制造的UV-Vis和拉曼光谱免疫平台
Summary
Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.
Abstract
免疫测定用于检测基于相关联的抗体的存在的蛋白质。因为他们的研究和临床广泛使用，免疫仪器和材料的大型基础设施都可以找到。例如，96和384孔聚苯乙烯板是市售的，并有一个标准设计，以容纳不同制造商紫外可见（UV-VIS）光谱法的机器。此外，各种各样的免疫球蛋白，检测标记和定制免疫测定的设计，如酶联免疫吸附测定（ELISA）阻断剂是可用的。
尽管现有的基础设施，标准ELISA试剂盒不符合所有研究的需要，要求个体免疫的发展，这可能是昂贵和费时的。例如，酶联免疫吸附试剂盒具有低复用（一次检测多于一种分析物的）的能力，因为它们通常依赖于荧光或栏orimetric方法进行检测。比色法和基于荧光分析已经有限的，由于宽光谱峰复用功能。与此相反，拉曼基于光谱的方法有用于复更大能力由于窄发射峰。拉曼光谱的另一个优点是，喇曼记者遇到显著少漂白比荧光标签1。尽管优势拉曼记者有超过荧光灯和比色标记，协议制造基于拉曼免疫是有限的。本文的目的是提供一种协议来制备官能化的探针结合使用以用于直接检测由UV-VIS分析和拉曼光谱的分析物的聚苯乙烯板。该协议将允许研究人员同时在预先建立的基础设施资本采取做它自己的方式为未来的多分析物检测。
Introduction
典型的三明治免疫测定法间接检测使用两种抗体的抗原的存在。捕获抗体结合至固体表面，并形成一种抗体 – 抗原时复杂接近适当的抗原。然后，检测抗体被引入并与抗原结合。洗涤后，将抗体/抗原/抗体复合物的遗体和由标记的检测抗体， 如图1A表明被检测到。典型的检测是通过荧光或比色检测完成后，限制了复用，由于宽光谱峰2,3 10分析物。相反，基于拉曼系统具有导致与源声称高达100分析物2,3的同时检测增强复用功能窄得多的发射峰。
许多文献资料都可以覆盖到免疫测定相关的重要方面4 – 6如一步一步细节打造个性化的ELISA试剂盒。不幸的是，这些协议是用于荧光或比色检测，限制定制免疫测定的复用能力。为了满足这一需求，我们提出了一个详细过程来制作， 如图1B所示此前7直接免疫公布的紫外-可见分光光度计/拉曼免疫。
这个协议包括官能化金基于纳米颗粒探针的制造中，在图2中所示，使拉曼/紫外可见探针通过拉曼记者结合金纳米颗粒的表面（金纳米粒子）开始的过程。该金纳米粒子，然后与使用聚乙二醇（PEG）相关抗体官能化。在金纳米粒子其余结合位点通过结合甲氧基聚乙二醇硫醇（MPEG-SH）至金纳米粒子，以防止在分析过程中后续的非特异性结合阻断。的制备金纳米粒子探针通过结合到抗原测试如在图1B所示固定到聚苯乙烯板的孔中。在洗涤所述板，所述金纳米粒子探针使用UV-Vis光谱检测到，同时用拉曼光谱检测相关的拉曼记者。结合紫外 – 可见和拉曼光谱数据提供分析的两种方法，从而增强该免疫的能力。
Protocol
Representative Results
Discussion
在详细的协议，有解决几个关键点。一个问题是拉曼记者和金纳米粒子的选择。虽然该协议被写入适于个人使用，拉曼记者DTTC用作一个例子。 DTTC是带正电的记者和结合至带负电荷的表面，如柠檬酸盐封端的金纳米粒子。该协议可以通过使用金纳米颗粒带有正表面电荷适于带负电荷的记者。例如，聚乙烯亚胺（PEI）封端的AuNPs提供正的表面电荷和带负电荷的记者更好结合。
维…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This work was supported by a Research Catalyst Award from Utah State University. The authors would like to thank Annelise Dykes, Cameron Zabriskie, and Donald Wooley for their contributions.
Materials
| 60nm Gold Nanoparticle | Ted Pella, Inc. | 15708-6 | These are citrate capped gold nanoparticles. Please see Discussion for relationship between Raman reporter and AuNP surface charge and its imporance to proper selection of AuNP and/or Raman reporter. |
| Sodium Bicarbonate | Fisher Scientific | S233-500 | |
| Methanol | Pharmco-Aaper | 339000000 | |
| Tris Buffered Saline (10X) pH 7.5 | Scy Tek | TBD999 | |
| Bottle Top Filtration Unit | VWR | 97066-202 | |
| Tween 20 (polysorbate 20) | Scy Tek | TWN500 | Used as an emulsifying agent for washing steps. |
| Phosphate Buffered Saline 10X Concentrate, pH 7.4 | Scy Tek | PBD999 | |
| Protein LoBind Tube 2.0 mL | Eppendorf Tubes | 22431102 | LoBind tubes prevent binding of proteins and AuNPs to surfaces of the tubes. |
| Protein LoBind Tube 0.5 mL | Eppendorf Tubes | 22431064 | LoBind tubes prevent binding of proteins and AuNPs to surfaces of the tubes. |
| Microplate Devices UniSeal | GE Healthcare | 7704-0001 | Used for sealing and storing functionalized plates. |
| Assay Plate, With Low Evaporation Lid, 96 Well Flat Bottom | Costar | 3370 | |
| HPLC grade water | Sigma Aldrich | 270733-4L | |
| 3,3′-Diethylthiatricarbocyanine iodide (DTTC) | Sigma Aldrich | 381306-250MG | Raman reporter |
| mPEG-Thiol, MW 5,000 – 1 gram | Laysan Bio, Inc. | MPEG-SH-5000-1g | |
| OPSS-PEG-SVA, MW 5,000 – 1 gram | Laysan Bio, Inc. | OPSS-PEG-SVA-5000-1g | OPSS-PEG-SVA has an NHS end. |
| Mouse IgG, Whole Molecule Control | Thermo Fisher Scientific | 31903 | Antigen |
| Goat anti-Mouse IgG (H+L) Cross Adsorbed Secondary Antibody | Thermo Fisher Scientific | 31164 | Antibody |
| Human Serum Albumin Blocking Solution | Sigma Aldrich | A1887-1G | Bovine serum albumin can be used instead. |
| In-house built 785nm inverted Raman microscope unit | N/A | N/A | An inverted Raman microscope is best for proper focusing onto surface of the well plate. Otherwise a very low magnification will be used due to height of the 96-well plate. An in-house built system was used as it was cheaper than buying from a vendor. However, any commercially available inverted Raman microscope system can be used. |
| Mini Centrifuge | Fisher Schientific | 12-006-900 | |
| UV-Vis Spectrophotometer | Thermo Scientific | Nanodrop 2000c | |
| UV-Vis Spectrophotometer | BioTek | Synergy 2 | |
| Desalting Columns | Thermor Scientific | 87766 |
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