Name: a polyaniline-based sensor of nucleic acids
Uploaded: 2016-11-01T10:45:00
Description: Watch this Scientific Journal Video about A Polyaniline-based Sensor of Nucleic Acids at JoVE.com

The authors have nothing to disclose.

1. Processable PANI Synthesis
<ol>
	<li>Dissolve aniline (1 ml, 11 mmol) completely in 60 ml of chloroform in a 250 ml round-bottom flask. Stir at 600 rpm for 5 min and cool to 0-5 &#176;C with ice. This usually takes 15-20 min (Figure 2A).</li>
	<li>Add sodium dodecyl benzene sulphonate (NaDBS) (7.44 g, 21 mmol) to the aniline solution in a round-bottomed flask while stirring at 600 rpm.</li>
	<li>Dissolve ammonium persulphate (APS) (3.072 g, 13.5 mmol) in 20 ml water and add all of it drop-wise over ...

<ol>
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A PANI-based sensor of nucleic acids requires solubilization of the polymer in water in order to interact with DNA and RNA. The dispersion of PANI in water is accomplished using surfactants, forming micelles as previously reported8. In addition to the NaDBS used here other anionic surfactants like dodecyl ester of 4-sulfophthalic acid, nonionic surfactants like nonyl phenol ethoxylate, or cationic surfactants like cetyltrimethyl ammonium bromide could also be used for synthesis of processable PANI9,10</su...

Conjugated polymers provide many options for molecular sensors. This includes fluorescence, electronic, and colorimetric responses1. There have been many efforts to incorporate conjugated polymers in nucleic acid sensors. However, most systems require secondary detection, limiting sensing options2. Recently, we reported a conjugated polymer-based sensor platform built on polyaniline (PANI) that exploits properties of this polymer, creating a label-free system3. PANI is an extensively conjugated electro-active polymer with properties such as fluorescence and resistance that are suitable for measuring biological systems4. The excitons within the structure are not localized leading to mobility of the positive charge between monomeric subunits. This provides a flexible scaffold of positive charges that can interact with the negatively charged backbone of DNA5,6. Importantly, electrostatically attached DNA is orientated such that nitrogenous bases can participate in base pairing. Association with DNA alters the electronic properties of PANI, an effect that can be enhanced by UV irradiation (Figure 1)3. Using this system, oligonucleotides complementary to target nucleic acids can be immobilized on PANI. Multiple studies have demonstrated that upon hybridization electrostatically adsorbed oligonucleotides dissociate from PANI or other cationic matrices due to conformational changes caused by the switch to a double-stranded DNA structure3,5,7.
In a sensor system where probe attachment modulates conjugated polymer properties, hybridization events can be transduced without labels or enzymatic modification of probes or target nucleic acids. Conjugated polymers offer great flexibility in detection methods, one of which is fluorescence. Through monitoring PANI fluorescence, concentrations of target nucleic acids as low as 10-11 M (10 pM) can be detected3. Detection is rapid, occurring within 15 minutes of hybridization, and specific where a single mismatch in a target molecule can be differentiated3. 
Fabrication of PANI-sensors is straightforward. High molecular weight PANI can be generated that is well-dispersed in water using standard synthesis procedures involving aniline monomer, surfactant, and controlled addition of an oxidant. Yield can be very high and unreacted oxidant removed by washing with water, ensuring no further PANI growth. PANI-probe association occurs spontaneously upon mixture, and complex formation is enhanced by mild UV exposure. Hybridization can be carried out immediately, and the changes in PANI fluorescence assayed following a short incubation. The simplicity of this technology makes it highly accessible to many laboratories.

<table border="0" cellpadding="0" cellspacing="0" width="1132">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">Aniline&#160;</td>
			<td style="width:131px;">Fisher Scientific&#160;</td>
			<td style="width:111px;">A7401-500&#160;</td>
			<td style="width:663px;">ACS, liquid, refrigerated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium peroxydisulfate</td>
			<td>Fisher Scientific&#160;</td>
			<td>A682-500&#160;</td>
			<td>ACS, crystalline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecylbenzene sulfonate</td>
			<td>Pfaltz &#38; Bauer&#160;</td>
			<td>D56340&#160;</td>
			<td>95% solid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloroform&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>MCX 10601&#160;</td>
			<td>Liquid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA primers&#160;</td>
			<td>MWG operon&#160;</td>
			<td>n/a&#160;</td>
			<td>custom DNA sequence ~20bps</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microplate&#160;</td>
			<td>USA Scientific&#160;</td>
			<td>1402-9800&#160;</td>
			<td>96 well, polypropylene as it is unreactive to chloroform</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microplate Adhesive Film</td>
			<td>USA Scientific&#160;</td>
			<td>2920-0000&#160;</td>
			<td>Reduces well-to-well contamination, sample spillage and evaporation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope Cover Glass</td>
			<td>Fisher Scientific&#160;</td>
			<td>12-544-D&#160;</td>
			<td>PANI coated on UV irradiated cover glass</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV crosslinker&#160;</td>
			<td>UVP&#160;</td>
			<td>HL-2000&#160;</td>
			<td>Energy: X100 &#956;J/cm2; Time: 2min</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hybridization Oven</td>
			<td>VWR&#160;</td>
			<td>01014705 T&#160;</td>
			<td>Temperature: 400C; with rocking for 15 min</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Apparatus&#160;</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Three necked round bottom flask for reaction; dropping funnel, stoppers, condenser, separating funnel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>Leica Microsystems&#160;</td>
			<td>Leica IMC S80</td>
			<td>Magnification 20X; Pseudo color 536 nm; Exposure 86 ms; Gain 1.0X; Gamma 1.6</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microplate Reader</td>
			<td>Molecular Devices&#160;</td>
			<td>89429-536</td>
			<td></td>
		</tr>
	</tbody>
</table>

a polyaniline-based sensor of nucleic acids

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies rely on enzymatic modification that can introduce bias and artifacts. A critical element of next generation detection platforms will be direct molecular sensing, thereby avoiding a need for amplification or labels. Advanced nanomaterials may provide the suitable chemical modalities to realize label-free sensors. Conjugated polymers are ideal for biological sensing, possessing properties compatible with biomolecules and exhibit high sensitivity to localized environmental changes. In this article, a method is presented for detecting nucleic acids using the electroconductive polymer polyaniline. Simple DNA "probe" oligonucleotides complementary to target nucleic acids are attached electrostatically to the polymer, creating a sensor system that can differentiate single nucleotide differences in target molecules. Outside the specific and unbiased nature of this technology, it is highly cost effective.

Figure 2A captures the reaction setup at the start of the polymerization process, i.e., before APS addition. Micelle formation is the initial step in the reaction process-PANI synthesis occurs at the micellar interface. Figure 2B shows a milky solution after 5 min. 30 min after APS is added the reaction turns to a slightly brown color. Figure 2C shows the color change associated with the formation of oligomers. Figure 2D<...

Watch this Scientific Journal Video about A Polyaniline-based Sensor of Nucleic Acids at JoVE.com

A Polyaniline-based Sensor of Nucleic Acids

Nucleic acids are common analytes for assessing biological systems; however, bias from enzymatic manipulation can cause concern. Here a method is described for label-free detection of nucleic acids using polyaniline. This sensitive, cost-effective sensor technology can distinguish single nucleotide differences between molecules.

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies ...

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.

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