols for studying the binding of gold cations (Au(III)) to various conformations of bovine serum albumin (BSA) as well as for characterizing the conformational dependent unique BSA-Au fluorescence are presented.

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em ...

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril

This work describes a rapid and highly sensitive method for the quantitative detection of an important biomarker, uric acid (UA), via surface-enhanced Raman spectroscopy (SERS) with a low detection limit of ~0.2 &#956;M for multiple characteristic peaks in the fingerprint region, using a modular spectrometer. This biosensing scheme is mediated by the host-guest complexation between a macrocycle, cucurbit[7]uril (CB7), and UA, and the subsequent formation of precise plasmonic nanojunctions within the self-assembled Au NP: CB7 nanoaggregates. A facile Au NP synthesis of desirable sizes for SERS substrates has also been performed based on the classical citrate-reduction approach with an option to be facilitated using a lab-built automated synthesizer. This protocol can be readily extended to multiplexed detection of biomarkers in body fluids for clinical applications.

Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril

A host-guest complex of cucurbit[7]uril and uric acid was formed in an aqueous solution before adding a small amount into Au NP solution for quantitative surface-enhanced Raman spectroscopy (SERS) sensing using a modular spectrometer.

This work describes a rapid and highly sensitive method for the quantitative detection of an important biomarker, uric acid (UA), via surface-enhanced ...