Ein Polyanilin basierenden Sensor von 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. verarbeitbaren PANI Synthese Auflösen Anilin (1 ml, 11 mmol) vollständig in 60 ml Chloroform in einem 250 ml Rundkolben. Rühre bei 600 Umdrehungen pro Minute für 5 Minuten und Abkühlen auf 0-5 ° C mit Eis. Dies dauert in der Regel 15 bis 20 min (2A). Hinzufügen Natriumdodecylbenzolsulfonat (NaDBS) (7,44 g, 21 mmol) zu der Anilin-Lösung in einem Rundkolben während bei 600 rpm gerührt wurde. Man löst Ammoniumpersulfat (APS) (3,072 g, 13,5 mmol) in 20 ml Wasser …

Representative Results

2A erfasst zu Beginn des Polymerisationsverfahrens des Reaktionsaufbau, also vor APS hinaus. Mizellenbildung ist der erste Schritt in dem Reaktionsverfahren-PANI – Synthese an der mizellaren Schnittstelle auftritt. 2B zeigt eine milchige Lösung nach 5 min. 30 min nach dem APS zugegeben wird die Reaktion dann auch in eine leicht braune Farbe. 2C zeigt die Farbänderung mit der Bildung von Oligomeren verbunden. 2D</strong…

Discussion

A PANI-basierten Sensor von Nukleinsäuren erfordert die Solubilisierung des Polymers in Wasser, um mit DNA und RNA zu interagieren. Die Dispersion von PANI in Wasser Tenside erreicht Verwendung bilden Mizellen wie zuvor ⁸ berichtet. Neben den hier NaDBS andere anionische Tenside wie dodecylester von 4-Sulfophthalsäure, nicht – ionische Tenside wie Nonylphenolethoxylat oder kationische oberflächenaktive Mittel wie Cetyltrimethylammoniumbromid könnte auch für die Synthese von verarbeitbaren PANI ^9,10<…

Materials

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 ~20bps
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: 2min
Hybridization Oven	VWR	01014705 T	Temperature: 400C; 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 ms; Gain 1.0X; Gamma 1.6
Microplate Reader	Molecular Devices	89429-536