The authors are grateful to Alzbeta Magdolenova for her help with flow cytometry. The authors acknowledge the financial support from GACR project Nr. 18-12533S. Microscopy was performed in the Laboratory of Confocal and Fluorescence Microscopy co-financed by the European Regional Development Fund and the state budget of the Czech Republic, projects no. CZ.1.05/4.1.00/16.0347 and CZ.2.16/3.1.00/21515, and supported by the Czech-BioImaging large RI project LM2015062.

1. Preparation of near-infrared emitting AuNCs (1)
<ol>
	<li>Add 7.8 mg (37.8 &#956;mol) thioctic acid (TA) and 60 &#956;L of 2 M NaOH to 23.4 mL of ultrapure water (resistivity 18.2 M&#937;.cm at 25 &#176;C) and stir (at least 1,000 rpm) until it dissolves completely (~15-20 min). For faster dissolution of TA, sonicate the mixture. For the synthesis, freshly prepared TA solution is recommended.</li>
	<li>Add 10.2&#160;&#956;L of HAuCl4&#183;3H2O (470 mg/mL) of aqueous solution to the solution.</li>
	<li>After 15 min, add 480 &#956;L of NaBH4 (1.9 mg/mL) under vigorous stirring (at least 1,000 rpm) and stir the reaction mixture under the same conditions overnight. 
	NOTE:&#160;Freshly prepare the NaBH4 solution in ice-cold ultrapure water and add to the reaction mixture immediately after preparation. 
	NOTE: The synthesis of AuNCs is easily scalable. Up to 2 L of AuNCs was synthesized in a single batch, without any change in the optical properties of the particles.</li>
	<li>(Critical) The next day, purify the solution by applying three cycles of centrifugation/filtration using a membrane filtration device with a molecular weight cut-off of 3 kDa. Without this purification procedure, the following step does not work properly.</li>
	<li>Add thiol-terminated polyethylene glycol (MW 2,000; 15.6 mg; 7.8 &#956;mol) to the solution, adjust the pH to 7-7.5 and stir the mixture overnight to obtain 1. Purify the dispersion by applying three cycles of centrifugation/filtration using a membrane filtration device with a molecular weight cut-off of 3 kDa. 
	NOTE: Adjustment of the pH to 7-7.5 is extremely important. Higher pH can result in a blue shift of emission maxima.</li>
</ol>
2. Conjugation of 3-(aminopropyl)triphenylphosphonium bromide (TPP) on the surface of 1
<ol>
	<li>Mix the 1 solution (24 mL) prepared in the previous step and 3-(aminopropyl)triphenylphosphonium bromide (12 mg, ~30 &#956;mol). Adjust the pH to 4.5 with 1 M HCl. 
	NOTE: 3-(Aminopropyl)triphenylphosphonium (TPP) bromide salt was prepared as described in the literature20.</li>
	<li>Start the reaction by adding an excess of N-(3-dimethyl-aminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride (EDC&#183;HCl) (60 mg, 312 &#956;mol). The pH of the solution will increase and should not be allowed to go beyond 6. Monitor the pH of the reaction mixture for the first hour. If the pH increases above 6, reduce it to 4.5&#8211;6 by adding 1 M HCl.</li>
	<li>Stir the reaction mixture overnight at room temperature.</li>
	<li>Purify the dispersion by applying three cycles of centrifugation/filtration using a membrane filtration device with a molecular weight cut-off of 3 kDa to obtain 2. Dilute 2 obtained here with ultrapure water to the initial volume of 24 mL. The concentration of Au in the solution is 200 &#956;g/mL.</li>
</ol>
3. Cell culture
<ol>
	<li>Culture HeLa cells (HPA culture collection) in Dulbecco&#8217;s modified Eagle&#8217;s Medium supplemented with 10% fetal bovine serum in 5% CO2 at 37 &#176;C.</li>
	<li>Split and passage the cells when they reach ~80% confluence. To minimize acquisition of new mutants, the number of cell propagations should not exceed 30.</li>
</ol>
4. AuNC internalization into HeLa cells
<ol>
	<li>Seed the cells in a 12-well plate at a density of 20,000 cells/mL (1 mL/well). The goal is to achieve ~50% confluence after 48 h.</li>
	<li>At 48 h post-seeding, aspirate the culture medium and add 400 &#181;L of complete culture medium (for untreated controls) or 500 &#181;g of nanoparticles in 400 &#181;L of complete cultured medium (for treated samples) to each well. Return the cultures to a 37 &#176;C incubator. 
	NOTE: Addition of high volumes of AuNC solution adversely affects the cell viability. AuNC solutions need to be concentrated. Thus 2 obtained in step 2.4 is concentrated 100 times. 40 mL AuNC was concentrated to 400 &#956;L. A 25 &#956;L aliquot of this concentrated solution was added to 400 &#181;L cell culture media to obtain the desired AuNC concentration.</li>
	<li>After 2 h of internalization, detach the cells by standard trypsinization according to the manufacturer&#8217;s protocol.</li>
	<li>Collect the samples in polypropylene microcentrifuge tubes and centrifuge for 5 min at 350 x g at 4 &#176;C.</li>
	<li>Prepare the following FCM buffer: pre-chilled phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM NaH2PO4, pH 7.4) supplemented with 2% bovine serum albumin at 4 &#176;C.</li>
	<li>Wash the pellets with 1 mL of FCM buffer and centrifuge for 5 min at 350 x g at 4 &#176;C.</li>
	<li>Resuspend the pellet in 500 &#181;L of FCM buffer and store samples at 4 &#176;C prior to analysis.</li>
</ol>
5. Flow cytometry analysis
<ol>
	<li>Filter all samples using a 5 mL polystyrene round-bottom tube with a cell-strainer cap.</li>
	<li>Before acquiring data using the instrument software, specify the cytometer configuration.</li>
	<li>Format all dot-plots and histograms for &#8216;Acquisitions&#8217;.</li>
	<li>Plot a two-parameter dot plot of the forward scatter area (FSC-A) and side scatter area (SSC-A) to show distribution of cells. To exclude doublets, create a two-parameter dot plot of FSC height (FSC-H) vs. FSC-A. To monitor the relative fluorescence intensity in the sample, plot a single-parameter histogram for the fluorescent channel area (FL-A). Use a linear scale to depict FSC and SSC data, and a logarithmic scale for all fluorescent parameters.</li>
	<li>Acquire untreated sample (without nanoparticles) at a low flow rate to minimize coincident events (if allowed by the instrument). During acquisition, adjust the photomultiplier tube (PMT) voltages to get the untreated population on scale on the FSC vs SSC plot. If necessary, adjust PMT voltages for the FL channel to place the unstained population on the left corner of the histogram.</li>
	<li>Select the specific &#8216;gate tab&#8217; in the software and draw an appropriate gate around the desired population. Cells inside the gate will move to the next checkpoint.</li>
	<li>Record 10,000 events per sample.</li>
	<li>Record all samples under the same instrument settings.</li>
	<li>Use an appropriate program to analyze the flow cytometry data. 
	NOTE: Some applications might require customized setup of filters. For filter exchange always follow the manufacture&#8217;s recommendations in the user guide. 
	NOTE: The experiment can be saved and reloaded to preserve the instrument settings and gating strategy. 
	NOTE: A side scatter height (SSC-H) vs. side scatter area (SSC-A) plot can be also used for doublet exclusion. This type of gating can be more sensitive as the FSC detector is not usually a PMT.</li>
</ol>
6. Internalization of 2 into HeLa cells for confocal laser scanning microscopy (CLSM)
<ol>
	<li>Seed the cells onto a 4-chamber glass bottom 35 mm dish at a density of 250,000 cells/mL (0.5 mL/chamber). Keep the chamber in a 37 &#176;C incubator with 5% CO2 atmosphere. The goal is to achieve ~50% confluence after 24 h.</li>
	<li>At 24 h post-seeding, add 100 &#181;g of 2 (or 10 &#181;L from the stock solution of 10 mg/mL) to each dish chamber containing 0.5 mL of medium with the cells (for treated samples).</li>
	<li>Return the dish to the incubator. Let the cells internalize the AuNCs for 24 h prior to using them for CLSM.</li>
	<li>After the internalization period, discard the medium and wash the cells with pre-warmed fresh medium for 5 min. Repeat the washing step once more. Then fill each chamber with 800 &#181;L of fresh medium.</li>
</ol>
7. CLSM Imaging of live Hela cells labeled with 2
<ol>
	<li>For microscopic imaging, use 63x oil (n = 1.518) objective lens (NA = 1.4) in a confocal microscope with Plan-Apochromat.</li>
	<li>Mount the dish on the microscope inverted stage with the chamber warmed to 37 &#176;C and supplied with humidified 5% CO2 atmosphere.</li>
	<li>To detect internalized AuNC, use a 405 nm laser set at 2% power with an appropriate beam splitter. Set the range of detection wavelengths between 650 and 760 nm.</li>
	<li>Set the resolution of the image to 2048 x 2048 pixels. In the acquisition speed setting, aim for a pixel dwell time around 4 &#181;s. Acquire the image with 2x averaging (line mode, averaging method mean). Set the pinhole to 1 Airy unit (for 405 nm light). For higher sensitivity, use photon-counting mode.</li>
	<li>For correct illumination in transmitted light with differential interference contrast (DIC), use K&#246;hler&#8217;s setting of the condenser and the field stop. For acquisition of transmitted light, use a 488 nm laser at 0.7% power without any fluorescence detector assigned. Set an appropriate beam splitter for the laser wavelength.</li>
	<li>Acquire two images for each track (red fluorescence and DIC). Track AuNCs by their red fluorescence; cell boundaries are easily determined in the transmitted light with DIC pictures.</li>
</ol>

Some portions of the methods and results were previously presented in the article by Pramanik et al.18 Here, these methods have been converted into practical point-by-point protocols. The authors declare no competing financial interests.

NIR-emitting AuNCs were synthesized using a bottom-up approach in which the gold precursor solution (HAuCl4) was treated with suitable thiol ligands, followed by reduction of Au3+. Reduction of metal ions in aqueous solution tend to aggregate and results in large nanoparticles rather than ultrasmall NCs21. To prepare ultrasmall (&#8804;2 nm) PL AuNCs, the synthetic conditions were adjusted to prevent formation of large particles and promote formation of ultrasmall clusters. T...

In the past decade, ultrasmall (&#8804; 2 nm) photoluminescent gold nanoclusters (PL AuNCs) have emerged as promising probes for both fundamental research and practical applications1,2,3,4,5,6,7,8,9,10. Their many desirable characteristics include high photostability, tunable emission maxima, long emission lifetimes, large Stokes shifts, low toxicity, good biocompatibility, renal clearance and facile bioconjugation. PL AuNCs can provide photoluminescence from the blue to the near-infrared (NIR) spectral region, depending on the number of atoms within the cluster11 and the nature of the surface ligand12. NIR (650-900 nm) emitting AuNCs are particularly promising for long-term in vitro and in vivo imaging of cells and tissues, as they offer high signal-to-noise ratio due to minimum overlap with intrinsic autofluorescence, weaker scattering and absorption, and high tissue penetration of NIR light13,14.
In recent years, various approaches that take advantage of Au-S covalent interactions have been developed to prepare NIR-PL AuNCs capped with a variety of thiol-containing ligands13,15,16,17. For biomedical applications, AuNCs must be functionalized with a biological component to facilitate binding interactions. Thus, AuNCs with high colloidal stability that are easily functionalizable in aqueous solvent are highly desirable. The overall goal of the current protocol is to describe a previously reported18 preparation of AuNCs with a functionalizable carboxylic acid group on the surface by employing thioctic acid and polyethylene glycol (PEG) in an aqueous environment in detail and their conjugation with molecules bearing a primary amine following the acid-amine coupling method. Because of the ease of synthesis and high reproducibility, this protocol can be used and adapted by researchers from non-chemistry backgrounds.
One of the key requisites for applications of AuNCs in biomedical research is the ability to observe and measure AuNCs inside cells. Among the methods available to monitor nanoparticle uptake by cells, flow cytometry (FCM) and confocal laser scanning microscopy (CLSM) offer robust, high-throughput methods which allow fast measurements of internalization of fluorescent nanomaterials in large number of cells19. Here, FCM and CLSM method for direct measurement and analysis of PL AuNCs inside cells, without the need for additional dyes, have also been presented.

<table><tbody><tr><td>1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride</td><td>TCI Chemicals</td><td>D1601</td><td>https://www.tcichemicals.com/eshop/en/eu/commodity/D1601/;jsessionid=3AD046E5389206AAE33C8AAB5036CDD6?gclid=CjwKCAjwiZnnBRBQEiwAcWKfYrO69K6Np3tYeSsAouqGndUvzzsy1hStBPuHG-X3cpTIsAqq9z0cDBoC76MQAvD_BwE</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A4161</td><td>https://www.sigmaaldrich.com/catalog/product/sigma/a4161?lang=en&amp;amp;region=CZ</td></tr><tr><td>Disodium hydrogen phosphate dihydrate</td><td>PENTA s.r.o.</td><td>15130-31000</td><td>https://www.pentachemicals.eu/soubory/specifikace/specifikace_281.pdf</td></tr><tr><td>DL-Thioctic acid, 98%</td><td>Alfa Aesar</td><td>L04711</td><td>https://www.alfa.com/en/catalog/L04711/</td></tr><tr><td>Hydrochloric acid 35%</td><td>PENTA s.r.o.</td><td>19350-11000</td><td>https://www.pentachemicals.eu/soubory/specifikace/specifikace_512.pdf</td></tr><tr><td>Hydrogen tetrachloroaurate(III) trihydrate, ACS, 99.99% (metals basis), Au 49.0% min</td><td>Alfa Aesar</td><td>36400</td><td>https://www.alfa.com/en/catalog/036400/</td></tr><tr><td>O-(2-Mercaptoethyl)-O&amp;prime;-methylpolyethylene glycol 2000</td><td>Sigma-Aldrich</td><td>743127</td><td>https://www.sigmaaldrich.com/catalog/product/aldrich/743127?lang=en&amp;amp;region=CZ</td></tr><tr><td>Potassium chloride</td><td>PENTA s.r.o.</td><td>16200-31000</td><td>https://www.pentachemicals.eu/soubory/specifikace/specifikace_346.pdf</td></tr><tr><td>Sodium borohydride</td><td>Sigma-Aldrich</td><td>452882</td><td>https://www.sigmaaldrich.com/catalog/product/aldrich/452882?lang=en&amp;amp;region=CZ&amp;amp;gclid=CjwKCAjwiZnnBRBQEiwAcWKfYuoZKvdK_fH24F1gGugG4pamF2FFZLd36YyZmRTdGgkbm5SbyGP0jBoCoo0QAvD_BwE</td></tr><tr><td>Sodium chloride</td><td>PENTA s.r.o.</td><td>16610-31000</td><td>https://www.pentachemicals.eu/soubory/specifikace/specifikace_376.pdf</td></tr><tr><td>Sodium dihydrogenphosphate dihydrate</td><td>PENTA s.r.o.</td><td>12330-31000</td><td>https://www.pentachemicals.eu/soubory/specifikace/specifikace_124.pdf</td></tr><tr><td>Sodium hydroxide pellets</td><td>PENTA s.r.o.</td><td>15740-31000</td><td>https://www.pentachemicals.eu/soubory/specifikace/specifikace_307.pdf</td></tr><tr><td>XTT (sodium 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt)</td><td>Thermo Fisher Scientific</td><td>X12223</td><td>https://www.thermofisher.com/order/catalog/product/X12223#/X12223</td></tr></tbody></table>

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.

NIR PL AuNCs were prepared from Au3+ in the presence of TA, and then thiol-terminated PEG (MW 2,000) was bound on the AuNC surface to obtain 1 following the workflow shown in Figure 1. Amidic coupling between 1 and 3-(aminopropyl) triphenylphosphonium (TPP) bromide provided 2. As expected, absorption spectra (Figure 2a) indicated that AuNCs 1 and 2 do not have a chara...

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Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

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 ...

synthesis-near-infrared-emitting-gold-nanoclusters-for-biological

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JoVE Journal

Bioengineering

7.7K Views.  Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences. Dit is een eenvoudig protocol voor de synthese van functionalizable, nabij-infrarood emitterende fotoluminescente gouden nanoclusters en hun detectie met behulp van een commerciële setup. Een van de belangrijkste voordelen van deze techniek is dat de bevestiging van thiol-gefunctionaliseerde ligand en de koppeling van amine-gefunctionaliseerde ligand op het oppervlak van gouden nanoclusters geen negatieve invloed heeft op de fotoluminescente eigenschappen en spoelstabiliteit. Het kan worden ...

Video: Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications including contrast enhancement in novel magnetomotive imaging modalities, simultaneous capture&#160;and detection of circulating tumor cells (CTCs),&#160;and multimodal molecular imaging combined with photothermal therapy of cancer cells. These applications have stimulated significant interest in development of protocols for synthesis of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared (NIR) region and a strong magnetic moment. Here, we present a novel protocol for synthesis of such hybrid nanoparticles that is based on an oil-in-water microemulsion method. The unique feature of the protocol described herein is synthesis of magneto-plasmonic nanoparticles of various sizes from primary blocks which also have magneto-plasmonic characteristics. This approach yields nanoparticles with a high density of magnetic and plasmonic functionalities which are uniformly distributed throughout the nanoparticle volume. The hybrid nanoparticles can be easily functionalized by attaching antibodies through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.

Here, we describe a protocol for synthesis of magneto-plasmonic nanoparticles with a strong magnetic moment and a strong near-infrared (NIR) absorbance. The protocol also includes antibody conjugation to the nanoparticles through the Fc moiety for various biomedical applications which require molecular specific targeting.

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications ...

Chemistry

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

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 ...

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

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 ...

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 ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

Neuroscience

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

This article presents the integration of a spectral-focusing module and a dual-output pulse laser, enabling rapid hyperspectral imaging of gold nanoparticles and cancer cells. This work aims to demonstrate the details of multimodal nonlinear optical techniques on a standard laser scanning microscope.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

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