Robust Fluorometric Aptamer Assay for Direct and Rapid Detection of Clinical Isolates of Candida spec.

Infections caused by yeasts of the genus Candida are likely to occur not only in immunocompromised patients but also in healthy individuals, leading to infections of the gastrointestinal tract, urinary tract, and respiratory tract. Due to the rapid increase in the frequency of reported Candidiasis cases in recent years, diagnostic research has become the subject of many studies, and therefore, we developed a polyclonal aptamer library-based fluorometric assay with high specificity and affinity towards Candida spec. to quantify the pathogens in clinical samples with high sensitivity. We recently obtained the specific aptamer library R10, which explicitly recognized Candida and evolved it by mimicking an early skin infection model caused by Candida using the FluCell-SELEX system. In the follow-up study presented here, we demonstrate that the aptamer library R10-based bioassay specifically recognizes invasive clinical Candida isolates, including not only C. albicans but also strains like C. tropcialis, C. krusei, or C. glabrata. The next-generation fluorometric bioassay presented here can reliably and easily detect an early Candida infection and could be used for further clinical research or could even be developed into a full in vitro diagnostic tool.

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.

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 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.

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.

Detection of cholera toxin with surface plasmon field-enhanced fluorescent spectroscopy.

In this work, a biosensor based on surface plasmon field-enhanced florescence spectroscopy (SPFS) method was successfully constructed to detect the truncated form of cholera toxin, that is, its beta subunit (CTX-B). CTX-B is a relatively small molecule (12 kDa) and it was chosen as model analyte for the detection of protein toxins originated from waterborne pathogens. Recognition layer was prepared on gold-coated LaSFN9 glasses modified with 11-mercaptoundecanoic acid (11-MUA). Biotin-conjugated anti-CTX-B polyclonal antibody (B-Ab) was immobilized on streptavidin (SA) layer constructed on the 11-MUA-modified surface. CTX-B amount was determined with direct assay using B-Ab in surface plasmon resonance (SPR) mode and with sandwich assay in SPFS mode using Cy5-conjugated anti-CTX-B polyclonal antibody. Minimum detected CTX-B concentrations were 10 and 0.01 µg/ml with SPR and SPFS, respectively, showing the sensitivity of the SPFS system over the conventional one. The detection was done in 2-6 h, which was faster than both culture and polymerase chain reaction (PCR)-based methods. Stability tests were performed with SA-coated sensors (excluding B-Ab). In this form, the layer was stable after 30 days of storage in phosphate-buffered saline (PBS; 0.01 M, pH = 7.4) at +4°C. B-Ab layer was formed immediately on them before each measurement.

Development of a specific troponin I detection system with enhanced immune sensitivity using a single monoclonal antibody.

Using an immunoassay in combination with surface plasmon fluorescence spectroscopy (SPFS), we report the rapid detection of troponin I, a valuable biomarker for diagnosis of myocardial infarction. We discuss the implementation of (i) direct, (ii) sandwich, and (iii) competitive assay formats, based on surface plasmon resonance and SPFS. To elucidate the results, we relate the experiments to orientation-dependent interaction of troponin I epitopes with respective immunoglobulin G antibodies. A limit of detection (LoD) of 19 pM, with 45 min readout time, was achieved using single monoclonal antibody that is specific for one epitope. The borderline between normal people and patients is 20 pM to 83 pM cTnI concentration, and upon the outbreak of acute myocardial infraction it can raise to 2 nM and levels at 20 nM for 6-8 days, therefore the achieved LoD covers most of the clinically relevant range. In addition, this system allows for the detection of troponin I using a single specific monoclonal antibody, which is highly beneficial in case of detection in real samples, where the protein has a complex form leading to hidden epitopes, thus paving the way towards a system that can improve early-stage screening of heart attacks.

Plasmonic biosensors relying on biomolecular conformational changes: Case of odorant binding proteins.

Plasmonic nanostructures serve in a range of analytical techniques that were developed for the analysis of chemical and biological species. Among others, they have been pursued for the investigation of odorant binding proteins (OBP) and their interaction with odorant molecules. These compounds are low molecular weight agents, which makes their direct detection with conventional surface plasmon resonance (SPR) challenging. Therefore, other plasmonic sensor modalities need to be implemented for the detection and interaction analysis of OBPs. This chapter provides a guide for carrying out such experiments based on two techniques that take advantage of conformation changes of OBPs occurring upon specific interaction with their affinity partners. First, there is discussed SPR monitoring of conformation changes of biomolecules that are not accompanied by a strong increase in the surface mass density but rather with its re-distribution perpendicular to the surface. Second, the implementation of surface plasmon-enhanced fluorescence energy transfer is presented for the sensitive monitoring of conformational changes of biomolecules tagged with a fluorophore at its defined part. Examples from our and other laboratories illustrate the performance of these concepts and their applicability for the detection of low molecular weight odorant molecules by the use of OBPs attached to the sensor surface is discussed.

UV-Laser Interference Lithography for Local Functionalization of Plasmonic Nanostructures with Responsive Hydrogel.

A novel approach to local functionalization of plasmonic hotspots at gold nanoparticles with biofunctional moieties is reported. It relies on photocrosslinking and attachment of a responsive hydrogel binding matrix by the use of a UV interference field. A thermoresponsive poly(N-isopropylacrylamide)-based (pNIPAAm) hydrogel with photocrosslinkable benzophenone groups and carboxylic groups for its postmodification was employed. UV-laser interference lithography with a phase mask configuration allowed for the generation of a high-contrast interference field that was used for the recording of periodic arrays of pNIPAAm-based hydrogel features with the size as small as 170 nm. These hydrogel arrays were overlaid and attached on the top of periodic arrays of gold nanoparticles, exhibiting a diameter of 130 nm and employed as a three-dimensional binding matrix in a plasmonic biosensor. Such a hybrid material was postmodified with ligand biomolecules and utilized for plasmon-enhanced fluorescence readout of an immunoassay. Additional enhancement of the fluorescence sensor signal by the collapse of the responsive hydrogel binding matrix that compacts the target analyte at the plasmonic hotspot is demonstrated.