Department of Biomedical Engineering, McGill University, Montreal, QC Canada
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Park, S., Chibli, H., Nadeau, J. Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count. J. Vis. Exp. (65), e3969, doi:10.3791/3969 (2012).
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.
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.
Representative results: Figure 1 shows an image of CdTe QDs under UV lamp illumination, and emission spectra before and after water solubilization, showing negligible change from the cap exchange. The size values are the core diameter measured by electron microscopy.
2. QD Conjugation to Antibiotic
This part of the protocol is applicable to any negatively-charged water-solubilized nanoparticle, including most commercial QDs, metal particles, and more19.
Representative results. In this example, PMB conjugation is characterized by changes in QD emission spectrum. Figure 2 shows the spectra of CdTe QDs with PMB addition.
3. Preparation of Bacteria for 96-well Screen; Determination of Antibiotic IC50
This is applicable to almost any bacterial strain grown in the appropriate medium18. The exact length of time the recordings should continue depends upon the bacterial growth rate. In our example, we use Escherichia coli grown in lysogeny broth (LB) medium.
where H is the Hill coefficient, ymax is the highest point of growth (ideally on a plateau), and ymin is the zero-point, also ideally on a plateau. It is unlikely that QDs alone will show much toxicity to the cells at the concentrations used, so a value will not be determined.
Representative results. At the end of the recording period, clear wells will indicate complete cell death, and a gradient of cell density should appear along increasing concentrations of the drug. The bacteria should show S-shaped growth curves (Figure 5 A); the location of the maximum plateau will vary greatly from strain to strain and also depends upon temperature. A given time point can be chosen as representative and the values plotted vs. Log[antibiotic] to give the IC50 (Figure 5 B). To evaluate QD toxicity, survival vs. Log[QD] may also be plotted, but achieving significant bacterial killing with QDs alone is rare (Figure 5 C).
4. Preparation of Bacteria for 96-well Screen with Antibiotic/QDs
Representative results. The combination of QDs and antibiotic may be less toxic than antibiotic alone; equally toxic; or more toxic. This can be quantified using the growth curves and IC50 measurements. Figure 7 shows an example of conjugates that equally toxic as antibiotic alone, and an example of conjugates that are more toxic.
5. Plate Count
Figure 8 shows an example CFU plate.
6. Representative Results
Figure 1. CdTe QDs. (A) Eight preparations of CdTe QDs illuminated with a UV wand (365 nm). (B) Absorbance and emission spectra of five selected sizes before and after water-solubilization. The dashed lines are spectra in toluene, the solid lines are in water.
Figure 2. Spectral and gel analysis of QD-PMB conjugates. Orange-emitting CdTe QDs were used for this example; the effects on other types of QDs will need to be evaluated for each experiment. (A) Typical absorbance (grey line) and emission spectra (black line) of the QDs before conjugation of PMB and the emission (dashed line) after the addition of 160 molar equivalents of PMB. (B) Relationship between the ratio of PMB and the QD emission intensity (squares) and peak wavelength location (triangles).
Figure 3. Suggested plate layout for control growth plate. A wide range of PMB and QD concentrations is represented. One half of the plate is irradiated (highlighted blue), and an identical half is protected from light. Click here to view larger figure.
Figure 4. Custom 96-LED lamp for uniform plate irradiation, showing appearance off and on. A typical hand-held UV lamp may also be used, but will not cover the entire plate uniformly.
Figure 5. Example results for control growth plate. (A) Representative bacterial growth curves with different drug concentrations, from 0 to complete cell death. The open symbols are PMB only with concentrations given; the solid symbols are CdTe-PMB without irradiation; and the half-filled symbols are CdTe-PMB with irradiation. Irradiation had no effect on the PMB-only samples, so these curves were omitted for clarity. All PMB-CdTe conjugates are 30:1 PMB:QD ratios. (B) Plots of growth curve values at 200 min vs. Log[PMB] and fits to Eq. (1). To control for the effects of light, a curve is done with antibiotic only with 30 min of light exposure. (C) Bacterial survival at 200 min vs. QD concentration, using CdTe QDs. Some toxicity is seen with light exposure, but too little to determine an IC50 value. Click here to view larger figure.
Figure 6. Suggested layout for conjugate test plate. The blue-highlighted half of the plate should be exposed to light, and the unhighlighted half is protected. Click here to view larger figure.
Figure 7. Example results for conjugate test plate. Growth curve values at 200 min were plotted and fit to Eq. (1). (A) CdTe-PMB conjugates show increased toxicity over PMB alone. (B) Gold nanoparticle Au-PMB conjugates show no increased toxicity over PMB alone.
Figure 8. Example of a CFU plate. E. coli seeded in a 96-well plate was treated with QD-PMB with or without irradiation for 30 min. then incubated at 32 °C for 4 hours. Serial dilutions of each bacterial sample were made with saline solution, and 10 μL of 100 X to 107 X dilutions were plated on agar plates. The plates were incubated at 37 °C and colonies were counted after 16 hours. The plate shows the dilutions along the rows as indicated; the columns are: (A) 0.06 μM PMB + 2 nM CdTe, (B) 0.12 μM PMB + 4 nM CdTe (C) 0.2 μM PMB + 6.7 nM CdTe, (D) 0.06 μM PMB + 2 nM CdTe irradiated, (E) 0.12 μM PMB + 4 nM CdTe irradiated, (F) 0.2 μM PMB + 6.7 nM CdTe irradiated.
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 dots. Many variations on this basic approach are possible, making it a versatile method for nanotoxicology.
No conflicts of interest declared.
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).
|Borate Buffer Component #1||Fisher||Boric acid A-74-1|
|Borate Buffer Component #2||Sigma-Aldrich||Sodium Tetraborate B9876|
|Vivaspin 500||GE Healthcare||28-9322||Various MWCO available|
|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|