对于未螯合钆基于核酸适配体传感器（III）
Summary
The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor.
Abstract
A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples of gadolinium-based contrast agents as a result of incomplete reactions between the ligand and the ion, or as a dissociation product. Since the ion is toxic, its detection is of critical importance. Herein, the design and usage of an aptamer-based sensor (Gd-sensor) for Gd3+ are described. The sensor produces a fluorescence change in response to increasing concentrations of the ion, and has a limit of detection in the nanomolar range (~100 nM with a signal-to-noise ratio of 3). The assay may be run in an aqueous buffer at ambient pH (~7 – 7.4) in a 384-well microplate. The sensor is relatively unreactive toward other physiologically relevant metal ions such as sodium, potassium, and calcium ions, although it is not specific for Gd3+ over other trivalent lanthanides such as europium(III) and terbium(III). Nevertheless, the lanthanides are not commonly found in contrast agents or the biological systems, and the sensor may therefore be used to selectively determine unchelated Gd3+ in aqueous conditions.
Introduction
在临床诊断中，这是由该技术的固有敏感性限制磁共振成像（MRI）的重要性日益增加，已导致在研究的快速增长到新的基于钆的造影剂（GBCAs）1的开发。 GBCAs是被给药，以提高图像质量的分子，并且它们通常具有配位的多齿配体的三价钆离子（钆3+）的化学结构。这络合作为未螯合的Gd 3+是有毒至关重要;它已经牵涉于肾源性系统纤维化的一些患者的肾疾病或衰竭2的开发。因此，检测该水性自由离子是确保GBCAs的安全工具。未螯合的Gd 3+的GBCA溶液存在往往是在配体和离子的复合物的解离，或者displacemen之间不完全反应的结果其他生物金属阳离子3吨 。
在目前用于确定的Gd 3+，那些在通用性和适用性4方面依靠色谱法和/或光谱法秩最高的存在下的几种技术。在他们的优势是较高的灵敏度和准确度，分析各种样品基质（包括人血清5，尿液和头发6，废水7和造影剂配方8），和多个钆3+络合物的同时定量（一个列表的能力的研究之前，2013是在全面审查Telgmann 等人 ）4所述。唯一的缺点是，一些这些方法需要仪器仪表（如电感耦合等离子体质谱）4，一些实验室可能无法访问。在小说GBCA发现在研究和论证的概念层面，AR上下文elatively更方便，快速和成本有效的基于分光法（如紫外吸收或荧光）可以作为一种有价值的替代。考虑到这些应用中，水的Gd 3+的荧光基于适体的传感器被开发9。
适体（钆适体）是与通过指数富集（SELEX）9的配体的系统进化的过程中分离出的碱基序列特异性的44碱基长的单链DNA分子。适应适体为荧光传感器，荧光团被连接到线料，然后将其与经由13互补碱基（ 图1）淬火链（QS）杂交的5'末端。了QS标记有在3'末端的暗淬灭剂分子。在不存在的Gd 3+，所述传感器（钆传感器）的，由1：分别钆适体和QS的2摩尔比，将具有最小的荧光发射是由于吨从荧光基团淬灭Ø能量转移。在加入含水的Gd 3+的将从钆适体置换的QS，从而增加荧光发射。
图 1. 传感器（GD-传感器），由标有荧光素（荧光基团），并标有DABCYL 13个碱基长的淬链（QS）的44个碱基长的适体（GD-适体）（暗淬灭剂）的。在不存在未螯合的Gd 3+的，传感器的荧光是最小的。与另外的Gd 3+的，发生的QS位移和增加的荧光发射是观察。 请点击此处查看该图的放大版本。
有目前，人们常用的基于分光法检测ING水的Gd 3+。该测定使用了分子二甲酚橙，其经历在其最大吸收波长从433至573纳米时螯合离子10的换档。这两个最大吸收的比率可以用来量化未螯合的Gd 3+的量。适体传感器是一种替代（也可以是互补的），以在二甲酚橙法，因为这两种方法具有不同的反应条件（如pH值和所使用的缓冲溶液的组合物），目标的选择性，定量的线性范围，和检测方式9。
Protocol
Representative Results
Discussion
使用基于适体的Gd-传感器，荧光发射的增加正比于观察未螯合的Gd 3+的浓度。为了最大限度地减少样品的使用量，该测定可以在384孔微量板以每孔45微升总样品体积运行。在此设计中，荧光素（FAM）和DABCYL（DAB）的选择主要基于试剂的成本;修改发射波长的荧光团不同的配对和猝灭剂，可以使用11。
要注意的是，以获得与该传感器最好的结果是很重要的，过程的…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
We would like to gratefully acknowledge Dr. Milan N Stojanovic from Columbia University, New York, NY for valuable scientific input. This work is supported by funding from the California State University East Bay (CSUEB) and the CSUEB Faculty Support Grant-Individual Researcher. O.E., T.C., and A.L. were supported by the CSUEB Center for Student Research (CSR) Fellowship.
Materials
| Gd-aptamer | IDTDNA | Input sequence and fluorophore modification in the order form | A fluorophore with a different emission wavelength may be used. The aptamer may also be ordered from another company. |
| Quenching strand | IDTDNA | Input sequence and quencher modification in the order form | A different quencher for optimal energy transfer from the fluorophore may be used. The aptamer may also be ordered from another company. |
| Molecular biology grade water | No specific manufacturer, both DEPC or non-DEPC treated work equally well | ||
| Gadolinium(III) chloride anhydrous | Strem | 936416 | Toxic |
| HEPES | Fisher Scientific | BP310-500 | |
| Magnesium chloride anhydrous | MP Biomedicals | 0520984480 – 100 g | |
| Sodium Chloride | Acros Organics | 327300025 | |
| Potassium chloride | Fisher Scientific | P333-500 | |
| Sodium hydroxide, pellets | Fisher Scientific | BP359 | Corrosive |
| Hydrochloric acid | Fisher Scientific | SA49 | Toxic and corrosive |
| 384-well low flange black flat bottom polystyrene NBS plates | Corning | 3575 | Plates which are suitable for fluorescence reading are required. |
| Nalgene Rapid-Flow sterile disposable bottle top filter | Thermo Scientific | 5680020 | The bottle top is fitted with 0.2 micron PES membrane |
| Disposable sterile bottles 250 mL | Corning | 430281 | A larger or smaller bottle may be used |
| 1.5 mL microcentrifuge tubes | No specific manufacturer, as long as they are DNAse and RNAse-free | ||
| 0.2 mL PCR tubes | No specific manufacturer, as long as they are DNAse and RNAse-free | ||
| Micropipets | No specific manufacturer | ||
| Pipet tips (non filter) of appropriate sizes | No specific manufacturer, as long as they are DNAse and RNAse-free | ||
| Name of Equipment | |||
| Plate reader | Biotek Synergy H1 | Plate readers from other manufacturers would work equally well |
Referencias
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