研究目的
To develop and apply a novel label-free method using AFM-IR for high-resolution analysis of molecular distribution and orientation in ultrathin, multicomponent model membranes, specifically to study the interactions and phase behavior in systems containing sphingomyelin, cholesterol, and cyclosporin A.
研究成果
AFM-IR is demonstrated as a powerful tool for nanoscale chemical mapping and orientation analysis in ultrathin model membranes, enabling direct observation of component distribution and molecular orientation without labels. It confirmed cyclosporin A's affinity for lipid rafts and provided insights into phase separation and ordering, with potential applications for studying other membrane-active molecules.
研究不足
The technique requires specialized equipment like AFM-IR microscopes and tunable lasers, which may not be widely accessible. Sample preparation involves precise LB film deposition, which can be time-consuming and sensitive to environmental conditions. The method is limited to surfaces that can be coated with gold for enhancement, and resolution is dependent on AFM tip quality. Additionally, the study focuses on model systems, which may not fully replicate complex biological membranes.
1：Experimental Design and Method Selection:
The study utilized AFM-IR (atomic force microscope infrared-spectroscopy) for nanoscale chemical characterization of Langmuir-Blodgett (LB) films. This method was chosen for its ability to provide label-free, chemoselective mapping and orientation analysis without the need for chemical modifications.
2：Sample Selection and Data Sources:
Model membranes were prepared using sphingomyelin (SM), cholesterol (Chol), and cyclosporin A (CsA) in specific molar ratios (e.g.,
3：
4：
5：30 and
6：
7：30). LB monolayers were transferred onto gold-coated silica wafers or mica substrates. List of Experimental Equipment and Materials:
Equipment included a NIMA 612D Langmuir-Blodgett trough, nanoIR2 AFM-IR microscope (Anasys Instruments) with a tunable quantum cascade laser (Daylight Solutions MIRcat), silicon and gold-coated AFM tips (Anasys Instruments models: standard silicon tip and PR-EX-nIR2-10), ATR-FTIR spectrometer (Nicolet Almega system), and software such as Opus and SPIP. Materials were high-purity chemicals: cyclosporin A and cholesterol from Sigma-Aldrich, sphingomyelin from Avanti Polar Lipids, chloroform from Sigma Aldrich, and ultrapure water from Milipore system.
8：Experimental Procedures and Operational Workflow:
Langmuir monolayers were formed by spreading solutions on water, compressed to 15 mN/m, and transferred to substrates using LB technique. AFM-IR spectra and maps were collected in contact mode with specific laser polarizations (p and s), covering spectral ranges 1929-1695 cm?1, 1695-1410 cm?1, and 1410-1145 cm?1. Data involved averaging multiple scans for signal-to-noise improvement.
9：Data Analysis Methods:
Spectra were processed using Opus software for smoothing and normalization. AFM and IR maps were analyzed with SPIP software, including normalization to laser power and extraction of intensity profiles for component distribution.
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