The overall goal of this procedure is to perform high throughput screening of antibacterial effects of nanoparticles with or without conjugation to antibiotics. Here, the antibacterial effects of poly mix and B, conjugated cadmium Telluride quantum dots are tested to accomplish this. The quantum dots are first solubilized in me, capto proprionic acid.
Next, the quantum dots are conjugated to poly mix and B, antibiotics plated bacteria are then treated with both antibiotic conjugated and unconjugated quantum dots, and half of the samples are subject to a radiation with 440 nanometer blue light for 30 minutes, which causes the quantum dots to generate exciton and reactive oxygen species to assess the effect of quantum dot treatments on bacterial growth and survival. Optical density readings are taken and colony counts are performed. The resulting data indicate that application of antibiotic conjugated cadium Telluride quantum dots, followed by I radiation yields the greatest tidal effects amongst the conditions tested.
Though this method can provide insight into the effect of cadmium telluride quantum dots and their conjugation to polyx in B on E coli growth. It can also be applied to other types of nanoparticles and antibiotics on various bacteria strains. Generally, individuals new to death method will struggle because any excess of MPA will make the quantum dots aggregate, and if they pipe out toline even a small amount, they will destroy the filter.
Although today we will demonstrate a method for assessing the antibacterial effects of antibiotic conjugated KE IDE quantum dots. Similar method could be used with other type of quantum dots such as indium phosphate, zinc sulfate, and cadmium ide zinc sulfate. Begin this procedure by preparing a solution of cadmium Telluride or CDTE quantum dots in toluene at 15 micromolar in a glass vial, measure the optical density or OD by absorbent spectroscopy using a pipette transfer 200 microliters of this stock into a glass vial.
Do not use plastic. Then add 800 microliters of toluene, one milliliter of 200 millimolar borate buffer, and two microliters of 11.5 molar MER capto proprionic acid or MPA cap the vial and shake it vigorously for 30 to 60 seconds. After shaking, the aqueous and organic solvent phases will separate spontaneously.
The aqueous phase will become the color of the quantum dots. To avoid the tiling layer, remove it, then remove the quantum dots phase and transfer it into a clean vial. To purify the solubilized quantum dots, transfer them to a 500 microliter centrifuge filter.
Add 100 microliters of 50 millimolar bore eight buffer and spin a 3000 times G for 13 minutes. Perform this wash four times after the final wash. Suspend the washed quantum dots in 500 microliters bore buffer, and store at four degrees Celsius.
They'll be stable for one to two weeks To estimate concentration of the quantum dots. Measure the absorbance and emission spectra. This figure shows CDTE quantum dots under UV lamp illumination and emission spectra before and after water solubilization showing negligible change in optical properties from the ligand exchange.
This part of the protocol is applicable to any negatively charged water solarized nanoparticle, including moss, commercial quantum dots, metal particles, and more. In this example, the antibiotic poly mixen B or PMB will be conjugated to the quantum dots At a molar ratio of 30 to one PMB is positively charged and self assembles without need for conjugation. Reagents dissolve PMB and water.
At 60 micromolar. For a 96 well plate with 0.3 milliliters bacteria per well in quad duplicates. 0.5 milliliters of 200 nano molar conjugate is sufficient to prepare this at 100 microliters of one micromolar quantum dots in 50 millimolar bore buffer to a micro centrifuge tube.
Next, add 50 microliters of the 10 x or 60 micromolar PMB antibiotic solution in water to the conjugate tube. Add 50 microliters water to the control tube. Bring up the volume to 0.5 milliliters with 50 millimolar ate buffer shield tubes from light with a aluminum foil and place them on a rotator for one hour.
If aggregation occurs, repeat the conjugation with lower concentrations of antibiotic. Titrate the concentration upwards until the particles do not aggregate to wash the conjugates. Pour the solutions through a 10, 000 molecular weight cutoff filter, which works for most antibiotics but not for proteins or antibodies.
Finally, estimate the number of antibiotic molecules per quantum. by absorbence or fluorescent spectroscopy gel electrophoresis or for your transform infrared spectroscopy. This figure shows the typical absorbance and emission spectra of the quantum dots before conjugation of PMB and the emission after the addition of 160 molar equivalents of PMB.
The relationship between the ratio of PMB and quantum dot emission intensity indicated by the squares and peak wavelength location indicated by the triangles is shown here. As the PMB concentration increases, the QD emission intensity decreases, resulting in a shift in the peak emission wavelength. The next part of the protocol is performed to determine the half max inhibitory concentration or IC 50 of the antibiotic and quantum toxicity in the evening two days before the conjugation experiment.
Seed 10 milliliters of LB medium from a fresh colony of e coli. The next day, one to two hours before the experiment fill each of the wells of a clear 96 well plate. With growth medium, the assay plate should be arranged as shown here.
Samples should be plated in triplicate and concentrations of antibiotics. Should span a wide enough range to include one concentration that inhibits the bacteria very little and one that kills them all wells containing quantum dots at the concentrations to be used in the conjugate Experiments should also be included in triplicate. Using a multi-channel pipette, see the appropriate wells with one to 50 microliters of bacterial culture.
The concentration will depend on how fast the particular strain grows and will need to be calibrated accordingly. To monitor growth, place the plate onto a plate reader and set it to read every 10 minutes for two hours at 600 nanometers. When the cells all reach an OD of 0.1 to 0.15, remove the plate from the plate reader and stop the recording.
Next, add the PMB in quantum dots to the appropriate wells and triplicate shield half of the plate with aluminum foil. Then place the plate under a custom 440 nanometer lamp made from 2.4 milliwatt LEDs for 30 minutes to expose the illuminated side of the plate After light exposure, put the plate into the plate reader and record OD 600 every 10 minutes for five to 12 hours depending upon growth rate. Keep the temperature less than 32 degrees Celsius if possible to avoid drying of the cultures using software.
Plop the growth curves at a selected time point versus log concentration and determine the IC 50 of the antibiotic using the hill equation shown on the screen where H is the hill coefficient. Y max is the highest point of growth and Y min is a zero point also ideally on a plateau. 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 as shown here. 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 versus log antibiotic to give the IC 50 to evaluate quantum dots.
Toxicity survival versus the log of the quantum dots concentration may also be plotted, but achieving significant bacterial killing with quantum dots alone at the concentration used is rare. Once the toxicity of the quantum dots and antibiotics alone has been determined, the same protocol can be applied to determine the toxicity of the antibiotic. Conjugated quantum dots shown.
Here is an example layout for the assay. Note that the conjugates are tested in increasing concentrations in quad duplicate a bacteria only control strip, a drug only strip and sample wells for quantum dots only should be included. OD 600 readings can then be used to generate growth curves, and I see 50 values as before.
This figure shows an example of conjugates that are as toxic as antibiotic alone and an example of conjugates that are more toxic. To determine the number of colony forming units or CFU for each condition, choose one or more wells of the treated 96 well plate to be used for performing plate counts. Make a serial dilution of each of the selected bacterial samples with saline solution.
To do this pipette 180 microliters saline solution to appropriate columns. Transfer 20 microliters of the bacterial solution and diluted with 180 microliters saline solution. Change the pipette tip mix and transfer 20 microliters of diluted bacterial solution to 180 microliters saline solution.
Repeat this process six times. Next, take 10 microliters each from the last six dilutions and spot plate them on a rectangular auger plate. 36 spots can be plated on a rectangular plate.
Allow the plate to dry for 15 minutes on the bench after 15 minutes, incubate the plate at 37 degrees Celsius for 16 hours following the incubation. Count the colonies to determine colony forming units per milliliter. Multiply the number of colonies by the dilution factor and divide by the volume plated to determine the effect of treatments on the bacterial growth.
Cell density of e coli was measured at OD 600. Shown here are representative bacterial growth curves with different drug concentrations from zero to complete cell death. The open symbols are PMB only with concentrations given.
The solid symbols are C-D-T-E-P-M-B without irradiation and the half filled symbols are C-D-T-E-P-M-B with irradiation. Irradiation had no effect on the PMB only samples, so these curves were omitted for clarity shown. Here are plots of growth curve values at 200 minutes versus log PMB and fits to this equation to determine IC 50 values to control for the effects of light.
A curve is done with antibiotic only with 30 minutes of light exposure. This figure indicates bacterial survival at 200 minutes versus QD concentration using CDTE qds. Some toxicity is seen with light exposure, but too little to determine an IC 50 value.
This indicates that the toxicity of CDTE alone at concentrations used in conjugation experiment is negligible to compare the effects of different treatment on cell growth growth curve values at 200 minutes were plotted and fit to the equation shown earlier as shown here. C-D-T-E-P-M-B conjugates were more toxic than PMB alone. In comparison, golden nanoparticle A-U-P-M-B conjugates show no increased toxicity over PMB alone to determine the effect of treatments on the bacterial survival.
The number of colony forming units was determined for e coli seated in a 96 well plate treated with P-M-B-Q-D-P-M-B or QD alone with or without I radiation for 30 minutes, then incubated at 32 degrees Celsius for four hours. Serial dilutions of each bacterial sample were made with saline solution and 10 microliters of 100 x to 10 to the seventh X solutions were plated on auger plates. The plates were incubated at 37 degrees Celsius and colonies were counted after 16 hours.
In this example, plate columns A through C are bacteria treated with increasing concentrations of CDTE. Conjugates without irradiation and D through F are bacteria treated with increasing concentrations of conjugates. Along with 30 minutes of irradiation, the dilutions are shown along the rows.
The data indicate that upon irradiation, the conjugates have greater inhibition on bacterial growth compared to the conjugates without irradiation. Don't forget that ca reagents are extremely toxic and precautions such as wearing gloves, lab coats, and goggles should always be taking while performing this procedure. After watching this video, you should have a good understanding of how to perform high throughput screening of the antibacterial effect of nanoparticles with or without conjugation to antibiotics.
