Localized Surface Plasmon Resonance Imaging to Detect Protein Secretions from a Single Cell
Published: January 31, 2024
Abstract
Source: Raghu, D. et al., A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions. J. Vis. Exp. (2015)
This video demonstrates localized surface plasmon resonance imaging to detect protein secretions from a single cell. Using a microfluidic setup and a peptide-bound plasmonic nanostructure chip, changes in reflected light intensity at the resonance angle reveal interactions with secreted proteins from a single cell.
Protocol
1. Nanostructure Fabrication Choose 25 mm diameter glass coverslips with an approximate thickness of 170 µm (No. 1.5) as substrates for nanofabrication. Immerse the coverslips in piranha solution (3:1 ratio of sulphuric acid and hydrogen peroxide) for at least 6 hr. Wash the piranha-soaked coverslip with copious amounts of ultrapure 18.2 MΩ deionized distilled water (DDW). CAUTION: Piranha acid reacts violently with organic materials an…
Representative Results
Figure 1. Optical Setup. The illuminated light from a halogen lamp is first filtered by a long pass filter (LP). The light is linearly polarized (P1) and illuminates the sample via a 40X/1.4 NA objective. The scattered light is collected by the objective and passed through a crossed polarizer (P2). A 50/50 beam splitter (BS) is inserted into the collected light path for…
Açıklamalar
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 |
Etiketler
Bu Makaleden Alıntı Yapın
Localized Surface Plasmon Resonance Imaging to Detect Protein Secretions from a Single Cell. J. Vis. Exp. (Pending Publication), e21923, doi: (2024).