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&#38;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&#8242;-methylpolyethylene glycol 2000</td>
			<td>Sigma-Aldrich</td>
			<td>743127</td>
			<td>https://www.sigmaaldrich.com/catalog/product/aldrich/743127?lang=en&#38;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&#38;region=CZ&#38;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...

Watch this Scientific Journal Video about Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications at JoVE.com

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. 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.

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

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

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 ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...