Localized Surface Plasmon Resonance Imaging to Detect Protein Secretions from a Single Cell

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…

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