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In this study, we present the coupling of chip-based electrochromatography to MS using a glass chip with a monolithically integrated nanoelectrospray emitter. As separation column, an acrylate-based porous polymer monolith is implemented into the glass chip by photopolymerization. For the establishment and development of this method, we used a test mixture detectable with both fluorescence and ESI-MS. After successful evaluation of the approach with the test solutes, it was applied exemplarily for drug analysis such as high-speed separations of benzodiazepines in pharmaceuticals.

In the present study, we introduce two-photon excitation at 532 nm for label-free fluorescence detection in chip electrochromatography. Two-photon excitation at 532 nm offers a promising alternative to one-photon excitation at 266 nm, as it enables the use of economic chip materials instead of fused silica. In order to demonstrate these benefits, one-photon and two-photon induced fluorescence detection are compared in different chip layouts and materials with respect to the achievable sensitivity in the detection of polycyclic aromatic hydrocarbons (PAHs). Customized chromatography chips with cover or bottom slides of different material and thickness are produced by means of a rapid prototyping method based on liquid-phase lithography. The design of thin bottom chips (180 μm) enables the use of high-performance immersion objectives with low working distances, which allows one to exploit the full potential of two-photon excitation for a sensitive detection. The developed method is applied for label-free analysis of PAHs separated on a polymer monolith inside polymer glass sandwich chips made from fused silica or soda-lime glass. The obtained limits of detection range from 40 nM to 1.95 μM, with similar sensitivities in fused silica thin bottom chips for one-photon and two-photon excitation. In deep-UV non- or less-transparent devices two-photon excitation is mandatory for label-free detection of aromatics with high sensitivity.

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The islet amyloid polypeptide (IAPP) is a regulatory peptide that can aggregate into fibrillar structures associated with type 2 diabetes. In this study, the IAPP 21−27 segment was modified with a biotin linker at the N-terminus (Btn-GNNFGAIL) to immobilize peptide fibrils on streptavidin-coated surfaces. Key residues for fibril formation of the N-terminal biotinylated IAPP 21−27 segment were identified by using an alanine scanning approach combined with molecular dynamics simulations, thioflavin T fluorescence measurements, and scanning electron microscopy. Significant contributions of phenylalanine (F23), leucine (L27), and isoleucine (I26) for the fibrillation of the short peptide segment were identified. The fibril morphologies of the peptide variants differed depending on their primary sequence, ranging from flexible and semiflexible to stiff and crystal-like structures. These insights could advance the design of new functional hybrid bionanomaterials and fibril-engineered surface coatings using short peptide segments. To validate this concept, the biotinylated fibrils were immobilized on streptavidin-coated surfaces under spatial control.