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

Summary

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.

Abstract

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 fluorescence^{1,2 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 considered^{16 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.

Protocol

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.

1. Prepare a solution of CdTe QDs in toluene at 15 μM (optical density = 2.5 at the first exciton peak).
2. Transfer 200 μL of this stock into a glass vial. Do not use plastic!
3. Add 800 μL toluene, 1 mL of 200 mM borate buffer (pH 9) and 2 μL of 11.5 M mercaptopropionic acid (MPA).
4. Cap the vial and shake it vigorously for 30-60 seconds.
5. The aqueous and organic-solvent phases will separate spontaneously. The aqueous phase will beco....

Discussion

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.......

Materials

List of materials used in this article

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