The overall goal of this procedure is to identify new soul gel based materials for microarray fabrication and to use these for nano volume enzyme assays. This is accomplished by first identifying materials that have sufficiently long ation times to avoid geling in the microarray printing pin during the printing process. The second step is to assess materials with long ation times for their ability to print as microarrays reproducibility resistance to cracking adhesion quality, undesirable phase separation, and ultimately high activity for entrapped enzymes.
Next, the enzyme of interest is printed as a microarray using the optimal materials and its ability to over spot the assay solution and generate a measurable enzymatic response is tested. The final step is to evaluate the materials for their ability to provide highly reproducible assays using the enzyme of interest and generate quantitative data. The final material for array fabrication is then chosen.
Ultimately, soul gel derived protein microarrays are used to identify small molecules that reduce enzyme activity. The main advantage of this method over existing methods like plate based screening, is that you can screen large numbers of materials in a systematic manner very rapidly using low volumes of reagents. Generally, individuals new to this technique will struggle because it is possible to have materials gel within the printing pins.
If these are then cleaned in properly, it will result in missed spots that can be interpreted as non printable materials. To begin, prepare the numerous additive solutions as described in the accompanying text protocol. Many of the solutions can be pre-made and stored for up to one month or longer under the correct conditions, but some must be made the day of the experiment.
Mix the sodium silicate based saws immediately before use, and be sure to keep them on ice. The SOS must be used within one hour of adding the water as longer waiting periods result in decreased and inconsistent DATION times. Next, prepare various combinations of buffers, salans polymers, and organs lanes as directed in the accompanying text protocol in order to identify which combinations can be used as printable material with dilation times greater than 2.5 hours.
Once a number of combinations have been identified, set the humidity within the printing chamber to 80 to 90%humidity of less than 80%may result in sample evaporation and inconsistencies in printing due to the small deposition volumes with the printer. Now ready, arrange the array patterns to be printed using the Chip Writer Pro program. Set the travel in the XY direction to 10 millimeters per second and the sample approach speed in the Z direction to two millimeters per second with the 2.5 second sample loading time using the saw gel combinations identified earlier.
Prepare 25 microliters of the base materials by combining corresponding polymers, organo lanes, and small molecule additives identified through the prescreening process. Using 50 millimolar heaps with a pH of 8.0 into individual wells of a 384 well microtiter plate. Then sonicate up to four slotted sheath pins of 100 micron diameter in double distilled water.
After 15 minutes, try them under a stream of nitrogen. Next, use a pipe cleaner to remove residual moisture from within the pin holder and carefully place the pin in the print head of the contact pin printing robot. Failure to remove residual moisture may hinder the pin from moving freely with the holder resulting in missed spots.
Then add 25 microliters of the respective SA to the well just prior to printing. Mix the solution using an up and down pipetting motion Repeated 50 times when mixing by pipette. Minimize the amount of air incorporated into the solution.
Air bubbles prevent complete loading of sample within the pin. Next, load the pin by lowering it into the sample. Once the pins are loaded, commence the printing process and print the sample onto one slide surface.
Pause the printing process before adding salt to the subsequent source plate. Well, with the process paused, remove the printing pin using a magnet and place a pipe cleaner in the print head to prevent moisture buildup in the print head. Then rinse the printing pin with double distilled water and sonicate it in clean double distilled water for 30 seconds.
Next, dry the printing pin under a stream of nitrogen and then place the pin back into the print head. Continue mixing, printing and cleaning for all samples remaining within the source plate up to 12, 000 spots, 100 microns in diameter can be deposited on a single slide. Once printing is finished, age the array for a minimum of 30 minutes and up to 24 hours within the printing chamber at 80 to 90%humidity.
Prepare 50 microliters of the positive control just prior to use by combining 25 microliters of one millimolar, acetylcholine iodide, and 0.14 microliters of five millimolar bodi pi, FIL cysteine, and 25 millimolar tris at pH 7.0 with 4%glycerol in the well of a 384 Well microtiter plate pipette the solution up and down slowly to mix without creating bubbles. Next, prepare the negative controls just prior to use by combining 0.14 microliters of five millimolar boai PI FIL cysteine to 49.86 microliters of 25 millimolar triss at pH 7.0 with 4%glycerol into another well of a 384 well micro titter plate and mix them gently over print the controls onto the aged microarrays using the same processes described in the previous section. However, instead of the 100 micron diameter pins, you slotted chief pins with a diameter of 235 microns.
This ensures that the solutions completely cover the previously deposited spots. Spots age the arrays in 80 to 90%humidity for one hour at room temperature due to the auto hydrolysis of acetylcholine. Longer incubation times may result in false positives due to enhanced enzyme activity.
Ensure that the alpha innotech novo array imager is turned on and equipped with a 478 plus or minus 17 nanometer excitation and 538 plus or minus 21 nanometer emission filter for measurement of the acetylcholine esterase microarrays. Then place the slide into the slide holder with the spots facing up. Set the number of preview sections so that at least one on either side of the microscope slide is previewed with a minimum resolution of four micrometers using auto exposure.
Once the parameters are found acceptable, acquire a slide image and save it as a TIFF file. Next, open the acquired slide image in image J 64. Click the Oval Selection Tool and select each spot.
Select a region slightly larger than the observed spot, and be sure to use a consistent size between spots to reduce the subjectivity of image J.Then measure the signal intensity of each spot using the measure option. Under the analyze tab. Average the intensity from 25 similar positive control spots and divide by the average intensity of 25 similar negative control spots to obtain positive control to negative control ratios for the individual material compositions.
Shown here is an example of the dynamic range of the resulting microarrays prepared using these methods. The high controls resulted in bright green regions, whereas low controls resulted in much dimmer spots. The intensity as measured by image J 64 shows the high controls to average about 22, 000 relative fluorescent units, and the low controls averaged close to 8, 000.
The dotted lines represent three standard deviations from the mean. Here are example microarrays for an honor array screening of synthetic analogs of ameral sier alkaloids identified as compound one and compound two. Both axes represent the percent of enzyme as determined by the fluorescent signal.
In duplicate the closeness of the dots to the diagonal represents high assay reproducibility. IC 50 plots of two different potential inhibitors are shown here. Compound one resulted in an IC 50 level of 16 micromolar, whereas the IC 50 of compound two was 21 micromolar Representative spots are displayed above to illustrate differences in signal proportional to inhibitor concentrations.
The array shown here was printed with four different kinases, P 38 map kinase, EGFR and GSK, and was overprinted with buffer alone buffer containing A TP and buffer containing A TP and starro sporin an inhibitor of enzyme activity. The results show a significant effect from the kinase inhibitor starro sporin based on the resultant fluorescence intensities as described here. The greatest difference seen at the concentration of stor sporin used here was from the P 38 spots.
Here, representative spots of a P 30 8:00 AM BP microarray are shown, which were over spotted with varying concentrations of stor sporin. As indicated, the IC 50 of this molecule was determined to be one micromolar and is shown in the graph on the right Once mastered. This technique can be done in a few hours if it is performed properly Following this procedure.
Other methods such as immunoassays or protein interaction assays can also be done to answer additional questions related to diagnostics or small molecule discovery.
