核酸的聚苯胺传感器

摘要

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.

摘要

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.

引言

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.

研究方案

1.可处理合成聚苯胺

1. 溶解苯胺（1毫升，11毫摩尔）完全在60毫升氯仿中的250毫升圆底烧瓶中。在搅拌600转5分钟，冷却至0-5℃，有冰冻。这通常需要15-20分钟（ 图2A）。
2. 十二烷基苯磺酸钠（NaDBS）（7.44克，21毫摩尔）添加到在圆底烧瓶中的苯胺溶液在600rpm搅拌。
3. 溶解过硫酸铵（APS）（3.072克，13.5毫摩尔）在20毫升水中，并添加所有的它逐滴在30分钟内，以避免过热的反应。
4. 进行在0-5℃反应24小时，并使其达到室温下再24小时。
5. 观察反应混合物最初打开乳白色15分钟后，再暗褐色后2小时，最后以24小时后（ 图2B-F）的深绿色。
6. 过滤用布氏漏斗聚苯胺-NaDBS解决方案。用80毫升氯仿和120ml水在作为混合eparation漏斗（ 图2G）。
7. 在室温下孵育24小时的溶液，并收集从分离漏斗暗绿色聚苯胺，留下未反应的NaDBS和APS在含水上清液。

2. PANI探针混合和紫外线照射

1. 稀聚苯胺溶液10倍，用氯仿 - 水（1:3体积/体积）并通过温和摇动用于在微量离心管15分钟，混合200微升稀释聚苯胺与探针DNA寡核苷酸的6.4微摩尔。
2. 照射在交联剂与1200μJ/ cm ²的紫外线的PANI-DNA溶液2分钟。至关重要的是，紫外线照射被限制为指定量。延长暴露于UV损害在聚苯胺的荧光的变化，可能是由于聚苯胺和DNA的共价交联。
3. 沉淀物通过离心在17000×g离心6分钟，并立即用磷酸盐缓冲盐水（PBS）中。再次颗粒，并在PBS再中止。

3. ^ h聚苯胺探头的ybridization

1. 添加8微升100微米的互补DNA寡核苷酸或靶核酸200微升聚苯胺探针复合物。
2. 在40摇动溶液混合物15分钟进行杂交 C。
3. 沉淀通过离心聚苯胺配合物在17000×g离心6分钟。用PBS洗涤，并在水中重新悬浮。

4.排放稳态荧光测量

1. 从不同处理在250nm处添加聚苯胺到96孔微量培养板，并测量在270-850纳米范围内发射荧光通过激发。对于聚苯胺的排放峰值应该围绕500纳米观察。

杂交复式5.荧光显微镜测量

1. 滴涂层聚苯胺上的硼硅玻璃盖玻片并干燥在40℃下48小时。
2. 在干燥的聚苯胺薄膜添加探头（8微升100μM），并用紫外线（1200微焦/厘米²照射它）2分钟。
3. 用PBS洗聚苯胺探针膜并干燥，在40℃48小时。
4. 通过加入靶核酸15分钟进行杂交。这可能是一个生物样品或控制目标寡核苷酸（8微升100μM的的）。用PBS洗涤跟随。
5. 获取荧光图像放大40倍，具有500纳米长通滤波器。

结果

图2A捕捉在聚合过程中， 即 ，APS添加前的开始时的反应设置。胶束的形成是在胶束界面反应中的初始步骤过程聚苯胺合成发生。 图2B示出了5分钟后乳状溶液。 30分钟的APS加到在反应后变成浅棕色颜色。 图2C示出了具有低聚物的形成相关的颜色的变化。 图2D示出了4小时后深棕色，表明短链聚苯胺的高浓度，具有一?...

讨论

核酸基于PANI-传感器需要在水中的聚合物增溶，以与DNA和RNA相互作用。聚苯胺在水中的分散体是使用表面活性剂来完成，形成微胶粒如先前报道^8。除了这里使用的其它阴离子表面活性剂一样的4-磺基邻苯二甲酸十二烷基酯的NaDBS，如壬基苯酚乙氧基化物，或类似的溴化十六烷基三甲铵阳离子表面活性剂的非离子表面活性剂也可用于加工的聚苯胺^9,10的合成。这里所描述的合成始于在2...

材料

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