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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.

Forensic genetic analysis of items possibly handled by a suspect or a victim is frequently inquired by the law enforcement authorities, since DNA left on touched objects can often be linked to an individual. Due to technical improvement, even poor traces, which seemed to be unsuitable for DNA analysis a few years ago, may be amplified successfully today. Yet, DNA can be transferred to a crime scene artificially or unintentionally without any primary contact between the individual and the object found at the crime scene, the so-called secondary transfer or indirect transfer in general. In this study, "secondary transfer" scenarios with cells and DNA of different origins under wet conditions were investigated. Transfer was simulated as either "washing by hand" in a washtub or as "machine laundry" in a washing machine. As expected, major differences were seen between blood stains and epithelial abrasions. DNA from blood donors could be detected clearly both on the donor and on the acceptor textile, regardless of washing method. Regarding epithelial abrasions, simulating worn clothes, after washing by hand, only little residual DNA was found, and partial profiles were displayed on the donor textile, while transfer to the acceptor textile occurred even less and not in noteworthy amount and quality. Single alleles could be found both on donor textiles and acceptor textiles after simulated machine wash, but no reliable DNA profile could be verified after laundry in machine. Therefore, a DNA transfer from one worn cloth (without blood stains) to another textile in the washing machine seems to be extremely unlikely.