The overall goal of this procedure is to select group-specific DNA aptamers to highly hydrophobic, small molecules and to use the selected aptamer to develop a sensitive electrochemical biosensor. This method can help to answer key questions in the aptamer selection field, about how to select and characterize group-specific aptamers when targets are highly hydrophobic molecules. While the most useful part of this method is the development of electrochemical biosensors, for the detection of titles.
These biosensors should also work for our aptamer affinity measurements. To begin the reaction, add 100 microliters of RNase-Free water to the purified aptamer PCR product. Vortex the mixture until the precipitate has dissolved.
Lambda exonuclease reaction is sensitive to the salt concentration and is a product should be proliferated by ethanol precipitation but another isopropanol. Place into each of five micro-centrifuge tubes, five microliters of the aptamer solution, 11 microliters of Rnase free water and two microliters of 10x reaction buffer. Add two microliters of Rnase free water and solutions of two, five, eight, and 10 units of lambda exonuclease in Rnase free water to one tube each.
Mix well with gentle pipetting. Incubate the tubes at 37 degrees Celsius for 35 minutes and at 80 degree Celsius for 15 minutes. Then hold the tubes at four degree Celsius and prepare a 12%native page gel.
Add to each tube one microliter of loading dye and four microliters of Rnase free water. Run the mixtures in the native page gel at 150 volts for 45 minutes. Identify the lowest amount of lambda exonuclease needed to completely generate single stranded DNA.
Perform a large scale reaction to generate single-stranded DNA. For each binding target to be tested, incubate 10 microliters of mean functionalized DBP coded magnetic beads with 500 microliters of one micromolar solution of DBP-1 for one hour at room temperature while rotating. Then discard the supernatant.
Wash the beads four times with 200 microliters portions of PAE binding buffer and resuspend the beads in 10 microliters of PAE binding buffer. Obtain 10 micromolar binding target test dispersions in PAE binding buffer. It's great go to dispersion of the hydrophobic targets in PAE binding buffer.
To ensure that, there is enrichment of the library, were selected under affinity tests. Add 10 microliters of DBP-1 coded beads to 110 microliters of each target test solution. Incubate the mixtures for one hour and collect the supernatants by magnetic separation.
Dilute the supernatants 100 fold and use three microliters for each quantitative PCR. Calculate the relative affinities by dividing the number of DBP-1 released in the presence of the test sample by the number of DBP-1 released in PAE binding buffer alone. Prior to performing the measurements synthesize and purify the DBP-1 based thiolated core sequence probe and the signaling probe.
Reconstitute the thiolated probe and the signaling probe and as 100 micromolar solutions in nuclease free water. Next polish a two millimeter diameter gold electrode to a mirror like surface with one, 0.3, and 0.5 micron alumina powder and a microfiber cloth for five minutes each. Sonicate the electrode in ultra pure water for five minutes after each polishing.
Immerse the polished electrode in a 0.5 molar solution of sulfuric acid. Clean the electrode with 35 successive cyclic voltammetry scans from minus 0.4 to positive 1.2 volts versus mercury calomel at 100 millivolts per second. Next prepare in a thin walled centrifuge tube a mixture of 0.5 micromolar thiolated probe DBP-1.
And 0.5 micromolar FC modified DBP-1 in 100 microliters of PBS. Heat the mixture in a water bath set to 95 degrees Celsius for 10 minutes. Then allow the mixture to cool to room temperature in the water bath.
Add one microliter of a 10 millimolar TCEP stock solution to the cooled mixture and maintain at room temperature for one hour. Then immerse the clean gold electrode in the mixture for 12 hours at room temperature. Rinse the electrode with PBS and then immerse the electrode in a one millimolar solution of thiolated PEG in PBS for one hour.
Thoroughly rinse the electrode with target free PAE binding buffer and immerse the electrode in the buffer. Next sonicate electrolytic cells of platinum counter electrode and a saturated calomel reference electrode for two minutes each in ultra pure water and PAE binding buffer in sequence. Open the square wave of voltammetry instrument software and enter the experiment parameters.
Fill a clean electrolytic cell with target free PAE binding buffer. Connect the three clean electrodes to a potentiostat and immerse the electrodes in the buffer. Acquire a background SWV scan.
Then immerse the gold working electrode in a 10 picamolar DEHP solution in PAE binding buffer for 30 minutes at room temperature. Thoroughly rinse the electrode with PAE binding buffer. Immerse all three electrodes in fresh PAE binding buffer and collect another SWV curve using the same parameters as before.
Repeat this process with increasing concentrations of DEHP to obtain a titration curve. The minimum amount of lambda exonuclease necessary to obtain only single strand DNA was found to be two units based on small scale reactions. The aptamer candidate DBP-1 was identified by SELEX followed by high throughput sequencing.
DBP-1 showed good group specificity to PAE congeners. An electrochemical aptasensor using DBP-1 selectively responded to DEHP over other common environmental pollutants. The aptasensor was highly sensitive to DEHP with the response at concentrations as low as 10 picamolar.
Laser particle where selection and characterization of aptamers for hazard hydrophobic small molecules. Characterization and volatilization of aptamers of hydrophobic small molecules like PAE is generally quite challenging, due to the extremely limited water solubility and the low molecule weight of the PAEs. After watching this video, you should have a good understanding of how to make an electrochemical aptasensors and how to use it to detect the titles.
