A Polyaniline-based Sensor of Nucleic Acids

Summary

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.

Abstract

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.

Introduction

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 DNA^5,6. 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 structure^3,5,7.

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.

Protocol

1. Processable PANI Synthesis

1. 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 °C with ice. This usually takes 15-20 min (Figure 2A).
2. 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.
3. Dissolve ammonium persulphate (APS) (3.072 g, 13.5 mmol) in 20 ml water and add all of it drop-wise over 30 min to avoid overheating the reaction.
4. Carry out the reaction at 0-5 °C for 24 hr, and allow it to reach room temperature for another 24 hr.
5. Observe the reaction mixture initially turn milky white after 15 min, then dark brown after 2 hr, and finally to dark green after 24 hr (Figure 2B-F).
6. Filter the PANI-NaDBS solution with a Buchner funnel. Mix with 80 ml chloroform and 120 ml water in a separation funnel (Figure 2G).
7. Incubate the solution for 24 hr at room temperature and collect the dark green PANI from the separation funnel, leaving unreacted NaDBS and APS in the aqueous supernatant.

2. PANI-probe Mixing and UV Irradiation

1. Dilute PANI solution 10x with chloroform-water (1:3 v/v) and mix 200 µl of diluted PANI with 6.4 µmol of probe DNA oligonucleotides by gentle rocking for 15 min in a microfuge tube.
2. Irradiate the PANI-DNA solution with 1,200 µJ/cm² of UV in a crosslinker for 2 min. 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-linking of PANI and DNA.
3. Pellet complexes by centrifugation at 17,000 x g for 6 min, and wash with phosphate buffered saline (PBS). Pellet again, and re-suspend in PBS.

3. Hybridization of PANI-probe

1. Add 8 µl of 100 µM complementary DNA oligonucleotides or target nucleic acids to 200 µl of PANI-probe complexes.
2. Perform hybridization by rocking solution mixture for 15 min at 40 °C.
3. Pellet the PANI complexes by centrifugation at 17,000 x g for 6 min. Wash with PBS and re-suspend in water.

4. Emission Steady State Fluorescence Measurement

1. Add PANI from different treatments into a 96 well microplate and measure emission fluorescence in the 270-850 nm range by excitation at 250 nm. An emission peak for PANI should be observed around 500 nm.

5. Fluorescence Microscopy Measurement of Hybridized Duplex

1. Drop coat PANI on a borosilicate glass coverslip and dry at 40 °C for 48 hr.
2. Add probe (8 µl of 100 µM) on a dried PANI film and irradiate it with UV light (1,200 µJ/cm²) for 2 min.
3. Wash the PANI-probe film with PBS and dry at 40 °C for 48 hr.
4. Perform hybridization for 15 min by adding target nucleic acids. This could be a biological sample or a control target oligonucleotide (8 µl of 100 µM). Follow with a PBS wash.
5. Obtain the fluorescent images at 40X magnification, with a 500 nm long pass filter.

Results

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<...

Discussion

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 PANI^9,10

Materials

Name	Company	Catalog Number	Comments
Aniline	Fisher Scientific	A7401-500	ACS, liquid, refrigerated
Ammonium peroxydisulfate	Fisher Scientific	A682-500	ACS, crystalline
Sodium dodecylbenzene sulfonate	Pfaltz & Bauer	D56340	95% solid
Chloroform	Fisher Scientific	MCX 10601	Liquid
DNA primers	MWG operon	n/a	custom DNA sequence ~20 bps
Microplate	USA Scientific	1402-9800	96 well, polypropylene as it is unreactive to chloroform
Microplate Adhesive Film	USA Scientific	2920-0000	Reduces well-to-well contamination, sample spillage and evaporation
Microscope Cover Glass	Fisher Scientific	12-544-D	PANI coated on UV irradiated cover glass
UV crosslinker	UVP	HL-2000	Energy: X100 μJ/cm2; Time: 2 min
Hybridization Oven	VWR	01014705 T	Temperature: 400 °C; with rocking for 15 min
Glass Apparatus	Fisher Scientific		Three necked round bottom flask for reaction; dropping funnel, stoppers, condenser, separating funnel
Microscope	Leica Microsystems	Leica IMC S80	Magnification 20X; Pseudo color 536 nm; Exposure 86 msec; Gain 1.0x; Gamma 1.6
Microplate Reader	Molecular Devices	89429-536