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

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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).

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