Name: solubilization and bio-conjugation of quantum dots and bacterial toxicity assays by growth curve and plate count
Uploaded: 2012-07-11T13:00:00
Description: Watch this Scientific Journal Video about Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count at JoVE.com

This work was funded by the NSERC Individual Discovery program, the NSERC/CIHR Collaborative Health Research Program (CHRP), and the NSERC CREATE Canadian Astrobiology Training Program (CATP).

1. QD Solubilization
This is a method appropriate for CdTe. Similar methods can be used with other types of QDs such as InP/ZnS20 and CdSe/ZnS 21.
<ol>
 <li>Prepare a solution of CdTe QDs in toluene at 15 &mu;M (optical density = 2.5 at the first exciton peak).</li>
 <li>Transfer 200 &mu;L of this stock into a glass vial. Do not use plastic!</li>
 <li>Add 800 &mu;L toluene, 1 mL of 200 mM borate buffer (pH 9) and 2 &mu;L of 11.5 M mercaptopropionic acid (MPA).</li>
 <li...

<ol>
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Nanoparticles represent a promising approach to creation of new anti-microbial agents. Growth curve analysis is a way to monitor bacterial cell density that distinguishes actively-growing cells from growth-inhibited cells. When coupled with plate counts, it allows for a thorough analysis of the antibiotic potential of a conjugate. The 96-well format permits relatively high-throughput variations of concentration and other conditions such as light exposure; the latter is crucial for light-activated agents such as quantum d...

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Borate Buffer Component #1</td>
			<td>Fisher</td>
			<td>Boric acid A-74-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Borate Buffer Component #2</td>
			<td>Sigma-Aldrich</td>
			<td>Sodium Tetraborate B9876</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MPA</td>
			<td>Sigma-Aldrich</td>
			<td>M5801</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vivaspin 500</td>
			<td>GE Healthcare</td>
			<td>28-9322</td>
			<td>Various MWCO available</td>
		</tr>
		<tr>
			<td>Glass vials</td>
			<td>Fisher</td>
			<td>03-338C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDC</td>
			<td>Sigma-Aldrich</td>
			<td>E6383</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polymyxin B</td>
			<td>Sigma-Aldrich</td>
			<td>P1004</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bacterial growth medium (LB) Component #1</td>
			<td>Fisher</td>
			<td>NaCl S271</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bacterial growth medium (LB) Component #2</td>
			<td>BD</td>
			<td>Tryptone 211705</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bacterial growth medium (LB) Component #3</td>
			<td>BD</td>
			<td>Yeast Extract 211929</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lamp for light exposure</td>
			<td>Custom</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Clear-bottom 96-well plates</td>
			<td>Fisher</td>
			<td>07-200-567 or 07-200-730</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescence spectrometer</td>
			<td>Molecular Devices</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Absorbance plate reader</td>
			<td>Molecular Devices</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BactoAgar for solid media</td>
			<td>Bioshop</td>
			<td>AGR001.1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Petri dishes round</td>
			<td>Fisher</td>
			<td>08-75-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Petri dishes rectangular</td>
			<td>Fisher</td>
			<td>08-757-11A</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

solubilization and bio-conjugation of quantum dots and bacterial toxicity assays by growth curve and plate count

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. QDs have attracted a lot of interest for imaging, diagnostics, and therapy due to their bright, stable fluorescence1,2 3,4,5. QDs can be conjugated to a variety of bio-active molecules for binding to bacteria and mammalian cells6.
QDs are also being widely investigated as cytotoxic agents for targeted killing of bacteria. The emergence of multiply-resistant bacterial strains is rapidly becoming a public health crisis, particularly in the case of Gram negative pathogens 7. Because of the well-known antimicrobial effect of certain nanomaterials, especially Ag, there are hundreds of studies examining the toxicity of nanoparticles to bacteria 8. Bacterial studies have been performed with other types of semiconductor nanoparticles as well, especially TiO2 9,10-11, but also ZnO12 and others including CuO 13. Some comparisons of bacterial strains have been performed in these studies, usually comparing a Gram negative strain with a Gram positive. With all of these particles, mechanisms of toxicity are attributed to oxidation: either the photogeneration of reactive oxygen species (ROS) by the particles or the direct release of metal ions that can cause oxidative toxicity. Even with these materials, results of different studies vary greatly. In some studies the Gram positive test strain is reportedly more sensitive than the Gram negative 10; in others it is the opposite 14. These studies have been well reviewed 15.
In all nanoparticle studies, particle composition, size, surface chemistry, sample aging/breakdown, and wavelength, power, and duration of light exposure can all dramatically affect the results. In addition, synthesis byproducts and solvents must be considered16 17. High-throughput screening techniques are needed to be able to develop effective new nanomedicine agents.
CdTe QDs have anti-microbial effects alone18 or in combination with antibiotics. In a previous study, we showed that coupling of antibiotics to CdTe can increase toxicity to bacteria but decrease toxicity to mammalian cells, due to decreased production of reactive oxygen species from the conjugates19. Although it is unlikely that cadmium-containing compounds will be approved for use in humans, such preparations could be used for disinfection of surfaces or sterilization of water.
In this protocol, we give a straightforward approach to solubilizing CdTe QDs with mercaptopropionic acid (MPA). The QDs are ready to use within an hour. We then demonstrate coupling to an antimicrobial agent.
The second part of the protocol demonstrates a 96-well bacterial inhibition assay using the conjugated and unconjugated QDs. The optical density is read over many hours, permitting the effects of QD addition and light exposure to be evaluated immediately as well as after a recovery period. We also illustrate a colony count for quantifying bacterial survival.

Watch this Scientific Journal Video about Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count at JoVE.com

Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count

Nanoparticles such as semiconductor quantum dots (QDs) can be used to create photoactivatable agents for anti-microbial or anti-cancer applications. This technique shows how to water-solubilize cadmium telluride (CdTe) QDs, conjugate them to an antibiotic, and perform a bacterial inhibition assay based upon growth curves and plate count.

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. ...

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