细胞间的通信为内和细胞外的控制各种生理活性的关键。本文描述了测量的单细胞的分泌物的时空性质的协议。为了实现这一目标,多学科的方法是使用集成了无标记纳米等离子体激感测与活细胞成像。
Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.
间的蜂窝通信是对许多生理活动的内部和细胞外部的调节是至关重要的。各种蛋白质和囊泡的可分泌其随后引发复杂的细胞过程如分化,伤口愈合,免疫应答,迁移和增殖。1-5故障的细胞间信号传导途径已牵涉许多疾病,包括癌症,动脉粥样硬化和糖尿病,仅举几例。
最优细胞分泌测定应当能够在空间和时间映射感兴趣的分泌蛋白而不破坏相应信号通路。以这种方式,浓度分布和接收单元的响应之间的因果关系可以推断。不幸的是,许多的最常用的荧光为基础的技术不符合这些标准。荧光融合蛋白可以用于标记分析物瓦特ithin的细胞,而且可以破坏分泌途径,或者如果分泌,结果,其中是难以量化单元外部的扩散辉光。荧光immunosandwich基测定法是最常用的技术,用于检测蜂窝分泌物但通常需要单个细胞的分离。6-11此外,引入感测抗体的通常停止或结束实验和的抗体标签的大小, 150 kDa的对IgG,是妨碍下游信号。
这些路障,因为它是优选无标记技术被用于图像蛋白分泌物和之间现有无标记技术,表面等离子体共振(SPR)和局域型表面等离子体共振(LSPR)传感器是优良的候选12-17这些传感器已被广泛地用于蛋白,外来体和其他生物标志物的分析物结合研究。在18-24 LSPR,电浆nanostr的情况下uctures可以将光刻图案化到玻璃盖玻片,并通过标准的宽视场显微镜的配置利用可见光激发。由于它们的纳米级尺寸,大多数的玻璃基板的可用于诸如亮场和荧光显微镜使这些探针非常适用于与活细胞显微术集成常见的成像技术。25-28我们已经证明了实时测量从使用官能化金电浆纳米结构的225毫秒和10微米,分别时空分辨率杂交瘤细胞抗体的分泌物。基本芯片配置示于图1。28显微镜的输出光路被分离用于成像的CCD照相机和一个光纤光学地耦合分光计用于定量测定纳米结构的给定阵列的分数占用(图2的间)。
该protoc醇在本文提出描述了实验设计为单细胞的分泌物的实时测量,同时监测使用标准明场显微术的细胞的响应。多学科方法包括纳米结构的细胞株的制造,纳米结构为高亲合力的分析物结合的官能化,这两个最小化非特异性结合和使用商业表面等离子体共振表征动力学速率常数(SPR)仪器表面优化,集成到衬底上,并且图像和光谱数据的分析。我们预计这种技术是一种使能技术进行细胞分泌物和接收单元的因果关系的时空映射。
The LSPR imaging technique described in this work has numerous advantages over more traditional methodologies for detecting cell secretions. First, the time resolution of our technique is on the order of seconds whereas the commercial alternative, an immunosandwhich assay known as EliSpot, has a typical time resolution of 2 to 3 days.7,32 As a result we were able to resolve sudden changes in the rate of protein secretion, such as that shown in Figure 6. Second, having arrays distributed over the chip allows for the secreted signal to be tracked in space and time which enables more rigorous comparisons to diffusion-based models of cell secretion. In addition, arrays like the control array shown in Figure 6 can be used to subtract out global changes in the image that typically arise from instrumental factors such as focus drift. Third, our technique requires no modification of the cells. If desired, the experiment can incorporate commonly used tags such as fluorescent proteins, but if there is concern that such tags may negatively affect cell viability or homeostasis the label-free nature of our approach does not require them. Fourth, using the spectroscopic data we have demonstrated that quantitative information regarding the fractional occupancy of surface bound ligands can be calculated.
There are numerous alternative methods to EBL for fabricating metallic nanoparticles. However, we have found that the EBL provides considerable flexibility for optimizing nanostructure and array dimensions to best suit the optics and the cells under investigation. Also critical is the fact that the chips can be readily regenerated by plasma ashing. In this way, a typical chip can be used dozens of times. Biofunctionalization details must be modified for the specific application. The protocol presented here conjugated the surface with relatively small c-myc peptide ligands. Larger ligands such as whole antibodies typically require more spacing and thus a higher SPO to SPN/SPC ratio. Regardless, a well formed SAM layer is essential for preventing non-specific binding in live-cell experiments. In general, larger molecular weight analytes are more readily detected by LSPR. Thus, in its single-cell manifestation, this technique may not be appropriate for detecting the secretion of small proteins, such as cytokines.
The current setup has been used for studying individual non-adherent cells. There are significant number of secreted signaling proteins and vesicles to which the results reported in this work are directly applicable. For example carcinoembryonic antigen (CEA) which for decades now has been a diagnostic marker for cancer. Colon cancer cells are known to secrete CEA at the rates of thousands of molecules/cell/hr and the molecular weight is 180 kDa which exceeds that of IgG antibodies. CEA is believed to be involved in autocrine and paracrine signaling pathways but the spatio-temporal nature of these secretions have never been measured. Our technique can directly address these signaling questions. An extension of this work will be to measure the spatio-temporal nature of CEA secretion from single cells.33 Future work will also focus on integrating LSPRi with two and three dimensional cell cultures of adherent cells. By incorporating multiplexed arrays capable of detecting a number of secreted proteins in parallel, this technique has the potential to open a new window into cell secretions and how they influence neighboring cells.
The authors have nothing to disclose.
The authors have nothing to disclose.
25mm diameter glass coverslips | Bioscience Tools | CSHP-No1.5-25 | 170±5 µm is optimal |
Poly-methyl methacrylate | Microchem | PMMA 950 A4 | |
Ethyl lactate methyl metacrylate | Microchem | MMA EL6 | |
Electron beam evaporator | Temescal | FC-2000 | |
Electron beam lithography | Raith | Series 150 | |
Ethanol | Sigma-Aldrich | 459836 | |
Acetone | Sigma-Aldrich | 320110 | |
CR-7 chromium etchant | Cyantek | CR-7 | |
Scanning electron microscope | Zeiss | Ultra 55 | |
Atomic force microscope | Veeco | Nanoscope III | |
Plasma ashing system | Technics | Series 85 RIE | |
SH-(CH2)8-EG3-OH (SPO) | Prochimia | TH 001-m8.n3-0.2 | |
SH-(CH2)11-EG3-COOH (SPC) | Prochimia | TH 003m11n3-0.1 | |
SH-(CH2)11-EG3-NH2 (SPN) | Prochimia | TH 002-m11.n3-0.2 | |
Surface plasmon resonance system | Biorad | XPR36 | |
Bare gold chip | Biorad | GLC chip | Plasma ashed to remove the monolayer |
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide | Thermo | 22980 | |
N-hydroxysuccinimide (NHS) | Thermo | 24510 | |
Pentylamine-Biotin | Thermo | 21345 | |
Ethanolamine | Sigma-Aldrich | E9508 | |
Neutraavidin | Thermo | 31000 | |
Phosphate buffered saline | Thermo | 28374 | |
Tween 20 | Sigma-Aldrich | P2287 | |
Inverted microscope | Zeiss | Axio Observer | Microscope is equipped with 40X oil immersion objective; CO2 and humidity incubation from Pecon GmbH |
CCD camera | Hamamatsu | Orca R2 | Thermoelectrically cooled (16 bit) |
Spectrometer | Ocean Optics | QE65Pro | |
Spectrasuite | Ocean Optics | version1.4 | |
c-myc peptide HyNic Tag | Solulink | SP-E003 | |
monoclonal anti-c-myc antibody | Sigma-Aldrich | M4439 | |
Hybridoma cell line | ATCC | CRL-1729 | |
Antibiotic Antimycotic Solution (100×) | Sigma-Aldrich | A5955 | |
Serum free media RPMI 1640 | Invitrogen | 11835-030 | |
Fetal bovine serum | ATCC | 30-2020 | |
Rhodamine DHPE | Life Technologies | L-1392 |