Name: luminophore formation in various conformations of bovine serum albumin by binding of gold(iii)
Uploaded: 2018-08-31T15:00:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII at JoVE.com

S.E. acknowledges the support from Duke Endowment Special Initiative Fund, Wells Fargo Fund, PhRMA Foundation, as well as Startup Funds from the University of North Carolina, Charlotte.

1. Synthesis of BSA-Au(III) Complex
<ol>
	<li>Dissolve 25 mg of BSA in 1 mL of high-performance liquid chromatography (HPLC) grade water in a 5 mL reaction vial. 
	NOTE: The solution should appear clear.</li>
	<li>Dissolve gold (III) chloride trihydrate (chloroauric acid) to a concentration of 5 mM in HPLC grade water. 
	NOTE: The solution should appear yellow. Chloroauric acid solution prepared at this concentration will result in a BSA to Au ratio of 1:13.
	<ol>
		<li>Alternatively, prepare a solution of chloroauric acid with a concentration of anywhere between 0.38 mM (BSA:Au = 1:1) to 20 mM (BSA:Au = 1 : 50) in HPLC grade water. 
		NOTE: Different ratios of BSA to gold will result in drastically different red fluorescence patterns of the excitation-emission map.</li>
	</ol>
	</li>
	<li>Place the reaction vial of BSA in a 37 &#176;C water bath and vigorously stir at 750 rpm using a magnetic stirrer.</li>
	<li>Immediately after the stirring begins, add 1 mL of chloroauric acid to the solution. The color of the solution should transform from clear to yellow.</li>
	<li>Stir the mixture for 2 min at 37 &#176;C and at 750 rpm using a magnetic stirrer.</li>
	<li>Into the reaction vial, add 100 &#956;L of 1 M NaOH to the solution to bring the pH to 12. 
	NOTE: Immediately after NaOH is added, the solution should darken slightly to a yellow-brown and then turn back to yellow.</li>
	<li>Continue to stir at 750 rpm for 2 h and at 37 &#176;C. The solution should slowly change from yellow to a dark yellow/brown color. This color change indicates the formation of the red fluorescing BSA-Au(III) complex.</li>
	<li>Allow the sample to sit at room temperature for 2 days, and the solution will continue to darken to an amber brown and the fluorescence intensity will increase.
	<ol>
		<li>Alternatively, let the sample continue to stir at 37 &#176;C for 12 more h as the color of the solution evolves to an amber brown.</li>
	</ol>
	</li>
</ol>
2. Synthesis of BSA-Cu(II)-Au(III)
<ol>
	<li>Dissolve 25 mg of BSA in 1 mL of HPLC grade water. The solution should appear clear.</li>
	<li>Dissolve copper (II) chloride dihydrate in HPLC grade water to a concentration of 5 mM. The solution should appear light blue.</li>
	<li>Add 1 mL of the aqueous BSA solution to a 5 mL reaction vial and place in a water bath at 37 &#176;C. Stir the mixture at 750 rpm.</li>
	<li>Immediately add 0.5 mL of the copper (II) chloride dihydrate solution to the reaction vial and mix for 2 min. The solution will remain light blue.</li>
	<li>Add 75 &#956;L of 1 M NaOH to bring the pH to 12 and allow to mix for 2 h. The solution will become purple.</li>
	<li>Dissolve chloroauric acid in HPLC grade water to a concentration of 5 mM.</li>
	<li>Add 0.5 mL of aqueous chloroauric acid to the reaction vial and adjust the pH back to 12 using 1 M NaOH.</li>
	<li>Stir the reaction mixture for 2 h. 
	NOTE: The solution should evolve to a brown color.</li>
</ol>
3. Synthesis of BSA-Ni(II)-Au(III)
<ol>
	<li>Dissolve 25 mg of BSA in 1 mL of HPLC grade water. The solution should appear clear.</li>
	<li>Dissolve nickel (II) chloride hexahydrate in HPLC grade water to a concentration of 5 mM. The solution should appear light green.</li>
	<li>Add 1 mL of the aqueous BSA solution to a 5 mL reaction vial and place in a water bath at 37 oC. Stir the mixture at 750 rpm.</li>
	<li>Immediately add 0.5 mL of the nickel (II) chloride hexahydrate solution to the reaction vial and mix for 2 min. 
	NOTE: The solution will remain light green.</li>
	<li>Add 75 &#956;L of 1 M NaOH to bring the pH to 12 and allow to mix for 2 h. 
	NOTE: The solution will become dark yellow.</li>
	<li>Dissolve chloroauric acid in HPLC grade water to a concentration of 5 mM.</li>
	<li>Add 0.5 mL of the aqueous chloroauric acid to the reaction vial and adjust the pH back to 12.</li>
	<li>Stir the reaction mixture for 2 h. 
	NOTE: The solution should evolve to a brown color.</li>
</ol>
4. Synthesis of [Cys34-capped-BSA]-Au(III)
<ol>
	<li>Dissolve 2 mg of N-ethylmaleimide (NEM) in 1 mL of phosphate buffered saline (PBS, pH 7.4).</li>
	<li>Dissolve 2 mg of BSA in 1 mL of PBS-NEM solution.</li>
	<li>Transfer the solution to a 5 mL reaction vial and stir at 20 &#176;C at 500 rpm for 1 h.</li>
	<li>Dialyze the solution using 12 kDa dialysis tubing in 500 mL of PBS, stirring at 50 rpm with a magnetic stirrer overnight to remove unreacted NEM.</li>
	<li>Dissolve chloroauric acid in PBS to a concentration of 0.4 mM. 
	NOTE: The solution will be a faint yellow.</li>
	<li>Transfer the reaction vial to a water bath at 37 &#176;C. Stir at 750 rpm.</li>
	<li>Immediately add 1 mL of chloroauric acid solution to the reaction vial and allow to mix for 2 min.</li>
	<li>Add 75 &#956;L of 1 M NaOH to the reaction vial to bring the pH to 12 and allow to mix for 2 h.</li>
</ol>
5. Synthesis of [all-thiol-capped-BSA]-Au(III)
<ol>
	<li>Prepare a solution of 2 M urea and 50 mM ammonium bicarbonate (NH4HCO3, pH 8.0) in HPLC grade water.</li>
	<li>Dissolve 3.3 mg of BSA in 1 mL of the above solution and transfer to a 5 mL reaction vial.</li>
	<li>Make a stock solution of 0.25 M tris(2-carboxyethyl)phosphine (TCEP) by dissolving 62.5 mg of TCEP in 1 mL of HPLC water.</li>
	<li>Add the stock solution of TCEP to the reaction vial until the final concentration of TCEP is 8 mM.</li>
	<li>Incubate the solution in a water bath for 1 h at 50 &#176;C. Stir at 500 rpm using a magnetic stirrer.</li>
	<li>Allow the solution to completely cool to room temperature.</li>
	<li>Prepare a stock solution of 100 mM NEM by dissolving 12.5 mg of NEM in 1 mL of HPLC grade water.</li>
	<li>Add the stock solution of NEM to the reaction vial until the final concentration of NEM is 16 mM.</li>
	<li>Allow the solution to combine for 2 h at 20 &#176;C. Stir at 500 rpm.</li>
	<li>Dialyze the solution using a 12 kDa dialysis tubing, stirring the solution at 50 rpm with a magnetic stirrer overnight in 500 mL of 50 mM NH4HCO3 to remove excess TCEP, NEM, and urea.</li>
	<li>Move the reaction vial to a water bath at 37 &#176;C and stir at 750 rpm.</li>
	<li>Dissolve chloroauric acid in HPLC grade water to a concentration of 0.66 mM.</li>
	<li>Immediately add 1 mL of chloroauric acid solution to the reaction vial and allow to mix for 2 min.</li>
	<li>Add 1 M NaOH until the pH of the solution is 12 and allow the solution to continue to mix for 2 h.</li>
</ol>

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The authors have nothing to disclose.

The BSA-Au(III) compounds prepared at pH 12 exhibit red fluorescence at an emission wavelength of &#955;em= 640 nm when excited with ultraviolet (UV) light &#955;ex= 365 nm (Figure 1A). The emergence of red fluorescence is a slow process and will take a few days at room temperature to increase to a maximum intensity. Running the reaction at 37 &#176;C will yield the optimum results, though higher temperature can be used to produce the red fluorescence faster. I...

A BSA-Au compound exhibiting an ultraviolet (UV)-excitable red fluorescence, with remarkable stokes shift, has been originally synthesized by Xie et al.1. The unique and stable red fluorescence can find various applications in fields such as sensing2,3,4, imaging5,6,7, or nanomedicine8,9,10,11,12,13. This compound has been studied extensively by many researchers in the field of nano-science in recent years14,15,16. The BSA-Au compound has been interpreted as Au25 nanoclusters. The goal of the presented method is to examine this compound in detail and to understand the origin of the red fluorescence. By following the presented approach, the presence of multiple Au binding sites, and the origin of fluorescence, alternative to the single-site nucleation of Au25 nanoclusters, can be illustrated. The same approach can be employed to study how other proteins17,18,19 complexed with Au(III) can change their intrinsic fluorescent properties.
The synthesis of the red-fluorescent BSA-Au compound requires a narrow control of the molar ratios of BSA to Au (BSA:Au) to maximize the intensity of the fluorescence and the location of the peaks in the excitation-emission map (EEM)20. It can be shown that multiple binding sites exist for Au(III) to bind, including the Asparagine fragment (or Asp fragment, the first four amino acid residues at the N-terminus of BSA)21,22. The 34th amino acid of BSA (Cys-34) is also shown to coordinate Au(III) and to be involved in the mechanism of the red fluorescence([Cys34-capped-BSA]-Au(III))20. Upon cleaving all Cys-Cys disulfide bonds and capping all thiols, red fluorescence is not produced ([all-thiol-capped-BSA]-Au(III)). This indicates the necessity of Cys-Cys disulfide bonds as the Au(III) binding site to produce the red fluorescence.
Protein chemistry techniques have not been widely used to study the BSA-Au(III) complexes in the nano-science community. However, it would be valuable to employ these techniques to understand certain aspects of these complexes, as well as to gain detailed understanding of the Au(III) binding sites in BSA. This article is intended to show some of these techniques.

<table><tbody><tr><td>Bovine Serum Albumin (BSA), 96%</td><td>Sigma-Aldrich</td><td>A5611</td><td></td></tr><tr><td>gold (III) chloride trihydrate, 99.9%</td><td>Sigma-Aldrich</td><td>520918</td><td></td></tr><tr><td>Copper (II) chloride dihydrate, 99.999%</td><td>Sigma-Aldrich</td><td>459097</td><td></td></tr><tr><td>Nickel (II) chloride hexahydrate, 99.9%</td><td>Sigma-Aldrich</td><td>654507</td><td></td></tr><tr><td>N-Ethylmaleimide (NEM), &gt;99.0%</td><td>Sigma-Aldrich</td><td>4259</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), &gt;98.0%</td><td>Sigma-Aldrich</td><td>C4706</td><td></td></tr><tr><td>Sodium hydroxide, &gt;98.0%</td><td>Sigma-Aldrich</td><td>S8045</td><td></td></tr><tr><td>Urea, 99.5%</td><td>Chem-Implex Int&#039;l</td><td>30142</td><td></td></tr><tr><td>Phospate buffered saline (PBS)</td><td>Corning</td><td>MT21040CV</td><td></td></tr><tr><td>Ammonium bicarbonate, 99.5%</td><td>Sigma-Aldrich</td><td>9830</td><td></td></tr></tbody></table>

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.

From the fluorescence of the BSA-Au(III) complex, it has been observed that the conversion of the intrinsic blue fluorescence of BSA (&#955;em = 400 nm) to red fluorescence (&#955;em = 640 nm) occurs at about pH 9.7 through an equilibrium transition (Figure 1). EEM of BSA-Au(III) at different BSA to Au molar ratios is shown in Figure 2, and this data shows how altering the molar ratios yiel...

Watch this Scientific Journal Video about Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII at JoVE.com

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.

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 ...

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6.8K Views.  University of North Carolina. 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.

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

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Biochemistry

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

Bioengineering

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a good source of macro- and micronutrients, bioactive peptides, and growth factors. Bovine colostrum is also a potential source of nutrition and bioactive because of its rich protein content that includes immunoglobulin G (IgG) and lactoferrin. However, the level of lactoferrin and IgG in bovine colostrum changes markedly during the lactation period. Therefore, monitoring the concentration of IgG and lactoferrin for the use of bovine colostrum as a protein source is an important question to study. Methods in this article describe how to determine protein content, as well as specific concentrations of lactoferrin and IgG. These methods include the following steps: Isolation of bovine colostrum proteins, Determination of protein concentration via Bicinchoninic acid assay (BCA), Visualization of proteins via SDS-PAGE, Determination of lactoferrin, and IgG concentration using an ELISA Assay.

Bovine colostrum is both a primary source of nutrients and immunological support for the newborn calf. The understanding of the level of therapeutic proteins (lactoferrin and IgG) is important for the bovine colostrum dosing and standardization for human consumption.

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a ...

Limited Proteolysis and Gel Electrophoresis in the Presence of Metal Cations: Au(III)-binding Luminescent Domain in Serum Albumins

The purpose of the presented protocols is to determine the domain of Au(III) binding in BSA. The BSA-Au(III) compound exhibits ultraviolet (UV)-excitable red luminescence (&#955;em = 640 nm), with unusual Stokes shifts compared to the innate UV/blue fluorescence arising from the aromatic residues. Red-luminescent complexes are formed in highly alkaline conditions above pH 10 and require a conformation change within the protein to occur. In addition, preservation of Cys-Cys disulfide bonds in BSA is necessary to obtain this red luminescence. In order to understand the mechanism of this luminescence, elucidation of the luminophore-forming Au(III) binding site is essential. A facile way to assess the luminophore-forming site would be to (1) predictably fragment the protein by enzymatic digestion, (2) react the obtained fragments with Au(III), then (3) perform gel electrophoresis to observe the well-separated fragment bands and analyze the in-gel red luminescence. However, due to the alkaline conditions and the reaction with metal cations, new limited proteolysis techniques and gel electrophoresis conditions must be applied. Particularly, the presence of metal cations in gel electrophoresis can make the band separations technically difficult. We describe this new protocol in steps to identify the red-luminophore-forming metal binding domain in BSA. This protocol can thus be applied for analyzing protein fragments that must remain in a non-denatured or a partially denatured state, in the presence of metal cations. Because the majority of proteins need metal cations to function, analyses of metal-bound proteins are often desired, which have relied on x-ray crystallography in the literature. This method, on the other hand, could be used in supplement to study the interactions of proteins with metal cations without requiring the protein crystallization and at a desired pH condition.

Limited Proteolysis and Gel Electrophoresis in the Presence of Metal Cations: AuIII-binding Luminescent Domain in Serum Albumins

We present a protocol for studying the binding domain of Au(III) in bovine serum albumin (BSA).

The purpose of the presented protocols is to determine the domain of Au(III) binding in BSA. The BSA-Au(III) compound exhibits ultraviolet (UV)-excitable ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Characterization of pH-Dependent Reversible Self-Assembly of Amyloid Beta 1-40-Coated Gold Colloids

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy and transmission electron microscopy (TEM). The observed pH-dependent shift of the SPR band of A&#946;1-40-coated 20 nm gold particles was correlated with alternation of aggregation and disaggregation observed in the TEM images. A pH of ~4&#160;induced an unfolded conformation, and a pH of ~10&#160;induced a folded conformation of A&#946;1-40 on the gold surface. This reversible aggregation process was observed by Raman imaging as the pH was gradually changed from pH 4 to pH 10. We observed a pH-dependent morphology change in A&#946;1-40 on the gold surface, where clear aggregates were observed at pH 4. However, we observed very subtle differences in the surface-enhanced Raman spectroscopy (SERS) spectrum between pH 4 and pH 10 conditions, with the most striking difference being spectral density in the region of 250 cm-1 and 1750 cm-1. Specifically, the mode analysis of the reversible aggregation indicated that the aggregates were formed by the unfolded conformation of A&#946;1-40 which involved the benzene ring section of Tyrosine and Phenylalanine. Conversely, the disassembly of the aggregates was associated with conformational changes in protein folding of A&#946;1-40 involving Histidine, Glutamine, Methionine, and Aspartic acid.

Here, we present a systematic method describing the quasi-reversible self-assembly of the amyloid beta 1-40 (A&#946;1-40)-coated 20 nm gold aggregates. The nano-size-dependent quasi-reversible networking between peptides was correlated with specific amino acids or sections of the A&#946;1-40 monomer.

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

Biology

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.