The overall goal of this procedure is to develop a microarray based high throughput biofilm screen for antifungal drug discovery. This is accomplished by first chemically modifying the surface of microscope glass slides. Next yeast cells are encapsulated in a collagen matrix.
Then a candida albicans biofilm chip or cab chip is prepared using a robotic microarray. Finally, drug susceptibility is estimated using the cab chip. Ultimately, a viability assay performed using a microarray scanner shows the susceptibility profile of antifungal drugs against nano biofilms formed on a cab chipp.
The main advantage of this microarray based technique over the existing methods such as the industry standard 9 0 6 well plate, is that this is truly high throughput. Rapid, robust use a smaller working volume and saves time To prepare functionalized slides Begin by placing microscope slides in a removable slide rack. Wash the slides twice by immersing them in a staining jar containing 99%histological grade.Ethanol.
Then use paper towels to wipe the slides and compress nitrogen gas to dry them while wearing safety goggles and chemical resistant gloves. Immerse the slides in sulfuric acid and incubate overnight at room temperature the next day, sonicate the slides for 30 minutes, then wash them with milq water for 30 minutes, followed by acetone for five minutes. This treatment exposes the Tylenol groups on the glass surface to coat the slides.
Immerse them in three amino propyl triethyl or A-P-T-E-S solution for 30 minutes. Then wash them three times in Milli Q water for 15 each before baking the slides in a furnace at 110 degrees Celsius for 15 minutes. To cross link the A-P-T-E-S to apply a mono layer of hydrophobic coating working under a fume hood pipette two milliliters of polystyrene coma and hydride or PSMA and toluene onto a clean glass slide mounted in a spin coder and spin a 3000 RPM for 30 seconds.
Slides can be stored for up to one month in a dry dust-free environment. At two to eight degrees Celsius. Prepare an overnight culture of sea albican strain SC 53 14 by inoculating a single colony into 10 to 20 milliliters of YPD.
Incubate the culture overnight in an orbital shaker at approximately 150 to 200 RPM and 30 degrees Celsius. The next day. Harvest the cells by centrifugation and wash twice for 10 minutes each.
And one milliliter of PBS centrifuge the cells at 1900 RPM for 10 minutes and resuspend them in one milliliter of reconstruction buffer. Using a hemo cytometer under a brightfield microscope, count the cells and adjust the cell density to five times 10 to the seventh cells per milliliter. Use 10 X-R-P-M-I 1640 and mop supplemented with L-glutamine to further dilute the suspension 10 times.
Mix the cell suspension with type one collagen for a final concentration of four times 10 to the sixth cells per milliliter. Keep the suspension on ice to prevent the collagen from gelling before printing. Using 70%ethanol, clean and disinfect all surfaces of the microarray, including the source plate station, wash and vacuum station, vacuum slide platter and printing chamber place and hold by vacuum the desired number of PSMA coated glass slides on the slide deck of the micro rare just before printing vigorously.
Vortex the cell suspension in collagen and transfer 100 microliters into a well of a 96 well plate. Place this source plate in the loading station to maintain a relative humidity of 100%throughout the printing process. Switch on the humidifier using a microarray spotter with conically tapered 190 micron orifice, ceramic tips and non-contact deposition.
Print 50 nanoliter spots of cell suspension, 1.2 millimeters apart in an array of 48 rows and 16 columns. Prime rinse and vacuum. Dry the tips twice after each round of printing immediately after printing.
Place the slide in an airtight humidified chamber and incubate at 37 degrees Celsius for 24 hours to allow for biofilm formation to test the biofilms against antifungal agents. Begin by preparing working solutions of the drugs in our PMI 1640 medium repair. Eight different concentrations of each compound by diluting the stock solutions in twofold.
Dilutions spanning a range with the estimated IC 50 of the compound in the middle. After 24 hours of biofilm growth, use the micro rare to print 50 nanoliters of eight different concentrations of the drugs along with positive and negative controls, and at least six replicates on top of the biofilms. Incubate the chip with the drugs in a humidified chamber for 24 hours at 37 degrees Celsius.
Next, wash off the drugs by submerging the cab chip three to five times in PBS for two minutes each. Then incubate the chip with 0.5 micromolar fund one for 30 minutes at 37 degrees Celsius. Wash the cab chip three to five times in PBS and dry it under a nitrogen stream.
Use a microray scanner to read the fluorescence intensity. Set the fluorescence intensity of the positive control and dead biofilm spots at 100%and 0%respectively. Determine the percentage inhibition by the reduction in the fluorescence intensity relative to the average of the control spots using the following equation where FF max and F zero are.
Raw fluorescence, intensities of drug treated, no drug control and bleach treated spots respectively. Finally, calculate the 50%inhibitory concentration or IC 50 by fitting the variable slope hill. Equation shown here is a representative candida albicans chip consisting of a 48 by 16 array of nano biofilms.
Brightfield microscopy shows an overall architectural feature of the biofilms and the scanning electron microscope images reveal that the fungal hyphae, which are two microns and 100 nanometers in diameter, embed themselves within the matrix of the collagen fibers. The fun one, stained microarray scanner images show the yeast and FL forms which are characteristic of fungal biofilms. The 2D and 3D confocal fluorescence images of fun.
One stained biofilms can be seen to have spatial heterogeneity with regions of metabolically active cells interspersed with extracellular matrix, which are not stained by the metabolic eye. The thickness of the biofilm was estimated to be approximately 50 microns. The cab chip was used to estimate the antifungal susceptibility of fluconazole and amphotericin B as shown here consistent with published reports on industry standard 96 well plate assays, the biofilms are resistant against fluconazole and the calculated IC 50 for amphotericin B is 0.3 micrograms per milliliter Once mastered this technique could be done in 48 hours if it is performed properly.
