Silver has been demonstrated to be a powerful cationization agent in mass spectrometry (MS) for various olefinic species such as cholesterol and fatty acids. This work explores the utility of metallic silver sputtering on tissue sections for high resolution imaging mass spectrometry (IMS) of olefins by laser desorption ionization (LDI). For this purpose, sputtered silver coating thickness was optimized on an assorted selection of mouse and rat tissues including brain, kidney, liver, and testis. For mouse brain tissue section, the thickness was adjusted to 23 ± 2 nm of silver to prevent ion suppression effects associated with a higher cholesterol and lipid content. On all other tissues, a thickness of at 16 ± 2 nm provided the best desorption/ionization efficiency. Characterization of the species by MS/MS showed a wide variety of olefinic compounds allowing the IMS of different lipid classes including cholesterol, arachidonic acid, docosahexaenoic acid, and triacylglyceride 52:3. A range of spatial resolutions for IMS were investigated from 150 μm down to the high resolution cellular range at 5 μm. The applicability of direct on-tissue silver sputtering to LDI-IMS of cholesterol and other olefinic compounds presents a novel approach to improve the amount of information that can be obtained from tissue sections. This IMS strategy is thus of interest for providing new biological insights on the role of cholesterol and other olefins in physiological pathways or disease.
Non-specific adsorption of the molecular components of biofluids is ubiquitous in the area of biosensing technologies, severely limiting the use of biosensors in real-world applications. The surface chemistries developed to prevent non-specific adsorption of crude serum are not necessarily suited for sensing in other biosamples. In particular, the diagnostic potential of differential expression of proteins in tissues makes cell lysate attractive for disease diagnostics using solid biopsies. However, crude cell lysate poses a significant challenge for surface chemistries because of a large concentration of highly adherent lipids. Contrary to the non-specific adsorption in crude serum being suppressed by hydrophilic surfaces, the surface plasmon resonance (SPR) analysis of serine-, aspartic-acid-, histidine-, leucine-, and phenylalanine-based peptide monolayers revealed that hydrophobic and positively charged peptides decreased non-specific adsorption when using lysate from HEK 293FT cells. A polyethylene glycol (PEG) monolayer resulted in 2-fold greater fouling than the best peptide [3-MPA-(His)2(Leu)2(Phe)2-OH] under the same conditions. Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis of the adsorbate from cell lysate confirmed that lipids are the main source of non-specific adsorption. Importantly, the mass spectrometry (MS) study revealed that both the number of lipids identified and their intensity decreased with decreasing non-specific adsorption. A peptide monolayer thus provides an efficient mean to suppress non-specific adsorption from this human cell lysate.
Characterization of protein adsorption to surfaces has implications from biosensing to protective biocoatings. While research studies have principally focused on determining the magnitude of protein adsorption to surfaces, the proteins involved in the process remains only broadly identified and has not been investigated on several surfaces. To further elucidate the nonspecific adsorption process of serum to surfaces, surface plasmon resonance (SPR) and matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) were used in combination to obtain quantitative and qualitative information about the process of protein adsorption to surfaces. To validate the technique, crude serum was nonspecifically adsorbed on four self-assembled monolayer (SAM) on gold: 16-mercaptohexadecanoic acid (16-MHA), 11-mercaptoundecane(ethylene glycol)3-COOH (PEG), 3-MPA-LHDLHD-OH, and 3-MPA-HHHDD-OH. Direct MS analysis of the nonspecifically adsorbed proteins suggested the presence of a variety of protein (BSA, IgG, and apolipoprotein A-1). Performing a trypsin digestion of the nonspecifically adsorbed proteins confirmed the presence of BSA and apolipoprotein A-1 and further revealed the complexity of the process by detecting the presence of complement C3, SHC-transforming protein 1, and kininogen 2. The level of nonspecific adsorption on different surfaces measured by SPR sensing directly correlated with the intensity of the serum protein and indirectly with the tryptic peptides measured by MS. Detailed analysis of the BSA peptides digested on 16-MHA and for BSA digested in solution was used to investigate the orientation of BSA on this surface. The combination of SPR and MS allows the quantitative and qualitative understanding of protein adsorption processes to surfaces.
A multi-channel system combining fluidics and micropatterned plasmonic materials with wavelength interrogation surface plasmon resonance (SPR) and fluorescence detection was integrated from the combination of a small and motorized fluorescence microscope mounted on a portable 4-channel SPR instrument. The SPR and fluorescent measurements were performed based on the same detection area in a multi-channel fluidic, with a sensing scheme for prostate-specific antigen (PSA) consisting of a sandwich assay with a capture anti-PSA immobilized onto the SPR sensor and a detection anti-PSA modified with horseradish peroxidase (HRP). In this dual-detection instrument, fluorescence was measured from the solution side of the micropatterned gold film, while the interface between the glass prism and the gold film served to interrogate the SPR response. The SPR sensors were comprised of microhole arrays fabricated by photolithography to enhance the instrumental response for PSA detection by approximately a factor of 2 to 3 and they were coated with a self-assembled monolayer of a peptide (3-MPA-HHHDD-OH) to minimize nonspecific adsorption. PSA was successfully detected at clinical concentrations from 10 pM to 50 nM with this integrated system in a single assay lasting 12 minutes, almost centering on the desired range for PSA diagnostic tests (>4 ng mL(-1) or >150 pM). The combination of two robust techniques in a single chip and instrument has led to a simple and effective assay that can be carried out on a small and portable instrument providing rapid biodetection of an important cancer biomarker with a dynamic range of nearly 4 orders of magnitude in the clinical range.
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