Simultaneous Visualization of Dual Intercellular Signal Transductions via SERS Imaging of Membrane Proteins Dimerization on Single Cells

Abstract

The visualization of protein dimerization on live cells is an urgent need and of vital importance for facile monitoring the signal transduction during intercellular communication. Herein, a highly sensitive and specific SERS strategy for simultaneously imaging dual homodimerizations of membrane proteins on single live cells was proposed by networking of AuNPs-based dual-recognition probes (dual-RPs) and SERS tags via proximity ligation-assisted catalytic hairpin assembly (CHA). The dual-RPs were prepared by comodifying hairpin-structured ssDNAs H1-Met and H1-TβRII on 50 nm AuNPs and two SERS tags for membrane proteins Met and TβRII were prepared respectively by labeling their corresponding Raman molecules and hairpin-structured single-stranded DNAs H2-Met or H2-TβRII on 15 nm AuNPs. The membrane proteins were ligated proximally by specific aptamers, and the dimerizations of proteins resulted in the proximity ligation-assisted CHA-based networking of dual-RPs and SERS tags to form 15Au–50Au network nanostructures with significantly enhanced SERS effect. The SERS strategy for visualizing the membrane protein dimerization was established and the good performance on simultaneously SERS imaging dual dimerizations of membrane proteins (i.e., Met-Met and TβRII-TβRII) was confirmed. Furthermore, the membrane protein dimerization-based signaling pathways between cancer cells and stromal cells or stem cells were observed by SERS, which indicates that the proposed SERS strategy is a good method for high-sensitivity monitoring of membrane proteins dimerizations-based multiple intercellular signal transductions in a natural and complex cellular microenvironment.

DNA-Programed Plasmon Rulers Decrypt Single-Receptor Dimerization on Cell Membrane

“…In this regard, our proposed method does not require further modification of the Raman label molecules as well as the combination with other amplification strategies, making it simpler and more convenient compared to SERS imaging. 26 Additionally, given that the interspacing of nanoparticle pairs can be precisely quantified by optical variations, the use of plasmon rulers could provide additional information about the proximity of biomolecules, 53 a property that made them particularly applicable to research on receptor assembly behavior. More importantly, this aptamer recognition strategy also enabled the results to be presented in a stoichiometric manner, in other words, a plasmon ruler corresponded to a single receptor or a pair of dimerized receptors, effectively increasing the precision of single-molecule analysis.…”

“…25 Second, the strong background fluorescence interference from intracellular components hinders the accuracy of the assay. 26 Third, the problem of fluorescence bleaching under continuous irradiation makes it difficult to achieve long-term monitoring. 27 As known, plasmonic nanomaterials feature better photostability and larger optical cross sections than fluorescent molecules, rendering them more suitable for long-term real-time tracking at the single-particle level.…”

“…First, the signals of conventional fluorescence methods are too weak to detect small amounts of cell membrane receptors, making imaging at the single-molecule level even harder to achieve . Second, the strong background fluorescence interference from intracellular components hinders the accuracy of the assay . Third, the problem of fluorescence bleaching under continuous irradiation makes it difficult to achieve long-term monitoring …”

DNA-mediated dynamic plasmonic nanostructures: assembly, actuation, optical properties, and biological applications

“…14c). 214 This SERS strategy was successfully applied to visual monitoring of cell signaling pathway during natural cellular communication. Furthermore, Wang and Song also proposed a novel theranostic nanosystem based on the target-induced AuNPs network-structured aggregates for cascaded SERS imaging of intracellular cancer-related miR-106a and miR-106a-triggered dual gene-silencing therapy of cancer cells (Fig.…”

Steric Hindrance On–Off Mass-Tagged Probe Set Enables Detection of Protein Homodimer in Living Cells

“…Currently, a few conventional methods exist to detect protein dimerization such as coimmunoprecipitation (co-IP), cross-linking mass spectrometry (MS) and proximity ligation assays (PLA), and surface-enhanced Raman scattering (SERS) strategy. − Classical co-IP and cross-linking MS methods normally require complex digestion and separation procedures, which may cause an unforeseeable protein information loss in physiological states such as living cells and may even destroy protein complexes . Comparatively, PLA is an effective method for the detection of protein dimers.…”