The overall goal of this polymer microarray technique is to identify synthetic polymers capable of modulating the attachment of bacteria to abiotic surfaces. This method can help accelerate progress in the discovery of simple, cost-effective biomaterials that inhibit bacterial attachment to medical devices. The main advantage of this technique compared with conventional biomaterial research of testing one material at a time is it's ability to screen hundreds of polymers simultaneously in a single asset.
To begin, prepare polymer solutions and add them to the wells of a 384-well plate, according to the text protocol. Create a routine to print polymers in quadruplicate consisting of 1536 spots, arranged as 48 rows and 32 columns, printing 32 polymer solutions at a time so that the printer can be stopped after printing each section to allow for cleaning of the pins. Now place the agarose coated slides on the platform and ensure a good vacuum seal to hold the slides in position.
To adjust the printhead so that all the pins are at the same height, manually lower the head onto a glass slide and confirm that the pins move up at the same time. Place the well plate on the plate holder with well A1 at the top right of the holder and ensure the well plate is firmly fixed. Confirm that the slide and well plate are in position when prompted.
Print one section at a time, allowing for cleaning of the pins between sections. Then use a paper towel dipped in acetone to clean the pins thoroughly. Followed by dry tissue paper to ensure the pins are fully dry.
When the microarrays are completely printed, place them in a slide holder and transfer them in a vacuum oven, set at 40 degrees celsius to dry, to remove the N&P in the polymer spots. Then use UV light to sterilize the plates for 30 minutes. Before inoculating slides with bacteria, take a measurement of background fluorescence according to the text protocol.
After culturing and preparing inocula for the microarrays, according to the text protocol, place the UV sterilized polymer microarrays in rectangular four-well plates and add six milliliters of mixed bacterial cultures. Incubate the plates at 37 degrees celsius with gentle agitation for five days. After the incubation, use PBS to gently rinse the microarrays twice.
Then add one microgram per milliliter of DAPI in PBS and incubate for 30 minutes. Next, transfer the microarrays to a fresh four-well plate, cover the microarrays with PBS then gently swirl. Then change the PBS once and repeat the wash.
After rinsing, dry the microarrays under a flow of air then apply sealing film and affix glass cover slips to the microarray slides. When the glue is dry, use 70%ethanol to sterilize the outer surface. Using a fluorescence microscope, fitted with a 20x subjective, capture single images for each polymer spot in bright field and strappy channels.
To coat cover slips with polymers, prepare solutions of HIT polymers in tetrahydrofuran or another appropriate solvent. Using a spin coater, spin coat circular glass cover slips of suitable size with the polymer solutions at 2000 RPM for ten seconds. Dry the coated cover slips in a convection oven at 40 degrees celsius overnight.
And use UV light to sterilize the cover slips for 30 minutes prior to inoculating with bacteria. Place the UV sterilized cover slips in a standard 12 or 24 well plate and incubate with bacteria as demonstrated earlier in this video. Following the incubation, use 0.1 molar cacodylate buffer to wash the coated cover slips twice and then with 2.5%weight per volume glutaraldehyde and 0.1 molar cacodylate buffer, fix the samples for two hours.
Post-fix the cover slips with 1%osmium tetroxide for one hour at room temperature. Before dehydrating them in a ethanol series for 30 minutes at each concentration. Then with a sputter coater and a 60 to 40%gold to platinum alloy mixture coat the samples using a current of 30 milliamps and a vacuum of 0.75 torr.
After taking images with SEM visually compare images of the uncoated cover slips and those coated with agarose or the HIT polymers to confirm bacterial binding or repelling abilities of the polymers. To evaluate various solvents for the compatibility with the end welling part of the catheter, as well as their ability to dissolve the HIT polymer, immerse the catheter pieces in the solvents and incubate for 12 hours before visually evaluating for integrity of the catheter and clarity of the solvent. To analyze bacterial attachment on catheters by SEM, prepare 10%polymer solution in acetone.
Press a 200 microliter micropipette tip into the midportion of the cut piece of the catheter to hold it and stick the piece in the polymer solution for approximately 30 seconds. Dry the coated piece in ambient conditions for 30 minutes then apply a second coating by immersing the catheter piece again into the polymer solution and dry overnight under ambient conditions. After incubating coated and uncoated catheter pieces in inoculated LB, according to the text protocol, use one milliliter of PBS to wash the catheters.
Then use 10%paraformaldehyde in PBS to fix the bacteria for 30 minutes. Following a wash with PBS, use DAPI to stain the bacteria on the catheter pieces for 20 minutes. Before washing with one milliliter of PBS.
Observe the arrangement of samples in the chamber. Using the following settings, take confocal images of the catheter pieces. Complete the confocal imaging by z-stacking 50 images across a 100 micrometer length of the catheter.
Following UV treatment and incubation with bacteria, use one milliliter of PBS to wash the pieces two times and transfer them into 48-well plates containing 10%formaldehyde in PBS for 30 minutes. After fixing the samples, perform another wash with PBS and dry over night at room temperature. Then mount on stubs with conductive carbon discs and use a sputter coater to gold coat.
Before imaging, using a scanning electron microscope. This figure shows bacterial attachment to a number of polymers as determined by microarray analysis. Spots printed without polymer act as the negative control as agarose strongly resists bacterial binding and thus fluorescence is very low.
The polymers displayed are alloy binding, although in most cases, the repellent properties of a polymer vary significantly between bacterial species tested. This reflects the wide differences between attachment mechanisms across different species. The low-binding polymers are easily identified by comparison with agarose.
High-binding polymers are likely to be identified in an array and can be used as positive controls for subsequent experiments. For polymers that do not show auto-fluorescence in the DAPI channel, qualitative comparison of the spot images can be made visually. Scale up experiments to test coating larger surfaces were performed to confirm the bacteria repellent properties of 22 appropriate polymers identified.
Shown here, are the best performing samples used to coat glass cover slips as analyzed by SEM. These figures show catheter slices analyzed by confocal microscopy and SEM. Both microscopic methods have the benefit of allowing direct cell counts on the surface providing unambiguous data since reduction in cell attachment is easily visible.
Following this procedure, serum or blood components could be tested to determine their impact on bacterial binding. An animal model could be used in order to assess the potential of the polymer coatings for clinical use. Don't forget that working with chemicals and pathogenic organisms can be hazardous.
And necessary controls should be in place during handling and disposal to mitigate personal and environmental risks.
