IMPATIENT-qPCR: monitoring SELEX success during in vitro aptamer evolution.

SELEX (Systematic Evolution of Ligands by Exponential enrichment) processes aim on the evolution of high-affinity aptamers as binding entities in diagnostics and biosensing. Aptamers can represent game-changers as constituents of diagnostic assays for the management of instantly occurring infectious diseases or other health threats. Without in-process quality control measures SELEX suffers from low overall success rates. We present a quantitative PCR method for fast and easy quantification of aptamers bound to their targets. Simultaneous determination of melting temperatures (Tm) of each SELEX round delivers information on the evolutionary success via the correlation of increasing GC content and Tm alone with a round-wise increase of aptamer affinity to the respective target. Based on nine successful and published previous SELEX processes, in which the evolution/selection of aptamer affinity/specificity was demonstrated, we here show the functionality of the IMPATIENT-qPCR for polyclonal aptamer libraries and resulting individual aptamers. Based on the ease of this new evolution quality control, we hope to introduce it as a valuable tool to accelerate SELEX processes in general. IMPATIENT-qPCR SELEX success monitoring. Selection and evolution of high-affinity aptamers using SELEX technology with direct aptamer evolution monitoring using melting curve shifting analyses to higher Tm by quantitative PCR with fluorescence dye SYBR Green I. KEY POINTS: • Fast and easy analysis. • Universal applicability shown for a series of real successful projects.

Aptamers as Novel Binding Molecules on an Antimicrobial Peptide-Armored Composite Hydrogel Wound Dressing for Specific Removal and Efficient Eradication of Pseudomonas aeruginosa.

Here we present for the first time a potential wound dressing material implementing aptamers as binding entities to remove pathogenic cells from newly contaminated surfaces of wound matrix-mimicking collagen gels. The model pathogen in this study was the Gram-negative opportunistic bacterium Pseudomonas aeruginosa, which represents a considerable health threat in hospital environments as a cause of severe infections of burn or post-surgery wounds. A two-layered hydrogel composite material was constructed based on an established eight-membered focused anti-P. aeruginosa polyclonal aptamer library, which was chemically crosslinked to the material surface to form a trapping zone for efficient binding of the pathogen. A drug-loaded zone of the composite released the C14R antimicrobial peptide to deliver it directly to the bound pathogenic cells. We demonstrate that this material combining aptamer-mediated affinity and peptide-dependent pathogen eradication can quantitatively remove bacterial cells from the "wound" surface, and we show that the surface-trapped bacteria are completely killed. The drug delivery function of the composite thus represents an extra safeguarding property and thus probably one of the most important additional advances of a next-generation or smart wound dressing ensuring the complete removal and/or eradication of the pathogen of a freshly infected wound.

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.

Increasing Antibiotic Susceptibility: The Use of Cationic Gold Nanoparticles in Gram-Negative Bacterial Membrane Models.

Antibiotic resistance will be one of the most prominent challenges to health-care systems in the coming decades, with the OECD predicting that up to 2.4 million deaths will be caused between 2015 and 2050 by drug-resistant bacterial infections in first-world countries alone, with infections costing health-care systems billions of dollars each year. Developing new methods to increase bacterial susceptibility toward drugs is an important step in treating resistant infections. Here, the synergistic effects of gold nanoparticles and the antibiotic drug colistin sulfate have been examined. A tethered lipid bilayer membrane was used to mimic a Gram-negative bacterial cell membrane. Exposing the membrane to gold nanoparticles prior to adding the antibiotic significantly increased the effect of the antibiotic on the membrane. Cationic gold nanoparticles could thus be used to enhance bacterial susceptibility to antibiotics, leading to a more potent treatment.

Solid-supported lipid bilayers - A versatile tool for the structural and functional characterization of membrane proteins.

The cellular membrane is central to the development of single-and multicellular life, as it separates the delicate cellular interior from the hostile environment. It exerts tight control over entry and exit of substances, is responsible for signaling with other cells in multicellular organisms and prevents pathogens from entering the cell. In the case of bacteria and viruses, the cellular membrane also hosts the proteins enabling invasion of the host organism. In a very real sense therefore, the cellular membrane is central to all life. The study of the cell membrane and membrane proteins in particular has therefore attracted significant attention. Due to the enormous variety of tasks performed by the membrane, it is a highly complex and challenging structure to study. Ideally, membrane components would be studied in isolation from this environment, but unlike water soluble proteins, the amphiphilic environment provided by the cellular membrane is key to the structure and function of the cell membrane. Therefore, model membranes have been developed to provide an environment in which a membrane protein can be studied. This review presents a set of tools that enable the comprehensive characterization of membrane proteins: electrochemical tools, surface plasmon resonance, neutron scattering, the surface forces apparatus and atomic force microscopy are discussed, with a particular focus on experimental technique and data evaluation.

Interaction Profiles and Stability of Rigid and Polymer-Tethered Lipid Bilayer Models at Highly Charged and Highly Adhesive Contacts.

Understanding interaction force versus distance profiles of supported lipid bilayers (SLBs) is relevant to a number of areas, which rely on these model systems, including, e.g., characterization of ligand/receptor interactions or bacterial adhesion. Here, the stability of 4 different SLB architectures was compared using the surface forces apparatus (SFA) and atomic force microscopy (AFM). Specifically, the outer envelope of the bilayer systems remained constant as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The inner layer was varied between DPPC and 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP) both on mica, and self-assembled monolayers (SAMs) of hexadecanethiol and the polymer-tethered diphytanylglycerol-tetraethylene glycol-lipoid acid (DPhyTL) on smooth gold surfaces. In that same order these gave an increasing strength of interaction between the inner layer and the supporting substrate and hence improved stability under highly adhesive conditions. Detachment profiles from highly charged and highly adhesive contacts were characterized, and approach characteristics were fitted to DLVO models. We find increasing stability under highly adhesive loads, approaching the hydrophobic limit of the adhesive energy between the inner and outer layers for the SAM-based systems. For all four SLBs we further compare AFM surface topographies, which strongly depend on preparation conditions, and the DLVO fitting of the SFA approach curves finds a strong charge regulation behavior during interaction, dependent on the particular model system. In addition, we find undulation characteristics during approach and separation. The increased stability of the complex architectures on a gold support makes these model systems an ideal starting point for studying more complex strongly adhesive/interacting systems, including, for example, ligand/receptor interactions, biosensing interactions, or cell/surface interactions.

Investigating the Structure of Self-Assembled Monolayers Related to Biological Cell Membranes.

Tethered bilayer lipid membranes are solid supported lipid membranes, where the inner leaflet is covalently linked to the solid supported substrate through anchorlipids. These anchorlipids form a self-assembled monolayer, which serves as the basis of the membrane and also provides submembrane space. The molecular structure and composition of this monolayer has thus significant influence on the membrane structural and functional properties. The density of the self-assembled monolayer can be tailored by adding small molecules to the monolayer. Here, the structure of fully tethered and sparsely tethered monolayers, where the anchorlipid has been diluted with a small surface-active thiol, has been analyzed using neutral impact collision ion scattering spectroscopy, X-ray photoelectron spectroscopy, and metastable induced electron spectroscopy. Combination of these three techniques allowed description of the self-assembly process in detail. The monolayers have been characterized in terms of layer thickness and orientation of the lipids.

A tethered bilayer lipid membrane that mimics microbial membranes.

A model membrane system has been developed, which mimics the outer membrane of Gram negative bacteria. The structure is based on a tethered monolayer which has been fused with vesicles containing lipopolysaccharide molecules. The effect of the composition of the monolayer and the lipids in the outer layer on the structural and electrical properties of the membrane has been investigated. By using electrochemical impedance spectroscopy as well as neutron scattering techniques, it could be shown that a relatively high tethering density and a small amount of diluting lipids in the outer membrane leaflet leads to the formation of a stable solid supported membrane. The influence of divalent ions on the membrane stability has been probed as well as the interaction of the bilayer with the antibiotic colistin. A number of different architectures were developed, suited to both the study of bacterial membrane proteins and the screening of antimicrobial activity of potential drug candidates.

Synthesis and Characterization of Novel Anchorlipids for Tethered Bilayer Lipid Membranes.

Tethered bilayer lipid membranes are versatile solid-supported model membrane systems. Core to these systems is an anchorlipid that covalently links a lipid bilayer to a support. The molecular structure of these lipids can have a significant impact on the properties of the resulting bilayer. Here, the synthesis of anchorlipids containing ester groups in the tethering part is described. The lipids are used to form bilayer membranes, and the resulting structures are compared with membranes formed using conventional anchorlipids or sparsely tethered membranes. All membranes showed good electrical sealing properties; the disulphide-terminated anchorlipids could be used in a sparsely tethered system without significantly reducing the sealing properties of the lipid bilayers. The sparsely tethered systems also allowed for higher ion transport across the membrane, which is in good correlation with higher hydration of the spacer region as seen by neutron scattering.

A Blautia producta specific gFET-based aptasensor for quantitative monitoring of microbiome quality.

The use of health-relevant bacteria originating from human microbiomes for the control or therapy of diseases, including neurodegenerative disorders or diabetes, is currently gaining increasing importance in medicine. Directed and successful engineering of microbiomes via probiotic supplementation requires subtle, precise as well as, more importantly, easy, fast and convenient monitoring of its success, e.g., in patients' gut. Based on a previously described polyclonal SELEX aptamer library evolved against the human gut bacterium Blautia producta, we finally isolated three individual aptamers that proved their performance concerning affinity, specificity and robustness in reliably labeling the target bacterium and in combination with "contaminating" control bacteria. Using biofunctionalization molecules on gFETs, we could specifically quantify 101-106 cells per mL, retrace their number in mixtures and determine aptamer Kd-values around 2 nM. These measurements were possible even in the context of a real human stool sample. Our results qualify gFETs in combination with BL2, BL7 and BL8 aptamers as a promising foundation for the construction of respective sensing devices, which will open new avenues towards developing an intended monitoring technique for probiotic therapy and microbiome engineering approaches.

Bridging basic science and applied diagnostics: Comprehensive viral diagnostics enabled by graphene-based electronic biosensor technology advancements.

This study presents a graphene field-effect transistor (gFET) biosensor with dual detection capabilities for SARS-CoV-2: one RNA detection assay to confirm viral positivity and the other for nucleocapsid (N-)protein detection as a proxy for infectiousness of the patient. This technology can be rapidly adapted to emerging infectious diseases, making an essential tool to contain future pandemics. To detect viral RNA, the highly conserved E-gene of the virus was targeted, allowing for the determination of SARS-CoV-2 presence or absence using nasopharyngeal swab samples. For N-protein detection, specific antibodies were used. Tested on 213 clinical nasopharyngeal samples, the gFET biosensor showed good correlation with RT-PCR cycle threshold values, proving its high sensitivity in detecting SARS-CoV-2 RNA. Specificity was confirmed using 21 pre-pandemic samples positive for other respiratory viruses. The gFET biosensor had a limit of detection (LOD) for N-protein of 0.9 pM, establishing a foundation for the development of a sensitive tool for monitoring active viral infection. Results of gFET based N-protein detection corresponded to the results of virus culture in all 16 available clinical samples and thus it also proved its capability to serve as a proxy for infectivity. Overall, these findings support the potential of the gFET biosensor as a point-of-care device for rapid diagnosis of SARS-CoV-2 infection and indirect assessment of infectiousness in patients, providing additional information for clinical and public health decision-making.

Q-lipid-containing membranes show high in-plane conductivity using a membrane-on-a-chip setup.

The light-driven reactions of photosynthesis as well as the mitochondrial power supply are located in specialized membranes containing a high fraction of redox-active lipids. In-plane charge transfer along such cell membranes is currently thought to be facilitated by the diffusion of redox lipids and proteins. Using a membrane on-a-chip setup, we show here that redox-active model membranes can sustain surprisingly high currents (mA) in-plane at distances of 25 µm. We also show the same phenomenon in free-standing monolayers at the air-water interface once the film is compressed such that the distance between redox centers is below 1 nm. Our data suggest that charge transfer within cell walls hosting electron transfer chains could be enabled by the coupling of redox-lipids via simultaneous electron and proton in-plane hopping, similar to conductive polymers. This has major implications for our understanding of the role of lipid membranes, suggesting that Q-lipid-containing membranes may be essential for evolving the complex redox machineries of life.

Native Function of the Bacterial Ion Channel SthK in a Sparsely Tethered Lipid Bilayer Membrane Architecture.

The plasma membrane protects the interiors of cells from their surroundings and also plays a critical role in communication, sensing, and nutrient import. As a result, the cell membrane and its constituents are among the most important drug targets. Studying the cell membrane and the processes it facilitates is therefore crucial, but it is a highly complex environment that is difficult to access experimentally. Various model membrane systems have been developed to provide an environment in which membrane proteins can be studied in isolation. Among them, tethered bilayer lipid membranes (tBLMs) are a promising model system providing a solvent-free membrane environment which can be prepared by self-assembly, is resistant to mechanical disturbances and has a high electrical resistance. tBLMs are therefore uniquely suitable to study ion channels and charge transport processes. However, ion channels are often large, complex, multimeric structures and their function requires a particular lipid environment. In this paper, we show that SthK, a bacterial cyclic nucleotide gated (CNG) ion channel that is strongly dependent on the surrounding lipid composition, functions normally when embedded into a sparsely tethered lipid bilayer. As SthK has been very well characterized in terms of structure and function, it is well-suited to demonstrate the utility of tethered membrane systems. A model membrane system suitable for studying CNG ion channels would be useful, as this type of ion channel performs a wide range of physiological functions in bacteria, plants, and mammals and is therefore of fundamental scientific interest as well as being highly relevant to medicine.

Activity of Single Insect Olfactory Receptors Triggered by Airborne Compounds Recorded in Self-Assembled Tethered Lipid Bilayer Nanoarchitectures.

Membrane proteins are among the most difficult to study as they are embedded in the cellular membrane, a complex and fragile environment with limited experimental accessibility. To study membrane proteins outside of these environments, model systems are required that replicate the fundamental properties of the cellular membrane without its complexity. We show here a self-assembled lipid bilayer nanoarchitecture on a solid support that is stable for several days at room temperature and allows the measurement of insect olfactory receptors at the single-channel level. Using an odorant binding protein, we capture airborne ligands and transfer them to an olfactory receptor from Drosophila melanogaster (OR22a) complex embedded in the lipid membrane, reproducing the complete olfaction process in which a ligand is captured from air and transported across an aqueous reservoir by an odorant binding protein and finally triggers a ligand-gated ion channel embedded in a lipid bilayer, providing direct evidence for ligand capture and olfactory receptor triggering facilitated by odorant binding proteins. This model system presents a significantly more user-friendly and robust platform to exploit the extraordinary sensitivity of insect olfaction for biosensing. At the same time, the platform offers a new opportunity for label-free studies of the olfactory signaling pathways of insects, which still have many unanswered questions.

Functional incorporation of the insect odorant receptor coreceptor in tethered lipid bilayer nanoarchitectures.

Membrane proteins are among the most important drug targets. To improve drug design, it is critical to study membrane proteins. However, due to the myriad roles fulfilled by the cellular membrane, it is a highly complex environment and challenging to study. Tethered membranes reproduce the basic physicochemical properties of the cellular membrane without its inherent complexity. The high electrical resistance and stability makes them ideal to study membrane proteins, particularly ion channels. However, due to the close proximity of the membrane to the support and the reduced fluidity and high packing density, they are unsuitable to study larger membrane proteins. We present here a tethered membrane system which adresses these challenges, allowing the functional reconstitution of the odorant receptor coreceptor from Drosophila melanogaster, a tetrameric ionotropic receptor was incorporated and its sensitivity to various ligands was examined via electrochemical impedance spectroscopy and atomic force microscopy.

A Polyclonal SELEX Aptamer Library Allows Differentiation of Candida albicans, C. auris and C. parapsilosis Cells from Human Dermal Fibroblasts.

Easy and reliable identification of pathogenic species such as yeasts, emerging as problematic microbes originating from the genus Candida, is a task in the management and treatment of infections, especially in hospitals and other healthcare environments. Aptamers are seizing an already indispensable role in different sensing applications as binding entities with almost arbitrarily tunable specificities and optimizable affinities. Here, we describe a polyclonal SELEX library that not only can specifically recognize and fluorescently label Candida cells, but is also capable to differentiate C. albicans, C. auris and C. parapsilosis cells in flow-cytometry, fluorometric microtiter plate assays and fluorescence microscopy from human cells, exemplified here by human dermal fibroblasts. This offers the opportunity to develop diagnostic tools based on this library. Moreover, these specific and robust affinity molecules could also serve in the future as potent binding entities on biomaterials and as constituents of technical devices and will thus open avenues for the development of cost-effective and easily accessible next generations of electronic biosensors in clinical diagnostics and novel materials for the specific removal of pathogenic cells from human bio-samples.

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.

Combination of Six Individual Derivatives of the Pom-1 Antibiofilm Peptide Doubles Their Efficacy against Invasive and Multi-Resistant Clinical Isolates of the Pathogenic Yeast Candida albicans

In previous studies, derivatives of the peptide Pom-1, which was originally extracted from the freshwater mollusk Pomacea poeyana, showed an exceptional ability to specifically inhibit biofilm formation of the laboratory strain ATCC 90028 as a model strain of the pathogenic yeast Candida albicans. In follow-up, here, we demonstrate that the derivatives Pom-1A to Pom-1F are also active against biofilms of invasive clinical C. albicans isolates, including strains resistant against fluconazole and/or amphotericin B. However, efficacy varied strongly between the isolates, as indicated by large deviations in the experiments. This lack of robustness could be efficiently bypassed by using mixtures of all peptides. These mixed peptide preparations were active against biofilm formation of all the isolates with uniform efficacies, and the total peptide concentration could be halved compared to the original MIC of the individual peptides (2.5 µg/mL). Moreover, mixing the individual peptides restored the antifungal effect of fluconazole against fluconazole-resistant isolates even at 50% of the standard therapeutic concentration. Without having elucidated the reason for these synergistic effects of the peptides yet, both the gain of efficacy and the considerable increase in efficiency by combining the peptides indicate that Pom-1 and its derivatives in suitable formulations may play an important role as new antibiofilm antimycotics in the fight against invasive clinical infections with (multi-) resistant C. albicans.

Association between uterine artery Doppler blood flow changes and arterial wall elasticity in pregnant women.

INTRODUCTION: Uterine artery (UtA) Doppler velocimetry changes and increased arterial stiffness are associated with preeclampsia. We aimed to investigate the relation between UtA velocimetry changes and arterial stiffness in pregnant women. METHODS: Doppler velocimetry and photoplethysmographic digital pulse wave analysis (DPA) were performed in 173 pregnant women in the second or the third trimester, where UtA Doppler pulsatility index (PI), diastolic notching, and UtA score (UAS) combining notching and high PI were calculated. DPA stiffness parameters representing large arteries were ejection elasticity index (EEI) and b/a, small arteries dicrotic index (DI) and d/a, and global stiffness the aging index (AI). RESULTS: One hundred and thirty women had normal Doppler and 43 had diastolic notching, of whom nine had high PI. DI indicated increased stiffness in small arteries when notching was present (p = 0.044) and showed a significant but weak correlation to UAS (p = 0.025, tau 0.12). EEI and b/a indicated increased large artery stiffness (p ≤0.014), d/a small artery stiffness (p = 0.023), and AI a systemic stiffness (p = 0.040) when high PI. CONCLUSION: High UtA PI was associated with increased systemic arterial stiffness, whereas notching was related to increased stiffness in small arteries only. This indicates pathophysiological differences between the two Doppler parameters.

Tethered and Polymer Supported Bilayer Lipid Membranes: Structure and Function.

Solid supported bilayer lipid membranes are model systems to mimic natural cell membranes in order to understand structural and functional properties of such systems. The use of a model system allows for the use of a wide variety of analytical tools including atomic force microscopy, impedance spectroscopy, neutron reflectometry, and surface plasmon resonance spectroscopy. Among the large number of different types of model membranes polymer-supported and tethered lipid bilayers have been shown to be versatile and useful systems. Both systems consist of a lipid bilayer, which is de-coupled from an underlying support by a spacer cushion. Both systems will be reviewed, with an emphasis on the effect that the spacer moiety has on the bilayer properties.