Saskia A. G. Lambrechts

Covalently assembled NIR nanoplatform for simultaneous fluorescence imaging and photodynamic therapy of cancer cells.

A highly efficient multifunctional nanoplatform for simultaneous upconversion luminescence (UCL) imaging and photodynamic therapy has been developed on the basis of selective energy transfer from multicolor luminescent NaYF(4):Yb(3+),Er(3+) upconversion nanoparticles (UCNPs) to photosensitizers (PS). Different from popular approaches based on electrostatic or hydrophobic interactions, over 100 photosensitizing molecules were covalently bonded to every 20 nm UCNP, which significantly strengthened the UCNP-PS linkage and reduced the probability of leakage/desorption of the PS. Over 80% UCL was transferred to PS, and the singlet oxygen production was readily detected by its feature emission at 1270 nm. Tests performed on JAR choriocarcinoma and NIH 3T3 fibroblast cells verified the efficient endocytosis and photodynamic effect of the nanoplatform with 980 nm irradiation specific to JAR cancer cells. Our work highlights the promise of using UCNPs for potential image-guided cancer photodynamic therapy.

Mechanistic Study of the Photodynamic Inactivation of Candida albicans by a Cationic Porphyrin

ABSTRACT The growing resistance against antifungal agents has renewed the search for alternative treatment modalities, and antimicrobial photodynamic inactivation (PDI) is a potential candidate. The cationic porphyrin 5-phenyl-10,15,20-Tris(N-methyl-4-pyridyl)porphyrin chloride (TriP[4]) is a photosensitizer that in combination with light can inactivate bacteria, fungi, and viruses. For future improvement of the efficacy of PDI of clinically relevant fungi such as Candida albicans, we sought to understand the working mechanism by following the response of C. albicans exposed to PDI using fluorescence confocal microscopy and freeze-fracture electron microscopy. The following events were observed under dark conditions: TriP[4] binds to the cell envelope of C. albicans, and none or very little TriP[4] enters the cell. Upon illumination the cell membrane is damaged and eventually becomes permeable for TriP[4]. After lethal membrane damage, a massive influx of TriP[4] into the cell occurs. Only the vacuole membrane is resistant to PDI-induced damage once TriP[4] passes the plasma membrane. Increasing the incubation time of C. albicans with TriP[4] prior to illumination did not increase the influx of TriP[4] into the cell or the efficacy of PDI. After the replacement of 100% phosphate-buffered saline (PBS) by 10% PBS as the medium, C. albicans became permeable for TriP[4] during dark incubation and the efficacy of PDI increased dramatically. In conclusion, C. albicans can be successfully inactivated by the cationic porphyrin TriP[4], and the cytoplasmic membrane is the target organelle. TriP[4] influx occurred only after cell death.

Effect of Monovalent and Divalent Cations on the Photoinactivation of Bacteria with meso-Substituted Cationic Porphyrins

Abstract It is well established that for successful photoinactivation (PI) of gram-negative bacteria a cationic photosensitizer is required. This requirement suggests a charge-dependent interaction between the photosensitizer and the gram-negative bacterium, which may be influenced by the presence of ions in the suspending medium. The aim of the present study was to investigate the effect of cations Na+ and Ca2+ on the efficacy of the PI of the gram-negative Pseudomonas aeruginosa and the gram-positive Staphylococcus aureus. The bacteria were suspended in buffer containing either meso-tetra(N-methyl-4-pyridyl)-porphyrin or meso-mono-phenyl-tri(N-methyl-4-pyridyl)-porphyrin as photosensitizer and various concentrations of Na+ or Ca2+. The cell suspensions were exposed to a broadband light dose of 9 J/cm2. In buffer without added cations, P. aeruginosa and S. aureus were equally sensitive to PI. Addition of cations strongly decreased the sensitivity of both bacteria to PI, with the PI of P. aeruginosa being much more decreased than that of S. aureus, and Ca2+ being more effective than Na+. The decreased sensitivity was accompanied by a reduced binding of the photosensitizers to the bacteria.

Oxidation Monitoring by Fluorescence Spectroscopy Reveals the Age of Fingermarks

No forensic method exists that can reliably estimate the age of fingermarks found at a crime scene. Information on time passed since fingermark deposition is desired as it can be used to distinguish between crime related and unrelated fingermarks and to support or refute statements made by the fingermark donors. We introduce a non-contact method that can estimate the age of fingermarks. Fingermarks were approached as protein-lipid mixtures and an age-estimation model was build based on the expected protein and lipid oxidation reactions. Two measures of oxidation are required from the fingermark to estimate its age: 1) the relative amount of fluorescent oxidation products 2) the rate at which these products are formed. Fluorescence spectroscopy was used to obtain these measures. We tested the method on 44 fingermarks and were able to estimate the age of 55% of the male fingermarks, up to three weeks old with an uncertainty of 1.9 days.

On the autofluorescence of fingermarks

The autofluorescence of fingermarks is used for their detection. The components responsible for this autofluorescence are largely unknown. Thin layer chromatography and fluorescence spectroscopy were used to identify autofluorescent components and evaluate their forensic value. Based on our results, tryptophan is hypothesized to be a major contributor to the autofluorescence when part of peptides or proteins, id est, not in its free form. Part of the autofluorescence could be assigned to a kynurenine derivative. Pheophorbide A, a metabolite of chlorophyll, is inferred as a red fluorescent fingermark component. Chlorophyll is a plant pigment which implies that dietary information can potentially be retrieved from fingermarks.

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.

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.

Fingermark age determinations: Legal considerations, review of the literature and practical propositions.

The question of the age of fingermarks is often raised in investigations and trials when suspects admit that they have left their fingermarks at a crime scene but allege that the contact occurred at a different time than the crime and for legal reasons. In the first part of this review article, examples from American appellate court cases will be used to demonstrate that there is a lack of consensus among American courts regarding the admissibility and weight of testimony from expert witnesses who provide opinions about the age of fingermarks. Of course, these issues are not only encountered in America but have also been reported elsewhere, for example in Europe. The disparity in the way fingermark dating cases were managed in these examples is probably due to the fact that no methodology has been validated and accepted by the forensic science community so far. The second part of this review article summarizes the studies reported on fingermark dating in the literature and highlights the fact that most proposed methodologies still suffer from limitations preventing their use in practice. Nevertheless, several approaches based on the evolution of aging parameters detected in fingermark residue over time appear to show promise for the fingermark dating field. Based on these approaches, the definition of a formal methodological framework for fingermark dating cases is proposed in order to produce relevant temporal information. This framework identifies which type of information could and should be obtained about fingermark aging and what developments are still required to scientifically address dating issues.

Techniques that acquire donor profiling information from fingermarks — A review

Fingermarks are among the most important types of evidence that can be encountered at the scene of a crime since the unique ridge pattern of a fingerprint can be used for individualization. But fingermarks contain more than the characteristic pattern of ridges and furrows, they are composed of a wide variety of different components that originate from endogenous and exogenous sources. The chemical composition can be used to obtain additional information from the donor of the fingermark, which in turn can be used to create a donor profile. Donor profiling can serve at least two purposes i) to enhance the evidential value of fingermarks and ii) to provide valuable tactical information during the crime scene investigation. Retrieving this additional information is not limited to fingermarks that have been used for individualization, but can also be applied on partial and/or distorted fingermarks. In this review we have summarized the types of information that can be obtained from fingermarks. Additionally, an overview is given of the techniques that are available addressing their unique characteristics and limitations. We expect that in the nearby future, donor profiling from contact traces, including fingermarks will be possible.

Immunolabeling of fingermarks left on forensic relevant surfaces, including thermal paper

The chemical composition of a fingermark contains donor profiling information. Immunolabeling is a technique that can be used to retrieve this chemical information from fingermarks. Additionally, immunolabeling can be used to (re)develop fingermarks. To be of interest in the forensic field, the applicability of immunolabeling should be highly diverse. Therefore, in this study we investigated the applicability of one such method: immunolabeling of fingermarks left on non-porous (aluminum foil, stainless steel keys, plastic sheets, different colored garbage bags, sandwich bags, Ziploc bags), semi-porous (tiles, laminated chipboard), and porous surfaces (thermal and copy paper). Successful immunolabeling of specific components in fingermarks was possible on all surfaces tested, except for laminated chipboards and copy paper. Additionally, high quality images could be obtained from the immunolabeled fingermarks. Surprisingly, fingermarks left on thermal paper showed improved visibility when developed with the immunolabeling method. In conclusion, one intrinsically similar immunolabeling method can visualize fingermarks left on non-porous, semi-porous and porous surfaces.