The overall goal of this procedure is to prepare and employ electrochemical DNA sensors. This is accomplished by first reducing the probe DNA, and then cleaning the gold electrodes upon which the sensors are built. The next step of the procedure is to deposit a thiol on gold self-assembled monolayer containing the probe, DNA, onto the gold electrodes.
Then the surface of the gold electrode is backfilled with mercaptoethanol. In order to ensure a complete well-formed monolayer is created, the final step of the procedure is to employ the sensors and samples such as buffer, urine, serum, or blood. Ultimately, results can be obtained that show analyte concentrations through changes in the peak current of square wave or alternating current volts.
This technique has wide ranging implications in molecular diagnostics because these sensors can detect many different biomarkers and drug molecules directly in urine or serum. Mr.Aaron Rowe, a graduate student in my research group, will be demonstrating the procedure. He'll be assisted by two postdoctoral fellows who work with us, Dr.Ryan White and Dr.Andrew Bonham.
To begin purchase the relevant probe, DNA from a custom oligonucleotide synthesis company, the probe is modified during synthesis by the addition of a six carbon thiol at its five prime end and a redox active methylene blue at its three prime end dissolve the probe DNA to yield a solution with a visible blue tint arising from the methylene blue moiety. Verify its concentration by measuring its absorbance at 260 nanometers, using a spectrophotometer to reduce any disulfide bonds that might be present in the probe DNA solution. Combine one microliter of the probe DNA stock solution with two microliters of freshly prepared TEP solution.
Gently mix the resulting solution with a pipet. Incubate the mixture for one hour in a dark refrigerated container. During the incubation, the blue solution should become clear as the TEP reversibly reduces the methylene blue.
Next, dilute the reduced DNA probe solution with PBS buffer to the desired concentration, which is usually between 25 and 1000. Ano molar sensor preparation begins with combining 0.05 micron Illumina powder with water on a fine polishing cloth. Use this cloth to polish a set of gold disc electrodes by pressing the gold surface firmly into the wet cloth and moving them in a figure eight pattern for approximately three minutes per electrode.
Rinse the polished electrodes with deionized water following the rinse. Immerse them in EOR tubes filled with deionized water. Then sonicate the electrodes for five minutes to remove any residual Illumina powder.
After sonication, place the electrodes into a 0.5 molar sulfuric acid solution. Then place a platinum counter electrode and a silver, silver chloride reference electrode into the solution and attach them to a potential stat. Then run a series of vol grams to electrochemically clean the surfaces of the electrodes.
The details of these procedures are included in the written supplement to this video and in a nature protocols paper previously published by this group. Following this, perform a second electrochemical cleaning in a solution of KCL in sulfuric acid according to the previously described procedure. Next, arrange a set of two milliliter einor tubes in Iraq and fill each with 200 microliters of the probe DNA solution.
The concentration of the probe, DNA in this solution will define the density with which the probe DNA is packed on the sensor surface and should be optimized for each new type of sensor. Rinse the gold disc electrodes with deionized water. Then immerse them in the relevant probe, DNA solution in an einor tube for one hour.
At this point, the probe DNA will attach to the gold electrode surface via the formation of a thiol on gold self-assembled monolayer. After rinsing the electrodes again with deionized water, immerse them in two millimolar mercaptoethanol in an einor tube. This backfills the surface ensuring a complete and stable self-assembled monolayer.
To prevent evaporation, seal the electrodes into the einor tube with paraform. Store the immersed electrodes in a dark place at room temperature for three hours to overnight to ensure complete formation of the self-assembled monolayer. To prepare the sensor for use after incubation, rinse the sensor with deionized water and then soak it in buffer for at least 10 minutes.
DNA detection with sensors is demonstrated using a 17 nucleotide probe strand affixed to a gold electrode via a thiol on its five prime end, the probe contains a methylene blue redox reporter at its three prime end. When the probe molecule hybridizes with a capture strand, the electrochemical current produced by the sensor decreases. To begin, rinse a fresh sensor with deionized water and immerse it into a blank sample.
In order to record the background signal, it produces run a square wave measurement from zero to negative 0.6 volts with an amplitude of 25 millivolts and a step voltage of one millivolt. The optimal square wave frequency will depend on the details of the probe architecture. A rounded peak should appear at approximately negative 0.25 volts, the redox potential of methylene blue.
The height of the baseline current to this peak is proportional to the efficiency of electron transfer between the methylene blue and the gold electrode. Save this background measurement. Next, move the electrodes to a solution that contains the target DNA molecule of interest and allow them to equilibrate.
Alternatively, target DNA can be added to the solution in which the sensors are immersed. Collect a second square wave volt Graham. The height of the peak at negative 0.25 volts will change from the initial background measurement.
The magnitude of this change is related to the concentration of the analyte. It is the main output data of the sensor. Following measurement, calculate the relative signal change.
This percentage is often more reproducible than measuring the absolute change in current as it corrects for electrode to electrode variations in surface area. After the procedure is complete, the sensor can be regenerated as described in the accompanying written procedure for antibody detection. Using the sensors methylene blue and thiol modified DNA serves as an anchor strand and is attached directly to the gold electrode.
This is then hybridized with a second recognition, DNA strand that has been covalently conjugated to the relevant antigen. This is then followed by addition of an incubation with a specific antibody target. Carry out the hybridization step by transferring a prefabricated sensor into an einor tube containing 100 nano molar of the relevant recognition.
DNA strand in PBS incubate the sensor for one hour. Follow this with immersion in PBS buffer to remove any un hybridized probe before moving into blank solution. Next, place the sensor in the relevant blank solution and place a platinum counter and a silver, silver chloride reference electrode into the solution.
Attach the electrodes to the potential stat. Perform square wave vol telemetry as described earlier, the optimal square wave frequency for the particular probe architecture used in this example is 60 hertz. A rounded peak should appear around negative 0.25 volts.
Save this background measurement. Transfer the electrodes to a solution containing the target analyte following a five to 60 minute incubation, collect a second square wave volta. If the target antibody is present, the peak at negative 0.25 volts will decrease.
The magnitude of this change is related to the antibody concentration. To demonstrate small molecule detection, the probe DNA on the sensor surface is an aptr. An aptr is a DNA or RNA molecule that has been selected in vitro to bind a specific molecular analyte.
Aptamers can often be re-engineered to change their structure. Upon such binding. The aptima employed here changes its confirmation upon binding to the drug cocaine.
To begin, rinse a fresh sensor with deionized water and immerse it into a blank sample, lacking the target in order to record the background signal it produces. Then place a platinum counter and a silver, silver chloride reference into the solution. Attach the electrodes to the leads of a potentials stat, perform square wave, or alternating current vault telemetry.
As was described earlier, the optimal square wave frequency for the particular probe architecture used here is 60 hertz. Again, a rounded peak should appear around negative 0.25 volts and should be saved as the background measurement. Then transfer the electrodes to a solution containing the target analyte.
After a 32nd incubation, collect a second square wave or alternating current volt Graham. The height of the peak at negative 0.25 volts will change. The magnitude of this change is related to the concentration of the target analyte.
When electrochemical, DNA biosensors are used to detect DNA with the probe architecture described here, the signal should decrease by at least 60%When equilibrated with a 200 nanomolar target after three brief rinses in deionized water, the signal should return to within 0.1 to 5%of its original value. Similarly, when the sensors described here are used to detect antibodies, the signal should decrease between 40 and 80%upon binding of antibody. Conversely, aptima based sensors for the detection of cocaine exhibit a signal increase of to 200%upon binding of small molecules.
The magnitude of this change depends on the electrochemical interrogation frequency and surface coverage on the sensors. For the cocaine sensor. A relatively low surface coverage is best Once mastered, the fabrication and use of these sensors can be performed in a single day.
It's important to remember when performing this procedure that parameters such as square way frequency, or probe density on the electrode surface greatly affect sensor performance. After watching this video, you'll know how to prepare electrochemical, DNA biosensors, aptamer biosensors, and scaffold biosensors, and you'll know how to use them for a wide variety of basic measurements. The only significant hazards in this protocol are the use of sulfuric acid, which obviously is caustic eye protection and gloves are mandatory.
