Abstract
Accurate detection and quantification of individual molecules is important for the development of improved diagnostic methods as well as biochemical characterization of disease progression and treatments. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a surface analysis technique capable of imaging the distribution of specific molecules on surfaces with a high spatial resolution (<1 μm) and high sensitivity. ToF-SIMS is particularly suitable for detection of molecules up to ∼2 kDa, including lipids, whereas larger molecules, such as peptides and proteins, are fragmented during analysis, which makes them difficult to identify. In this study, an approach for extending the molecular detection capability of ToF-SIMS is presented, based on the specific binding of functionalized liposomes to molecular targets on the sample surface and subsequent detection of the liposomes by ToF-SIMS. Furthermore, by using different recognition elements conjugated to liposomes with different lipid compositions, simultaneous detection of different targets was accomplished. This multiplexing capability was investigated for two types of recognition elements (antibodies and cholera toxin) and for target molecules immobilized on surfaces using two frequently applied surface functionalization strategies: a supported lipid bilayer aimed to mimic a cell membrane and a polyethylene glycol modified surface, commonly employed in bioanalytical sensor applications. The efficacy of the conjugation protocols and the specificity of the recognition mechanism were confirmed using quartz crystal microbalance with dissipation monitoring, while fluorescence microscopy was used to validate the ToF-SIMS data and the reliability of the freeze-drying step required for ToF-SIMS analysis. The results demonstrated specific binding of the two types of liposomes to each target and showed a concentration-dependent binding to the targets on the different model surfaces. In particular, the possibility to use the contrasts in the mass spectra of SIMS to identify the concentration dependent coverage of different liposomes opens up new opportunities for multiplexed detection and quantification of molecules at biotechnology relevant interfaces.
Highlights
Imaging mass spectrometry is a powerful approach for biomolecular detection that allows for label-free identification of many different molecular species in parallel.2–8 the different techniques applied, such as matrixassisted laser desorption/ionization or time-of-flight secondary ion mass spectrometry (ToF-SIMS), are typically limited with respect to the types of biomolecular species that can be detected at the same time, and in addition, the sensitivity is typically inferior to methods that rely on detection of single target molecules
0.1% PLL-g-polyethylene glycol (PEG)-biotin, the results show that reducing the liposome concentration from 0.1 to 0.01 mg/ml results in a moderate signal reduction of approximately 50%, whereas the signal at 0.001 mg/ml drops to only 11% and 16% (ToF-SIMS) of the signal intensities at 0.1 mg/ml
A novel approach was evaluated for multiplexed biomolecular detection based on specific binding of liposomes to target molecules on surfaces and subsequent liposome detection by ToF-SIMS
Summary
Imaging mass spectrometry is a powerful approach for biomolecular detection that allows for label-free identification of many different molecular species in parallel. the different techniques applied, such as matrixassisted laser desorption/ionization or time-of-flight secondary ion mass spectrometry (ToF-SIMS), are typically limited with respect to the types of biomolecular species that can be detected at the same time, and in addition, the sensitivity is typically inferior to methods that rely on detection of single target molecules. The approach is based on specific binding of liposomes to target molecules on the surface, using a recognition element (such as an antibody) that is conjugated to the liposome, and subsequent detection and imaging of the liposomes using ToF-SIMS Potential advantages of this approach compared to conventional optical imaging include (1) high sensitivity since each binding event is monitored by the detection of a single liposome, (2) high spatial resolution in the 1–2 lm range, and (3) high multiplexing potential. The potential and general applicability of the approach are demonstrated by the parallel detection of target molecules on two commonly applied target-presenting surfaces: self-assembled monolayers of poly(L-lysine) (PLL)-g-polyethylene glycol (PEG) polymers and supported lipid bilayers (SLBs), respectively. 03B413-3 investigated with respect to specific binding of liposomes conjugated with either antibiotin antibodies or protein cholera toxin subunit B (Chtx), which is known to bind to GM1 (Ref. 14) [see Fig. 1(b)]
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