A Polyclonal Aptamer Library for the Specific Binding of the Gut Bacterium Roseburia intestinalis in Mixtures with Other Gut Microbiome Bacteria and Human Stool Samples.

Roseburia intestinalis has received attention as a potential probiotic bacterium. Recent studies have demonstrated that changes in its intestinal abundance can cause various diseases, such as obesity, enteritis and atherosclerosis. Probiotic administration or fecal transplantation alter the structure of the intestinal flora, offering possibilities for the prevention and treatment of these diseases. However, current monitoring methods, such as 16S rRNA sequencing, are complex and costly and require specialized personnel to perform the tests, making it difficult to continuously monitor patients during treatment. Hence, the rapid and cost-effective quantification of intestinal bacteria has become an urgent problem to be solved. Aptamers are of emerging interest because their stability, low immunogenicity and ease of modification are attractive properties for a variety of applications. We report a FluCell-SELEX polyclonal aptamer library specific for R. intestinalis isolated after seven evolution rounds, that can bind and label this organism for fluorescence microscopy and binding assays. Moreover, R. intestinalis can be distinguished from other major intestinal bacteria in complex defined mixtures and in human stool samples. We believe that this preliminary evidence opens new avenues towards aptamer-based electronic biosensors as new powerful and inexpensive diagnostic tools for the relative quantitative monitoring of R. intestinalis in gut microbiomes.

BSA Hydrogel Beads Functionalized with a Specific Aptamer Library for Capturing Pseudomonas aeruginosa in Serum and Blood.

Systemic blood stream infections are a major threat to human health and are dramatically increasing worldwide. Pseudomonas aeruginosa is a WHO-alerted multi-resistant pathogen of extreme importance as a cause of sepsis. Septicemia patients have significantly increased survival chances if sepsis is diagnosed in the early stages. Affinity materials can not only represent attractive tools for specific diagnostics of pathogens in the blood but can prospectively also serve as the technical foundation of therapeutic filtration devices. Based on the recently developed aptamers directed against P. aeruginosa, we here present aptamer-functionalized beads for specific binding of this pathogen in blood samples. These aptamer capture beads (ACBs) are manufactured by crosslinking bovine serum albumin (BSA) in an emulsion and subsequent functionalization with the amino-modified aptamers on the bead surface using the thiol- and amino-reactive bispecific crosslinker PEG4-SPDP. Specific and quantitative binding of P. aeruginosa as the dedicated target of the ACBs was demonstrated in serum and blood. These initial but promising results may open new routes for the development of ACBs as a platform technology for fast and reliable diagnosis of bloodstream infections and, in the long term, blood filtration techniques in the fight against sepsis.

FluCell-SELEX Aptamers as Specific Binding Molecules for Diagnostics of the Health Relevant Gut Bacterium Akkermansia muciniphila.

Based on their unique properties, oligonucleotide aptamers have been named a gift of biological chemistry to life science. We report the development of DNA aptamers as the first high-affinity binding molecules available for fast and rapid labeling of the human gut bacterium Akkermansia muciniphila with a certain impact on Alzheimer´s disease. Fast and reliable analyses of the composition of microbiomes is an emerging field in microbiology. We describe the molecular evolution and biochemical characterization of a specific aptamer library by a FluCell-SELEX and the characterization of specific molecules from the library by bioinformatics. The aptamer AKK13.1 exerted universal applicability in different analysis techniques in modern microbiology, including fluorimetry, confocal laser scanning microscopy and flow cytometry. It was also functional as a specific binding entity hybridized to anchor primers chemically coupled via acrydite-modification to the surface of a polyacrylamide-hydrogel, which can be prototypically used for the construction of affinity surfaces in sensor chips. Together, the performance and methodological flexibility of the aptamers presented here may open new routes not only to develop novel Akkermansia-specific assays for clinical microbiology and the analyses of human stool samples but may also be an excellent starting point for the construction of novel electronic biosensors.

Azulitox-A Pseudomonas aeruginosa P28-Derived Cancer-Cell-Specific Protein Photosensitizer.

Azulitox as a new fusion polypeptide with cancer cell specificity and phototoxicity was generated and is composed of a photosensitizer domain and the cell-penetrating peptide P28. The photosensitizer domain (EcFbFP) was derived from a bacterial blue-light receptor, which belongs to the family of light-oxygen-voltage proteins and produces reactive oxygen species (ROS) upon excitation. P28 is derived from the cupredoxin protein azurin that is known to specifically penetrate cancer cells and bind to the tumor suppressor protein p53. We show that the P28 domain specifically directs and translocates the fused photosensitizer into cancer cells. Under blue-light illumination, Azulitox significantly induced cytotoxicity. Compared to the extracellular application of EcFbFP, Azulitox caused death to about 90% of cells, as monitored by flow cytometry, which also directly correlated with the amount of ROS produced in the cells. Azulitox may open new avenues toward targeted polypeptide-photosensitizer-based photodynamic therapies with reduced systemic toxicity compared to conventional photosensitizers.

Polyclonal aptamer libraries as binding entities on a graphene FET based biosensor for the discrimination of apo- and holo-retinol binding protein 4.

Oligonucleotide DNA aptamers represent an emergently important class of binding entities towards as different analytes as small molecules or even whole cells. Without requiring the canonical isolation of individual aptamers following the SELEX process, the focused polyclonal libraries prepared by this in vitro evolution and selection can directly be used to label their dedicated targets and to serve as binding molecules on surfaces. Here we report the first instance of a sensor able to discriminate between loaded and unloaded retinol-binding protein 4 (RBP4), an important biomarker for the prediction of diabetes and kidney disease. The sensor relies on two aptamer libraries tuned such that they discriminate between the protein isoforms, requiring no further sample labelling to detect RBP4 in both states. The evolution, binding properties of the libraries and the functionalization of graphene FET sensor chips are presented as well as the functionality of the resulting biosensor.

Polyclonal Aptamers for Specific Fluorescence Labeling and Quantification of the Health Relevant Human Gut Bacterium Parabacteroides distasonis.

Single-stranded DNA aptamers as affinity molecules for the rapid, reliable detection of intestinal bacteria are of particular interest to equip health systems with novel robust and cheap diagnostic tools for monitoring the success of supplementation strategies with selected probiotic gut bacteria in the fight against major widespread threats, such as obesity and neurodegenerative diseases. The human gut bacterium Parabacteroides distasonis (P. distasonis) is positively associated with diseases such as obesity, non-alcoholic fatty liver disease and multiple sclerosis with reduced cell counts in these diseases and is thus a promising potential probiotic bacterium for future microbial supplementation. In this paper we report on the evolution of a specific polyclonal aptamer library by the fluorescence based FluCell-SELEX directed against whole cells of P. distasonis that specifically and efficiently binds and labels P. distasonis. The aptamer library showed high binding affinity and was suited to quantitatively discriminate P. distasonis from other prominent gut bacteria also in mixtures. We believe that this library against a promising probiotic bacterium as a prototype may open new routes towards the development of novel biosensors for the easy and efficient quantitative monitoring of microbial abundance in human microbiomes in general.

Diffusion-controlled release of the theranostic protein-photosensitizer Azulitox from composite of Fmoc-Phenylalanine Fibrils encapsulated with BSA hydrogels.

Hydrogels offer a promising potential for the encapsulation and regulated release of drugs due to their biocompatibility and their tunable properties as materials. Only a limited number of systems and procedures enable the encapsulation of sensitive proteins. N-terminally fmoc-protected phenylalanine has been shown to self-assemble into a transparent, stable hydrogel It can be considered a supergelator due to the low amount of monomers necessary for hydrogelation (0.1% w/v), making it a good candidate for the encapsulation and stabilization of sensitive proteins. However, application options for this hydrogel are rather limited to those of many other fibril-based materials due to its intrinsic lack of mechanical strength and high susceptibility to changes in environmental conditions. Here, we demonstrate that the stability of a fibrillary system and the resulting release of the protein-photosensitizer Azulitox can be increased by combining the hydrogel with a tightly cross-linked BSA hydrogel. Azulitox is known to display cell-penetrating properties, anti-proliferative activity and has a distinctive fluorescence. Confocal microscopy and fluorescence measurements verified the maintenance of all essential functions of the encapsulated protein. In contrast, the combination of fibrillary and protein hydrogel resulted in a significant stabilization of the matrix and an adjustable release pattern for encapsulated protein.

Antimicrobial Peptides Pom-1 and Pom-2 from Pomacea poeyana Are Active against Candidaauris, C. parapsilosis and C. albicans Biofilms.

Recently two peptides isolated from the Cuban freshwater snail Pomacea poeyana (Pilsbry, 1927) were described to have antimicrobial activity against bacterial pathogens. Here we show considerable activities of Pom-1 and Pom-2 to reduce the viability of C. albicans, C. parapsilosis and the less common species C. auris measured as the decrease of metabolic activity in the resazurin reduction assay for planktonic cells. Although these activities were low, Pom-1 and Pom-2 turned out to be highly potent inhibitors of biofilm formation for the three Candida species tested. Whereas Pom-1 was slightly more active against C. albicans and C. parapsilosis as representatives of the more common Candida species Pom-2 showed no preference and was fully active also against biofilms of the more uncommon species C. auris. Pom-1 and Pom-2 may represent promising lead structures for the development of a classical peptide optimization strategy with the realistic aim to further increase antibiofilm properties and other pharmacologic parameters and to generate finally the first antifungal drug with a pronounced dedication against Candida biofilms.