Inter-cellulær kommunikation er kritisk for styring af forskellige fysiologiske aktiviteter i og uden for cellen. Dette papir beskriver en protokol til måling af spatio-temporale karakter af encellede sekreter. For at opnå dette, er en tværfaglig tilgang, der anvendes som integrerer label-fri nanoplasmonic sensing med levende celle billeddannelse.
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.
Inter-cellulær kommunikation er afgørende for reguleringen af mange fysiologiske aktiviteter både i og uden for cellen. En række proteiner og vesikler kan udskilles som efterfølgende udløse komplekse cellulære processer, såsom differentiering, sårheling, immunrespons, migration og proliferation. 1-5 Fejl af inter-cellulære signalveje er blevet impliceret i en lang række lidelser, herunder cancer, aterosklerose og diabetes, for at nævne et par stykker.
Den optimale celle sekretion assay bør være i stand til rumligt og tidsligt kortlægge udskilt protein af interesse uden at forstyrre de relevante signalveje. På denne måde kan udledes årsagssammenhænge mellem koncentrationen profiler og respons de modtagende celler. Desværre har mange af de mest almindeligt anvendte fluorescerende teknikker ikke opfylder disse kriterier. Fluorescerende fusionsproteiner kan anvendes til at mærke analytten wnden for cellen, men kan forstyrre den sekretoriske pathway, eller hvis udskilt, resulterer i en diffus glød uden for cellen, som er vanskelig at kvantificere. Fluorescerende immunosandwich-baserede assays er de mest almindeligt anvendte teknikker til påvisning af cellulær sekreter, men kræver typisk isolering af individuelle celler. 6-11 Hertil kommer, at indførelsen af sensing antistof typisk stop eller slutter eksperimentet og størrelsen af antistof etiketter, 150 kDa for IgG, er en hindring for nedstrøms signalering.
På grund af disse forhindringer er det at foretrække, at en etiket-fri teknik anvendes til at afbilde protein sekreter og blandt eksisterende label-free teknologier, overfladeplasmonresonans (SPR) og lokaliseret overfladeplasmonresonans (LSPR) sensorer er fremragende kandidater. 12-17 Disse sensorer er ofte blevet brugt til analytbindingsmiddel studier af proteiner, exosomer og andre biomarkører. 18-24 I tilfælde af LSPR den plasmoniske nanostructures kan mønstrede litografisk på dækglas og ophidset ved hjælp af synligt lys via standard wide-field mikroskopi konfigurationer. På grund af deres nanoskala fodaftryk, størstedelen af glassubstratet er tilgængelige for almindelige billedteknik såsom lysfeltsbillede og fluorescensmikroskopi gøre disse prober velegnet til integration med live-cell mikroskopi. 25-28 Vi har vist realtid måling af antistof sekreter fra hybridomaceller hjælp funktionaliseret guld plasmoniske nanostrukturer med rumlige og tidslige resolutioner 225 msek og 10 um, hhv. Den grundlæggende chip konfiguration er illustreret i figur 1. 28 udgangslysvej af mikroskopet er delt mellem et CCD-kamera anvendes til billeder og en fiber-optisk koblet spektrometer til kvantitativ bestemmelse af fraktioneret belægning af et bestemt opstilling af nanostrukturer (figur 2 ).
Den protocol præsenteres i dette papir beskriver den eksperimentelle design til real-time måling af encellede sekreter samtidig overvåger cellernes reaktion ved hjælp af standard lyse-felt mikroskopi. Den tværfaglige tilgang omfatter fremstilling af nanostrukturer, funktionalisering af nanostrukturer til høj affinitet binding af analytter, overflade optimering for både at minimere ikke-specifik binding og karakterisering kinetiske hastighedskonstanter anvendelse af et kommercielt overfladeplasmonresonans (SPR) instrument, integration af cellelinier på substratet, og analyse af billeder og spektrale data. Vi forventer denne teknik for at være en nødvendig teknologi for spatio-temporale kortlægning af celle sekreter og deres årsagssammenhænge med som får celler.
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 |