高密度条码抗体芯片的水流模式的指导下制作
Summary
这个协议概括了大规模的制造中，复用的二维DNA或抗体阵列，在细胞信号研究和生物标记物检测的应用潜力。
Abstract
Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.
Introduction
抗体微阵列已被广泛用于蛋白质组学研究了数十年来检查定位的蛋白质，包括蛋白生物标志物1-3的存在。虽然这个领域目前正面临着来自其他高通量技术的巨大挑战，如质谱（MS），仍然有足够的空间用于抗体芯片的效用，主要是因为这些设备提供简单的数据解释和易用的界面与其他实验。近年来，微阵列集成到微芯片的支架提供了抗体微阵列的新契机兴旺4-7。例如，条形码芯片集成到一个单细胞的微芯片已被用于在小区通信研究8,9。该技术具有比其他可用的芯片技术优势明显。它具有数组元素在10-100微米，比典型的150微米大小的常规芯片的Elemen使用更小TS。较小的阵列元件的构造是利用系统的流动图案形成方法来实现，这产生了紧凑的微阵列，可以检测单细胞分泌的蛋白质和细胞内蛋白质。另一个优点是使用简单，仪器免费安装的。这是特别重要的，因为大多数实验室和小公司可能无法访问微阵列核心设备。这种条码抗体微阵列具有增强测定的吞吐量和可用于在单细胞进行高多重分析，同时实现高灵敏度和特异性与常规夹心酶联免疫吸附试验的可比性（ELISA 8）。这项技术已发现许多应用中检测来自成胶质细胞瘤9-11 T细胞的蛋白质，12和循环肿瘤细胞13。或者，条形码DNA微阵列单独已用于神经元和星形胶质细胞对模拟天生的精确定位荷兰国际集团的脑组织14的 体内组装。
这个协议只聚焦于实验步骤和二维（2-D）条码抗体微阵列具有在流体样品中的生物标志物的检测的潜在应用集结块和单个细胞。该技术是基于一个寻址单链的，一维（1-D）的DNA微阵列构造使用被在玻璃基板上的空间图案正交的寡核苷酸。一维图案时形成平行流动通道中的流动图案化步骤中使用，而这样的图像呈现为离散的频带视觉上相似于1-D的通用产品代码（UPC）条形码。的2-D（n×m个 ）的抗体阵列的构成-让人想起的2-D快速响应（QR）码矩阵-需要更复杂的图案化的策略，但允许抗体在较高密度8,15的固定。该制造需要两个DNA图案化步骤，与第一图案垂直于第二。这两种模式的交叉点构成的阵列的n×m个元素。通过策略性选择的单链DNA（ssDNA）的流动图案化利用的序列，在一给定阵列的每个元素被分配一个特定的地址。这个空间参考是必要的，在微阵列载玻片荧光信号之间进行区分。的单链DNA阵列被转换为抗体阵列通过互补DNA抗体偶联物掺入，形成了所谓的DNA编码的抗体库（DEAL 16）的平台。
此视频协议描述了在两个取向创建n×m个抗体阵列，其中包括制备聚二甲基硅氧烷（PDMS）条码模具，流动图案化单链DNA，制备抗体-寡核苷酸缀合物DEAL，和3×3 DNA阵列转换成一个3的关键步骤×3抗体芯片。
Protocol
Representative Results
Discussion
流量模式的设计是在制造的2-D芯片的第一个关键步骤。为了产生在玻璃基板上两个重叠的DNA模式，所述第一设计的信道特征应垂直于与第二（图1A-B）的 。该设计还考虑到芯片的下游应用。在单细胞分析的情况下，微阵列被用于检测来自单个细胞包封在微容器的蛋白质，因此所述通道尺寸都是用，与2-D阵列对准微容器兼容。每个设计呈现在光掩模和标准光刻?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge the startup fund from SUNY Albany and the access of facilities at the University at Albany Cancer Research Center.
Materials
| Sylgard 184 silicone elastomer base | Dow Corning | 3097366-1004 | |
| Sylgard 184 silicone elastomer curing agent | Dow Corning | 3097358-1004 | |
| SU-8 2025 photoresist | MicroChem | Y111069 | |
| Silicon wafers | University Wafers | 452 | |
| Poly-L-lysine coated glass slides | Thermo Scientific | C40-5257-M20 | |
| Oligonucleotides | Integrated DNA Technologies | *Custom-ordered from Integrated DNA Technologies, see table below | |
| Poly-L-lysine adhesive stock solution | Newcomer Supply | 1339 | |
| Bis (sulfosuccinimidyl) suberate (BS3) | Thermo Scientific | 21585 | |
| 1x Phosphate buffered saline, pH 7.4 | Quality Biological | 114-058-101 | |
| Äkta Explorer 100 Fast Protein Liquid Chromatography (FPLC) System | GE (Amersham Pharmacia) | 18-1112-41 | |
| Superose 6 10/300 GL column | GE Healthcare Life Sciences | 17-5172-01 | |
| Capture antibodies | various | various | *Antibody selection depends on application |
| Succinimidyl-6-hydrazino-nicotinamide (S-HyNic) | Solulink | S-1002 | |
| Succinimidyl-4-formylbenzamide (S-4FB) | Solulink | S-1004 | |
| N,N-dimethylformamide | Sigma-Aldrich | 227056 | |
| Citric acid, anhydrous | Acros | 42356 | |
| Sodium hydroxide | Fisher Scientific | S318 | |
| Amicon Ultra spin filter 10 kDa MWCO | EMD Millipore | UFC201024 | |
| Spin coater | Laurell Technologies | WS-650-MZ | |
| Biopsy punch with plunger (0.50 mm diameter) | Electron Microscopy Sciences | 57393 | |
| Diamond scribe (Style 60) | SPI supplies | 6004 | |
| Trimethylchlorosilane | Sigma Aldrich | 92361 |
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