DFT Study of Imine-Exchange Reactions in Iron(II)-Coordinated Pincers.

The imine bond is among the most applied motifs in dynamic covalent chemistry. Although its uses are varied and often involve coordination to a transition metal for stability, mechanistic studies on imine exchange reactions so far have not included metal coordination. Herein, we investigated the condensation and transimination reactions of an Fe2+ -coordinated diimine pyridine pincer, employing wB97XD/6-311G(2d,2p) DFT calculations in acetonitrile. We first experimentally confirmed that Fe2+ is strongly coordinated by these pincers, and is thus a justified model ion. When considering a four-membered ring-shaped transition state for proton transfers, the required activation energies for condensation and transimination reaction exceeded the values expected for reactions known to be spontaneous at room temperature. The nature of the incoming and exiting amines and the substituents on the para-position of the pincer had no effect on this. Replacing Fe2+ with Zn2+ or removing it altogether did not reduce it either. However, the addition of two ethylamine molecules lowered the energy barriers to be compatible with experiment (19.4 and 23.2 kcal/mol for condensation and transimination, respectively). Lastly, the energy barrier of condensation of a non-coordinated pincer was significantly higher than found for Fe2+ -coordinating pincers, underlining the catalyzing effect of metal coordination on imine exchange reactions.

Improving the visualization of fingermarks using multi-target immunolabeling.

The development of fingermarks is an important step in visualizing ridge patterns for individualization purposes. Immunolabeling can be applied to fingermarks to selectively and sensitively detect antigens in fingermarks, and can be used as a developing method to visualize fingermarks. In this study we investigated single (the detection of one antigen) and multiple targeting approaches (the detection of multiple antigens simultaneously) to improve fingermark development. The detection of dermcidin, an antimicrobial peptide, was used as the gold standard to compare single and multi-target detection of keratins, albumin and/or dermcidin. Single detection of dermcidin and albumin mostly resulted in clear ridge details and/or pore detection, whereas the single keratin detection resulted in a poor visualization of the fingermarks. The multi-target approach in which both dermcidin and albumin were targeted, resulted in improved fingermark development compared to single dermcidin detection. Therefore, we recommend the use of multi-target detection consisting of anti-dermcidin and anti-albumin when using immunolabeling as fingermark development technique. Additionally, the optimized multi-target approach was tested as a pre- and post-development technique in combination with powder dusting and cyanoacrylate fuming. Immunolabeling has not been implemented yet in forensic case work, however we expect that immunolabeling can be used to redevelop poorly developed and/or smudged fingermarks in the nearby future. Currently, an ongoing pilot-study is being conducted in collaboration with the Dutch police.

Detection of extracellular vesicles in plasma and urine of prostate cancer patients by flow cytometry and surface plasmon resonance imaging.

Large (> 1 µm) tumor-derived extracellular vesicles (tdEVs) enriched from the cell fraction of centrifuged whole blood are prognostic in metastatic castration-resistant prostate cancer (mCRPC) patients. However, the highest concentration of tdEVs is expected in the cell-free plasma fraction. In this pilot study, we determine whether mCRPC patients can be discriminated from healthy controls based on detection of tdEVs (< 1µm, EpCAM+) and/or other EVs, in cell-free plasma and/or urine. The presence of marker+ EVs in plasma and urine samples from mCRPC patients (n = 5) and healthy controls (n = 5) was determined by flow cytometry (FCM) and surface plasmon resonance imaging (SPRi) using an antibody panel and lactadherin. For FCM, the concentrations of marker positive (+) particles and EVs (refractive index <1.42) were determined. Only the lactadherin+ particle and EV concentration in plasma measured by FCM differed significantly between patients and controls (p = 0.017). All other markers did not result in signals exceeding the background on both FCM and SPRi, or did not differ significantly between patients and controls. In conclusion, no difference was found between patients and controls based on the detection of tdEVs. For FCM, the measured sample volumes are too small to detect tdEVs. For SPRi, the concentration of tdEVs is probably too low to be detected. Thus, to detect tdEVs in cell-free plasma and/or urine, EV enrichment and/or concentration is required. Furthermore, we recommend testing other markers and/or a combination of markers to discriminate mCRPC patients from healthy controls.

Identification and detection of protein markers to differentiate between forensically relevant body fluids.

The identification of body fluids at a crime scene is an important aspect of forensic casework analysis, being a source for investigative leads and contributing to case evidence. Yet, current methods for the forensic identification of body fluids suffer from several limitations, ranging from poor sensitivity and specificity, to sample destruction and interference with subsequent DNA analysis. Moreover, current identification assays target only one body fluid at the time. Besides being inefficient in terms of time, money and sample consumption, poor identification methods can also negatively influence the outcome of a (court) case. In this study, eleven potential protein biomarkers and antibodies were selected and assessed on their suitability for serving as identification markers, as a first step towards the development of a new multiplex protein-based body fluid identification assay relying on antigen-antibody interactions. Performing antibody-based dot blot assays, the specificity of the biomarkers for their target body fluids was evaluated, and biomarker detection was studied in diluted, mixed, aged and simulated casework samples. Hereby, nine out of eleven markers were identified as promising biomarkers to identify blood, semen, saliva, urine and sweat. With the identification of these targets and detection antibodies, a major step forward has been taken towards the development of a highly sensitive and specific, fast and non-labour-intensive protein-based body fluid identification assay, suitable for on-site analysis and able to test for multiple body fluids in a single reaction.

Simultaneous labeling of multiple components in a single fingermark.

A fingermark contains important forensic information of the donor, not only in its ridge pattern, but also in the chemical composition of its secretion. Detection and identification of these secretions can be done by immunolabeling. In this study, we describe for the first time a reproducible immunolabeling method that allows the simultaneous detection of multiple components of interest. This method not only reduces the manipulation of fingermarks, but also different types of information can be obtained about the donor in one labeling session. To prove the concept of this technique, we selected two general components as antigens of interest, dermcidin and the human serum albumin. Conjugation of both antibodies to two different synthetic fluorophores, followed by simultaneous incubation of both conjugated antibodies, resulted in successful multiple immunolabeling of fingermarks left on a porous nitrocellulose membrane and on a non-porous glass slide surface. In order to minimize false positives to prevent non-specific binding of antibodies to fingermarks and surface carriers, careful blocking and washing steps were found crucial. With this reproducible protocol, high quality images could be obtained from the multiple labeled fingermarks. In conclusion, simultaneous multiple immunolabeling of antibodies in fingermarks can identify specific components in the secretion of the fingermark, including components related to hygiene, diet, time of day, contacts gender and drug use. Multiple immunolabeling therefore has the potential to make a major impact in the forensic field.

The compatibility of fingerprint visualization techniques with immunolabeling.

The chemical composition of a fingermark potentially holds a wealth of information about the fingermark donor, which can be extracted by immunolabeling. Immunolabeling can be used to detect specific components in fingermarks; however, to be applicable in the forensic field, it should be compatible with commonly used fingerprint visualization techniques. In this study, the compatibility of immunolabeling with two different fingerprint visualization techniques, magnetic powdering and ninhydrin staining, was investigated on fingermarks deposited on glass and on nitrocellulose membranes. With dermcidin as antigen of interest, immunolabeling was performed successfully on all developed fingermarks. We can conclude that immunolabeling is compatible with magnetic powdering and ninhydrin staining, which can be of great forensic value.