تقنية تسمية خالية لتصوير المكانية والزمانية لإفرازات خلية واحدة
Summary
الاتصالات بين الخلايا أمر بالغ الأهمية للسيطرة على مختلف الأنشطة الفسيولوجية داخل وخارج الخلية. وتصف هذه الورقة بروتوكول لقياس طبيعة المكانية والزمانية لإفرازات خلية واحدة. ولتحقيق ذلك، يستخدم نهج متعدد التخصصات الذي يجمع بين nanoplasmonic التسمية خالية الاستشعار مع تصوير الخلايا الحية.
Abstract
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.
Introduction
الاتصالات بين الخلايا هو أمر حاسم لتنظيم العديد من الأنشطة الفسيولوجية داخل وخارج الخلية. مجموعة متنوعة من البروتينات والحويصلات يمكن يفرز التي تؤدي فيما بعد العمليات الخلوية المعقدة مثل التمايز، والتئام الجروح، الاستجابة المناعية، والهجرة، والانتشار. قد تورطت 1-5 الأعطال من مسارات الإشارات بين الخلايا في أمراض عديدة منها السرطان وتصلب الشرايين ، ومرض السكري، على سبيل المثال لا الحصر.
يجب أن تكون الخلية الأمثل إفراز فحص قادرة على مكانيا وزمانيا رسم خرائط بروتين يفرز من الفائدة دون تعطيل مسارات الإشارات ذات الصلة. وبهذه الطريقة يمكن الاستدلال على العلاقات السببية بين ملامح التركيز واستجابة الخلايا المستقبلة. لسوء الحظ، فإن العديد من التقنيات التي تعتمد الفلورسنت الأكثر استخداما لا تلبي هذه المعايير. البروتينات الفلورية الانصهار يمكن استخدامها لعلامة الحليلة ثithin الخلية ولكن يمكن أن يعطل مسار إفرازية، أو إذا يفرز نتائج في توهج منتشر خارج الخلية التي يصعب قياسها كميا. المقايسات القائم على immunosandwich الفلورسنت هي الأكثر استخداما التقنيات للكشف عن الإفرازات الخلوية ولكنها تتطلب عادة عزل الخلايا الفردية. 6-11 وبالإضافة إلى ذلك، وإدخال من الأجسام المضادة الاستشعار عادة ما يوقف أو ينهي التجربة وحجم التسميات الأجسام المضادة، 150 كيلو دالتون للمفتش، وعائقا أمام إشارات المصب.
بسبب هذه الحواجز فمن الأفضل أن تقنية خالية من التسمية أن تستخدم لإفرازات البروتين الصورة وبين تقنيات خالية من التسمية الحالية، سطح صدى مأكل (SPR) والمترجمة صدى مأكل السطح (LSPR) أجهزة الاستشعار مرشحين ممتازة. 12-17 هذه وقد استخدمت أجهزة الاستشعار على نطاق واسع للدراسات ملزمة تحليلها من البروتينات، exosomes والمؤشرات الحيوية الأخرى. 18-24 وفي حالة LSPR، وnanostr plasmonicuctures يمكن منقوشة lithographically على coverslips الزجاج ومتحمس باستخدام الضوء المرئي عبر تكوينات المجهر واسعة المجال القياسية. ونظرا لحضورها النانوية، والغالبية العظمى من الركيزة الزجاج متاحة لتقنيات التصوير الشائعة مثل مشرق الميدان والفحص المجهري مضان جعل هذه التحقيقات أيضا مناسبة للتكامل مع المجهر الخلية الحية. 25-28 لقد أثبتنا قياس الوقت الحقيقي من إفرازات الجسم المضاد من خلايا هجين استخدام الذهب functionalized النانو plasmonic مع القرارات المكانية والزمانية من 225 ميللي ثانية و 10 ميكرون، على التوالي. ويتضح تكوين رقاقة الأساسي في الشكل 1. 28 ينقسم مسار الضوء الناتج من المجهر بين كاميرا CCD المستخدمة في التصوير ومطياف جانب الألياف بصريا لتحديد الكمي للشغل كسور من مجموعة معينة من النانو (الشكل 2 ).
وprotocرأ الواردة في هذه الورقة يصف التصميم التجريبي لقياس الوقت الحقيقي من إفرازات خلية واحدة في حين رصد في الوقت نفسه استجابة الخلايا باستخدام معيار المجهر مشرق الميدان. يتضمن نهج متعدد التخصصات لتصنيع النانو، functionalization من النانو لتقارب عالية ملزم من التحاليل، والتحسين السطح لكل من التقليل غير محددة وملزمة وتميز الثوابت معدل الحركية باستخدام سطح التجاري بلازمون الرنين (SPR) الصك، دمج خطوط الخلايا على الركيزة، وتحليل الصور والبيانات الطيفية. نتوقع هذه التقنية أن تكون التكنولوجيا ملائمة لرسم الخرائط المكانية والزمانية من إفرازات الخلية وعلاقاتها السببية مع الخلايا المستقبلة.
Protocol
Representative Results
Discussion
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.
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
The authors have nothing to disclose.
Materials
| 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 |
Referenzen
- Ludwig, A. -. K., Giebel, B. Exosomes: Small vesicles participating in intercellular communication. The International Journal of Biochemistry & Cell Biology. 44, 11-15 (2012).
- Friedl, P., Gilmour, D. Collective cell migration in morphogenesis, regeneration and cancer. Nature Reviews Molecular Cell Biology. 10, 445-457 (2009).
- Letterio, J. J., Roberts, A. B. Regulation of immune responses by TGF-beta. Annual Review of Immunology. 16, 137-161 (1998).
- Werner, S., Grose, R. Regulation of wound healing by growth factors and cytokines. Physiological Reviews. 83, 835-870 (2003).
- Werner, S., Krieg, T., Smola, H. Keratinocyte-fibroblast interactions in wound healing. Journal of Investigative Dermatology. 127, 998-1008 (2007).
- Bailey, R. C., Kwong, G. A., Radu, C. G., Witte, O. N., Heath, J. R. DNA-encoded antibody libraries: A unified platform for multiplexed cell sorting and detection of genes and proteins. Journal of the American Chemical Society. 129, 1959-1967 (2007).
- Gazagne, A., et al. A Fluorospot assay to detect single T lymphocytes simultaneously producing multiple cytokines. Journal of Immunological Methods. 283, 91-98 (2003).
- Han, Q., et al. Polyfunctional responses by human T cells result from sequential release of cytokines. Proceedings of the National Academy of Sciences of the United States of America. 109, 1607-1612 (2012).
- Han, Q., Bradshaw, E. M., Nilsson, B., Hafler, D. A., Love, J. C. Multidimensional analysis of the frequencies and rates of cytokine secretion from single cells by quantitative microengraving. Lab on a Chip. 10, 1391-1400 (2010).
- Ma, C., et al. A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells. Nature Medicine. 17, 738-743 (2011).
- Shirasaki, Y., et al. Real-time single-cell imaging of protein secretion. Scientific Reports. 4, (2014).
- Milgram, S., et al. On chip real time monitoring of B-cells hybridoma secretion of immunoglobulin. Biosensors and Bioelectronics. 26, 2728-2732 (2011).
- Abbas, A., Linman, M. J., Cheng, Q. A. New trends in instrumental design for surface plasmon resonance-based biosensors. Biosensors & Bioelectronics. 26, 1815-1824 (2011).
- Ermakova, A., et al. Detection of a Few Metallo-Protein Molecules Using Color Centers in Nanodiamonds. Nano Letters. 13, 3305-3309 (2013).
- Haes, A. J., Van Duyne, R. P. A nanoscale optical blosensor: Sensitivity and selectivity of an approach based on the localized surface plasmon resonance spectroscopy of triangular silver nanoparticles. Journal of the American Chemical Society. 124, 10596-10604 (2002).
- Horowitz, V. R., Aleman, B. J., Christle, D. J., Cleland, A. N., Awschalom, D. D. Electron spin resonance of nitrogen-vacancy centers in optically trapped nanodiamonds. Proceedings of the National Academy of Sciences of the United States of America. 109, 13493-13497 (2012).
- Sepulveda, B., Angelome, P. C., Lechuga, L. M., Liz-Marzan, L. M. LSPR-based nanobiosensors. Nano Today. 4, 244-251 (2009).
- Barbillon, G., et al. Biological and chemical gold nanosensors based on localized surface plasmon resonance. Gold Bulletin. 40, 240-244 (2007).
- Endo, T., et al. Multiple label-free detection of antigen-antibody reaction using localized surface plasmon resonance-based core-shell structured nanoparticle layer nanochip. Analytical Chemistry. 78, 6465-6475 (2006).
- Endo, T., Kerman, K., Nagatani, N., Takamura, Y., Tamiya, E. Label-free detection of peptide nucleic acid-DNA hybridization using localized surface plasmon resonance based optical biosensor. Analytical Chemistry. 77, 6976-6984 (2005).
- Haes, A. J., Hall, W. P., Chang, L., Klein, W. L., Van Duyne, R. P. A localized surface plasmon resonance biosensor: First steps toward an assay for Alzheimer’s disease. Nano Letters. 4, 1029-1034 (2004).
- Jonsson, M. P., Jonsson, P., Dahlin, A. B., Hook, F. Supported lipid bilayer formation and lipid-membrane-mediated biorecognition reactions studied with a new nanoplasmonic sensor template. Nano Letters. 7, 3462-3468 (2007).
- Park, J. H., et al. A regeneratable, label-free, localized surface plasmon resonance (LSPR) aptasensor for the detection of ochratoxin A.. Biosensors & Bioelectronics. 59, 321-327 (2014).
- Mayer, K. M., Hao, F., Lee, S., Nordlander, P., Hafner, J. H. A single molecule immunoassay by localized surface plasmon resonance. Nanotechnology. 21, (2010).
- Endo, T., Yamamura, S., Kerman, K., Tamiya, E. Label-free cell-based assay using localized surface plasmon resonance biosensor. Analytica Chimica Acta. 614, 182-189 (2008).
- Huang, Y. X., Cai, D., Chen, P. Micro- and Nanotechnologies for Study of Cell Secretion. Analytical Chemistry. 83, 4393-4406 (2011).
- Oh, B. R., et al. Integrated Nanoplasmonic Sensing for Cellular Functional Immunoanalysis Using Human Blood. ACS Nano. 8, 2667-2676 (2014).
- Raphael, M. P., Christodoulides, J. A., Delehanty, J. B., Long, J. P., Byers, J. M. Quantitative Imaging of Protein Secretions from Single Cells in Real Time. Biophysical Journal. 105, 602-608 (2013).
- Raphael, M. P., et al. A New Methodology for Quantitative LSPR Biosensing and Imaging. Analytical Chemistry. 84, 1367-1373 (2011).
- Raphael, M. P., et al. Quantitative LSPR imaging for biosensing with single nanostructure resolution. Biophysical Journal. 104, 30-36 (2013).
- Raphael, M. P., et al. A new methodology for quantitative LSPR biosensing and imaging. Analytical Chemistry. 84, 1367-1373 (2012).
- Henn, A. D., et al. Modulation of single-cell IgG secretion frequency and rates in human memory B cells by CpG DNA, CD40L, IL-21, and cell division. Journal of Immunology. 183, 3177-3187 (2009).
- Bramswig, K. H., et al. Soluble Carcinoembryonic Antigen Activates Endothelial Cells and Tumor Angiogenesis. Krebsforschung. 73, 6584-6596 (2013).
