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

For cleaning and regenerating the localized surface plasmon resonance, or LSPR chips, plasma ash at a power of 40 watts in a 300-milliliter mixture of 5% hydrogen and 95% argon for 45 seconds. Functionalize the gold surface and the nanostructures immediately after plasma-ashing by immersing the chip in a two-component ethanolic thiol solution consisting of a 3-to-1 ratio of SPO and SPC. After leaving the chip in the thiol solution overnight to form a self-assembled monolayer, rinse it with ethanol, and dry with nitrogen gas.

Load the LSPR chip within a custom-made microfluidic holder by placing the chip on an aluminum bottom piece. Then, sandwich the chip between this bottom piece, and a silicone gasket, and a clear plastic top piece using four screws to clamp the assembly.

For a typical SPC-based thiol application, drop-coat 300 microliters of a 1-to-1 mixture of 133 millimolar EDC and 33 millimolar NHS in deionized distilled water to activate the carboxyl groups of the SPC thiol component. After waiting for 10 minutes, manually rinse the surface with 10 millimolar PBS.

Following this, conjugate the activated carboxyl group with the ligand of interest by drop-coating 300 microliters of the ligand solution. After waiting for 1 hour and rinsing the surface with 10 millimolar PBS, drop-coat 300 microliters of 0.1 molar ethanolamine in PBS on the chip to minimize nonspecific binding. After waiting for 10 minutes, wash the ethanolamine with PBS, containing 0.005% Tween 20 or PBS-T20.

Next, place a quartz piece above the chip to reduce fluctuations in the data related to a changing meniscus. Keep the chip wet with PBS-T20 buffer while mounting on the microscope. Attach the microfluidics tubing to the assembly and flow buffer until a steady state is reached. Then, place the LSPR tip assembly firmly into the heated stage sample holder.

The optical setup for this method is shown here. The light from the halogen lamp is passed through a 593-nanometer-long pass filter to eliminate wavelengths, which do not contribute to the nanoplasmonic response. The light is then linearly polarized, and the sample is illuminated, via a 40x 1.4 numerical aperture objective for the excitation of the plasmonic gold nanostructures. The scattered light is collected by the objective, and passed through a crossed polarizer to reduce the background signal from the glass substrate. A 50/50 beam splitter is inserted into the collected light path for simultaneous spectroscopic and imagery analysis.

After allowing the assembly and microscope to equilibrate for at least two hours, align the chip using the joystick so that the central array is aligned with the fiber optic for spectroscopy. After culturing and pelleting hybridoma cells by centrifugation, harvest them. Then, test for viability before introducing them onto the LSPR chips. Following this, introduce 50 microliters of the cell solution manually onto the LSPR chips with a micropipette. Finally, wash away the remaining cells in solution with fresh serum-free media using a microfluidic perfusion system to prepare the LSPR chips for imaging.