Microfluidically Partitioned Dual Channels for Accurate Background Subtraction in Cellular Binding Studies by Surface Plasmon Resonance Microscopy

Abstract

Unlike conventional surface plasmon resonance (SPR) using an antifouling film to anchor biomolecules and a reference channel for background subtraction, SPR microscopy for single-cell analysis uses a protein-or polypeptide-modified gold substrate to immobilize cells and a cell-free area as the reference. In this work, we show that such a substrate is prone to nonspecific adsorption (NSA) of species from the cell culture media, resulting in false background signals that cannot be correctly subtracted. To obtain accurate kinetic results, we patterned a dual-channel substrate using a microfluidic device, with one channel having poly-L-lysine deposited in situ onto a preformed polyethylene glycol (PEG) self-assembled monolayer for cell immobilization and the other channel remaining as PEG-covered for reference. The two 2.0 mm-wide channels are separated by a 75 μm barrier, and parts of the channels can be readily positioned into the field of view of an SPR microscope. The use of this dual-channel substrate for background subtraction is contrasted with the conventional approach through the following binding studies: (1) wheat germ agglutinin (WGA) attachment to the N-acetyl glucosamine and N-acetylneuraminic acid sites of glycans on HFF cells, and (2) the S1 protein of the COVID-19 virus conjugation with angiotensinconverting enzyme 2 (ACE2) on the HEK293 cells. Both studies revealed that interferences by NSA and the surface plasmon polariton wave diffracted by cells can be excluded with the dual-channel substrate, and the much smaller refractive index changes caused by the injected solutions can be correctly subtracted. Consequently, sensorgrams with higher signal-to-noise ratios and shapes predicted by the correct binding model can be obtained with accurate kinetic and affinity parameters that are more biologically relevant. The affinity between S1 protein and ACE2 is comparable to that measured with recombinant ACE2, yet the binding kinetics is different, suggesting that the cell membrane does impose a kinetic barrier to their interaction.

Live Cells versus Fixated Cells: Kinetic Measurements of Biomolecular Interactions with the LigandTracer Method and Surface Plasmon Resonance Microscopy

“…The cell-covered and reference areas were partitioned in a manner similar to that described earlier. 36 Briefly, the gold-coated glass chips (Biosensing Instrument Inc.) were modified with an HS-PEG self-assembled monolayer (SAM) by soaking each chip in a 2 mM HS-PEG solution for 24 h in the dark. Into the well of a PDMS device (Xona Microfluidics, Rayleigh, NC) placed onto the PEGmodified chip, 0.45 μM PLL was added and allowed to stand for 2 h. The PEG SAMs and the PEG−PLL films were characterized with an UVISEL Plus ellipsometer (HORIBA, Kyoto, Japan).…”

“…Before measurements, a diluted running buffer (90%, v/v) was injected to calibrate the instrument. The cell-covered and reference areas were partitioned in a manner similar to that described earlier . Briefly, the gold-coated glass chips (Biosensing Instrument Inc.) were modified with an HS-PEG self-assembled monolayer (SAM) by soaking each chip in a 2 mM HS-PEG solution for 24 h in the dark.…”

Research on mine moving target detection method based on deep learning

“…In the field of motion target detection, traditional techniques include frame differencing [1], multi-frame differencing [2], background differencing [3], and optical flow [4]. Frame differencing extracts moving targets by performing differencing operations on adjacent frame images and has the characteristics of speed and robustness, making it suitable for simple target scenes.…”

Large-scale validation of a plasmonic sensor for SARS-CoV-2 pseudo-neutralization with a cohort of food and retail workers

“…[2][3][4][5][6] Among them, surface plasmon resonance (SPR) sensing is a particularly tantalizing alternative to the current standard clinical assays, as SPR provides quantitative biomolecular information and is amenable to portable and POC testing formats. 7 The pandemic facilitated the testing of new detection platforms in plasmonics such as SPR interferometry, 8 thermoplasmonics sensing, 9 the use of 2D materials such as MXenes, 10 metasurfaces, 11 a new background subtraction strategy, 12 and the use of plasmonics for the detection of target biomarkers in saliva and diluted whole blood. 13 Furthermore, the COVID-19 pandemic enabled the validation of portable sensing platforms such as fiber-optics-based SPR sensors, 14 smartphone-based LSPR detection synchronized with machine learning (ML), 15 and lightweight SPR sensors with simple graphical user interface.…”