The overall goal of this procedure is to combine a simple method for visually detecting multi-nucleotide polymorphisms with a pneumatic droplet manipulation platform. The DNA detection platform is on an open surface and is straight forward to use for biochemical researchers. We investigated a simple tool to repeatedly detect genetic mutations and genetic diseases.
It has greater potential helping us understand how genetic variation leads to disease. We combined two novel and simple methods. The multi-nucleotide polymorphism can be detected on the pneumatic droplet manipulation platform with the naked eye and without any additional instrument.
The method for detecting multi-nucleotide polymorphism will be demonstrated by Tzu-Ming Wang, the post doctor in our group. To begin this procedure, prepare a 100 micromolar solution of probe DNA as indicated in the text protocol. Then, add the probe DNA to a one OD per milliliter solution of gold nanoparticles.
Bring the final concentration of SDS to 0.01%and of PBS to 0.01 molar. Allow the solution to incubate for 20 minutes. Next, increase the concentration of sodium chloride to 0.05 molar by adding a solution of two molar sodium chloride and 0.01 molar PBS.
Maintain the SDS concentration at 0.01%and let the solution incubate for anoher 20 minutes. Increase the concentration of sodium chloride in increments of 0.1 molar over 20 minute intervals until a final concentration of one molar is reached. Incubate the solution at 23 degrees Celsius overnight on a rotator.
After the DNA samples and gold nanoparticles have become conjugated, centrifuge the solution at 7, 000 times g for 30 seconds. Remove the supernatant. Next, prepare a 25 nanomolar gold nanoparticle probe solution by resuspending the pellet in deionized water.
Prepare target DNA samples as outlined in the text protocol. Add six microliters of gold nanoparticle probe solution into each DNA sample with sodium chloride. Using a vortex mixer, shake each sample solution for five minutes at 23 degrees Celsius.
Then, add six microliters of 400 millimolar hydroxyl amine and six microliters of 25.4 millimolar chloroauric acid to each DNA sample solution. Set these samples aside as a steady change in coloration will occur over the next hour. the size, shape-dependent optical properties of the nanoparticle, the number of the mismatch in a sample DNA fragment typical can be discerned by eye.
To begin fabricating the droplet manipulation platform, utilize a computer numerical controlled machine equipped with a 0.5 millimeter drill bit to produce PMMA-based mastermolds with microstructures. For the drill, use a feed speed of seven millimeters per second and a rotation rate of 26, 000 rpm, and inspect the molds after drilling. Use an air blower and deionized water to clean the surface of the mastermolds and remove any PMMA scrap.
Following this, prepare a 10 to one mixture of PDMS base and curing agent. Degas the mixture in a desiccator until all air bubbles are removed. Then, pour the PDMS mixture into the mastermold.
Bake for three hours at 60 degrees Celsius to obtain the inverse microstructures in the air chambers and air channels. After baking is complete, remove the PDMS layer from the mastermold, and punch holes in it for the air suction inlets. Use an oxygen plasma treatment system to clean the PDMS membrane and PDMS layer.
After treatment, bond the PDMS membrane and the PDMS layer, and then punch holes in this conjugated structure. Next, bond the PDMS and glass substrate together using a hot plate at 90 degrees Celsius for 10 minutes. Once the layers are bonded together, place the composite plate into a laser cutting machine PDMS membrane-side up.
Import the dwg file to define the super hydrophobic area, and note this will be directly engraved into the PDMS membrane. Clean the newly engraved superhydrophobic surface with deionized water. This will wash away any carbonized residues.
After the surface has been cleaned, connect the air suction inlet and vacuum pump through a solenoid valve. Then, connect the solenoid valve to a computer to control it with a digital IO USB module. Once the pneumatic droplet manipulation platform has been set up, add 10 microliters of 0.5 micromolar target DNA on the superhydrophobic area.
Next, add 10 microliters of freshly hybridized 25 nanomolar gold nanoparticle probe solution next to the target DNA drop. Use a simple program to control air suction to the platform through a solenoid valve. Set the pressure to negative 80 kilopascals, and the driving frequency to five hertz.
Provide suction along the droplet's vibration paths, causing them to collide and coalesce. Next, mix the coalesced droplet by manipulating the suction to roll the sample for three minutes. Add two microliters of 400 milimolar hydroxylamine, and two microliters of 25.4 milimolar chloroauric acid to the superhydrophobic area of the pneumatic droplet manipulation platform.
Manipulate the suction so that the droplets collide and merge. Then, mix the merged droplet by rolling it for one minute. Observe the color of the final droplet with the naked eye.
In this study, multi nucleotide polymorphisms are detected colorometrically using DNA hybridization mediated growth of gold nanoparticle probes. Here, the DNA samples possess different affinities to the probe DNA, causing differences in the growth of the gold nanoparticles, seen as a change in color. The three base pair mismatched DNA sample has less hybridization than the fully complimentary DNA, which can be seen as noticeable distinctions in color, especially at higher concentrations.
The six base mismatched DNA has weak hybridization affinity to the probe, and thus the growth size of the gold nanoparticles was small. This colorometric analysis is shown to be repeatable when samples are manipulated via a pneumatic suction platform, as the results of DNA mismatch are readily observable by the naked eye. Once mastered, the entire procedure takes less than five minutes from the addition of the sample and the reagent until result of them, which is much quicker than the traditional method.
The total in each operation is only 10 microliter, which is significantly less than what is required in a large system. Following this procedure, once the sequence variation of the space of DNA fragment is confirmed to be responsible for a specific disease. The specifically designed product can be used to acquire a number of mismatch of the sample DNA.
The pneumatic droplet manipulation platform is more biocompatible than other methods such as optical weighting, dye electro fluorescence, electro weighting, and shoomoo's capillary actuation. The proposed multinucleotide polymorphism detection method was conducted under pneumatical droplet manipulation platform, which means the samples and reagents can be directly loaded and collected with pipette. After its development, this technique has a high potential for DNA screening, and other chemical and biomatical applications.
