Carbodiimide-mediated immobilization of acidic biomolecules on reversed-charge zwitterionic sensor chip surfaces.

The carbodiimide-mediated amine coupling of protein ligands to sensor chips coated with anionic polycarboxylate hydrogels, such as carboxymethyl dextran, is the predominant covalent immobilization procedure utilized in optical biosensors, namely surface plasmon resonance (SPR) biosensors. Usually, electrostatic interactions at a slightly acidic pH and low ionic strength are employed to efficiently accumulate neutral and basic ligands on the chip surface, which are then covalently coupled by surface-bound active N-hydroxysuccinimide (NHS) esters. Unfortunately, this approach is not suitable for acidic proteins or other ligands with low isoelectric points (IEPs), such as nucleic acids, because the charge density of the polycarboxylates is greatly reduced at acidic pH or because electrostatic attraction cannot be achieved. To overcome these drawbacks, we have established a charge-reversal approach that allows the preconcentration of acidic proteins above their IEPs. A precisely controlled amount of tertiary amines is applied to reverse the previous anionic surface charge while maintaining carbodiimide compatibility with future protein immobilization. The mechanism of this reversed-charge immobilization approach was demonstrated employing protein A as a model protein and using attenuated total reflectance Fourier transform infrared spectroscopy, dynamic contact angle measurements, colorimetric quantification, and SPR analysis to characterize surface derivatization. Furthermore, even though it had previously proven impossible to preconcentrate DNA electrostatically and to covalently couple it to polyanionic chip surfaces, we demonstrated that our approach allowed DNA to be preconcentrated and immobilized in good yields. Graphical abstract Principle of the covalent immobilization of acidic ligands on reversed-charge zwitterionic sensor chip surfaces.

Absorption and isomerization of caffeoylquinic acids from different foods using ileostomist volunteers.

BACKGROUND: Polyphenols are thought to play important roles in human nutrition and health but these health effects are dependent on their bioavailability. This study is one of a series with the aim of determining possible effects of food matrices on caffeoylquinic acid (CQA) bioavailability using ileostomy volunteers. METHODS: After a CQA-free diet, ileostomists consumed coffee (746 µmol total CQA), and CQAs in excreted ileal fluid were subsequently identified and quantified with HPLC-diode array detection and HPLC-ESI-MS/MS. In our previous studies, other food sources such as cloudy apple juice (CAJ) (358 µmol CQA) and apple smoothie (AS) (335 µmol CQA) were investigated with the same model. RESULTS: Interesterification of CQA from both apple matrices was observed during gastrointestinal passage, whereas CQA consumed in coffee was not influenced by interesterification reactions. In total, 74.3, 22.4, and 23.8 % of the CQA from CAJ, AS, and coffee, respectively, were absorbed or degraded. CONCLUSION: Our results show that variations in food matrices and variations in phenolic composition have a major influence on intestinal bioavailability and interesterification of the investigated subclass of polyphenols, the CQAs.