The overall goal of this procedure is to synthesize water dispersed polyaniline that could be used to detect nucleic acids in a label-free fashion. This technique is a cost-effective method for detecting nucleic acids. Labs that adopt it will find it easy, robust and sensitive.
The main advantage of the technique is that it is label free and can be used to directly detect RNAs and DNAs without the need for enzymatic manipulation. This technique has potential for new diagnostic technologies and since it doesn't use a label, it can be used for processing patient samples with little manipulation. Visual demonstration will help demystify the sensor.
This platform includes polymer science and biology, which seem very exotic but is in fact, extraordinarily easy to implement. Dissolve aniline completely in 60 ml of chloroform in a 250 ml round-bottom flask. After stirring at 600 RPM for five minutes, cool to zero to five degrees Celsius with ice.
This usually takes 15 to 20 minutes. Next, add sodium dodecylbenzenesulfonate to the aniline solution in a round-bottom flask while stirring at 600 RPM. Dissolve ammonium persulfate in 20 ml of water and add it dropwise over 30 minutes to avoid overheating the reaction.
Carry out the reaction at zero to five degrees Celsius for 24 hours. Observe the reaction mixture initially turn milky-white after 15 minutes then dark brown after two hours and finally to dark green after 24 hours. Then allow it to reach room temperature for another 24 hours.
Filter the PANI sodium dodecylbenzenesulfonate solution with a buchner funnel. Mix the filtrate with 80 ml of chloroform and 120 ml of water in a separation funnel. Incubate the solution for 24 hours at room temperature then collect the dark green PANI from the separation funnel, leaving unreacted sodium dodecylbenzenesulfonate and APS in the aqueous supernatant.
Dilute PANI solution 10 X with chloroform water then mix 200 microliters of diluted PANI with 6.4 micromoles of probe DNA oligonucleotides by gentle rocking for 15 minutes in a microfuge tube. Irradiate the PANI DNA solution with 1200 microjoules per square centimeter of UV in a crosslinker for two minutes. It is critical that UV exposure is limited to the indicated amount.
Extended exposure to UV compromises the fluorescence change in PANI, likely due to covalent cross-leaking of PANI and DNA. Pellet complexes by centrifugation at 17, 000 x g for six minutes and wash with phosphate buffered saline or PBS. After pelleting a second time, resuspend in PBS.
Add eight microliters of 100 micromoler complementary DNA oligonucleotides or target nucleic acids to 200 microliters of PANI probe complexes. Perform hybridization by rocking the solution mixture for 15 minutes at 40 degree Celsius. After pelleting the PANI complexes by centrifugation at 17, 000 x g for six minutes, wash with PBS and resuspend in water.
Add PANI from different treatments into a 96-well microplate and measure emission fluorescence in the 270 to 850 nanometer range by excitation at 250 nanometers. An emission peak for PANI should be observed around 500 nanometers. To perform fluorescence microscopy measurement of the hyberdized duplex, drop coat PANI on a borosilicate glass cover slip and dry at 40 degrees Celsius for 48 hours.
Add eight microliters of 100 micromoler probe on a dried PANI film then irradiate it with UV light for two minutes. Following the radiation, wash the PANI probe film with PBS and dry at 40 degrees Celsius for 48 hours. Perform hybridization for 15 minutes by adding target nucleic acids.
This could be a biological sample or a controlled target oligonucleotide. Follow with a PBS wash. Obtain the fluorescent images at 40 x magnification with a 500 nanometer long pass filter.
Shown here is the electrostatic interaction of PANI and DNA probes enhanced by UV irradiation. Hybridization of the target nucleic acid triggers duplex formation, changing structural confirmation, which results in dissociation of the probe target from the PANI surface. This figure shows the change in PANI fluorescence in the absence or presence of probe, both in solution and solid coating medium.
The increase in fluorescence is due to electron flux from negatively charged DNA, increasing polymer electron density. Upon hybridization of complimentary target oligonucleotides, probes detach, leading the restoration of basil fluorescence. Conversely, hybridization with single mismatched target oligonucleotides does not result in this restoration of basil fluorescence.
The main novelty of this technique is that it uses mild UV for an exact interaction and that actually helps it to distinguish non-specific and light interaction. Once mastered, this technique could take up to two hours excluding the senses time and the drying time of the coatings. While trying this procedure, it's important to remember that the DNA dissolves in water and the acidic tea actually changes the electronic property of the PANI.
After watching this video, viewers should have good understanding of how to synthesize water dispersed polyaniline and use its fluorescent properties to detect nucleic acids. They will be able to immobilize Probe DNAs and perform hybridization to detect targets. Working with the monomer, it's important to remember that this synthesis is hazardous and should be done in a hood with the protective equipments like safety glasses, gloves and lab coats.
