Accurate calculation of affinity changes to the close state of influenza A M2 transmembrane domain in response to subtle structural changes of adamantyl amines using free energy perturbation methods in different lipid bilayers.

Experimental binding free energies of 27 adamantyl amines against the influenza M2(22-46) WT tetramer, in its closed form at pH 8, were measured by ITC in DPC micelles. The measured Kd's range is ~44 while the antiviral potencies (IC50) range is ~750 with a good correlation between binding free energies computed with Kd and IC50 values (r = 0.76). We explored with MD simulations (ff19sb, CHARMM36m) the binding profile of complexes with strong, moderate and weak binders embedded in DMPC, DPPC, POPC or a viral mimetic membrane and using different experimental starting structures of M2. To predict accurately differences in binding free energy in response to subtle changes in the structure of the ligands, we performed 18 alchemical perturbative single topology FEP/MD NPT simulations (OPLS2005) using the BAR estimator (Desmond software) and 20 dual topology calculations TI/MD NVT simulations (ff19sb) using the MBAR estimator (Amber software) for adamantyl amines in complex with M2(22-46) WT in DMPC, DPPC, POPC. We observed that both methods with all lipids show a very good correlation between the experimental and calculated relative binding free energies (r = 0.77-0.87, mue = 0.36-0.92 kcal mol-1) with the highest performance achieved with TI/MBAR and lowest performance with FEP/BAR in DMPC bilayers. When antiviral potencies are used instead of the Kd values for computing the experimental binding free energies we obtained also good performance with both FEP/BAR (r = 0.83, mue = 0.75 kcal mol-1) and TI/MBAR (r = 0.69, mue = 0.77 kcal mol-1).

(R)evolution of the Standard Addition Procedure for Immunoassays.

A new method to transfer the standard addition procedure for concentration determination to immunoassays with non-linear calibration curves was developed. The new method was successfully applied to simulated data and benchmarked against a state-of-the-art algorithm, showing a significantly improved performance with improvement factors between 2 and 192. The logit function was used to transform the immunoassay signal response of test samples spiked with known analyte concentrations. The relationship between logit(signal) and log-transformed estimated total analyte concentration is linear if the estimated total analyte concentration is correct. Finally, the new method was validated experimentally using different assays in varying, relevant complex matrices, such as serum, saliva, and milk. Different concentrations of testosterone and amitriptyline between 0.05 and 3.0 µg L-1 were quantified using a binding inhibition assay in combination with reflectometric interference spectroscopy (RIfS) as the transduction principle. The sample concentration was calculated using a numerical method. Samples could be quantified with recoveries between 70 and 118%. The standard addition method accounts for individual matrix interference on the immunoassay by spiking the test sample itself. Although the experiments were carried out using RIfS, the method can be applied to any immunoassay that meets the analytical requirements.

Tools to compare antibody gold nanoparticle conjugates for a small molecule immunoassay.

Antibody gold nanoparticle conjugates as recognition elements are essential for the overall performance of lateral flow assays. When immobilizing antibodies on gold nanoparticles, the challenge is to prevent aggregation and to ensure that the antibodies are correctly oriented so that they remain functional and their paratopes remain accessible. There are many methods available, and it is difficult to decide which one to use. To help selecting the most appropriate conjugate production method, different synthetic routes of binding antibodies to gold nanoparticles are systematically investigated for the purpose of a quantitative lateral flow test for small molecules. The direct comparison of different conjugate syntheses shows how to select a suitable conjugate for a lateral flow assay. The syntheses examined are direct adsorption of antibody, direct adsorption of reduced antibody, covalent binding to polyethylene glycol linker, and binding via biotin-streptavidin interaction. The conjugates are characterized using UV-Vis spectroscopy and dynamic light scattering to determine their stability. Their performance on structured lateral flow test strips is examined using calibrations for different amitriptyline concentrations. It was shown that the best conjugate for quantification of amitriptyline was realized by direct adsorption of an UV-light irradiated antibody to gold nanoparticles. The methods employed can serve as a guide for selecting the most appropriate conjugate for an application and enhance the performance of lateral flow assays.

Parallelized label-free monitoring of cell adhesion on extracellular matrix proteins measured by single colour reflectometry.

The understanding of the initial cell adhesion to biomaterials is crucial for the survival of implants. The manifold possibilities to tailor an implant surface and the diverse requirements for different implant applications necessitate a timesaving and highly parallelized analytical methodology. Due to its intrinsic advantages (label-free, time-resolved, robust against temperature fluctuations, and particularly the multiplexing possibilities), single colour reflectometry (SCORE) is used for the first time to investigate cell adhesion to different extracellular matrix protein-coated surfaces. The excellent correlation between the novel SCORE technology and well-established reference methods proves that the results obtained by using this direct optical method are able to reflect the cell binding processes at the transducer surface. Additionally, the high time resolution of SCORE revealed the differences in the adhesion behaviour of the cells on the different extracellular matrix protein-coated glass slides during the initial adsorption phase and during the spreading of the cells on the surfaces. Therefore, we conclude that SCORE is a perfectly suited methodology for studying the entire cell adsorption process, including morphological changes, and shows great potential for other cell-based sensing applications.

Comparison of methods for quantitative biomolecular interaction analysis.

In order to perform good kinetic experiments, not only the experimental conditions have to be optimized, but the evaluation procedure as well. The focus of this work is the in-depth comparison of different approaches and algorithms to determine kinetic rate constants for biomolecular interaction analysis (BIA). The different algorithms are applied not only to flawless simulated data, but also to real-world measurements. We compare five mathematical approaches for the evaluation of binding curves following pseudo-first-order kinetics with different noise levels. In addition, reflectometric interference spectroscopy (RIfS) measurements of two antibodies are evaluated to determine their binding kinetics. The advantages and disadvantages of the individual approach will be investigated and discussed in detail. In summary, we will raise awareness on how to evaluate and judge results from BIA by using different approaches rather than having to rely on "black box" closed (commercial) software packages.

An integrated device for fast and sensitive immunosuppressant detection.

The present paper describes a compact point of care (POC) optical device for therapeutic drug monitoring (TDM). The core of the device is a disposable plastic chip where an immunoassay for the determination of immunosuppressants takes place. The chip is designed in order to have ten parallel microchannels allowing the simultaneous detection of more than one analyte with replicate measurements. The device is equipped with a microfluidic system, which provides sample mixing with the necessary chemicals and pumping samples, reagents and buffers into the measurement chip, and with integrated thin film amorphous silicon photodiodes for the fluorescence detection. Submicrometric fluorescent magnetic particles are used as support in the immunoassay in order to improve the efficiency of the assay. In particular, the magnetic feature is used to concentrate the antibody onto the sensing layer leading to a much faster implementation of the assay, while the fluorescent feature is used to increase the optical signal leading to a larger optical dynamic change and consequently a better sensitivity and a lower limit of detection. The design and development of the whole integrated optical device are here illustrated. In addition, detection of mycophenolic acid and cyclosporine A in spiked solutions and in microdialysate samples from patient blood with the implemented device are reported.

Fibronectin adsorption on oxygen plasma-treated polyurethane surfaces modulates endothelial cell response.

Fibronectin coating increases implant biocompatibility by enhancing surface endothelialization via integrin-mediated binding. Surface properties determine the fibronectin orientation and conformation, dictating which ligands are presented, and therefore altering the bioactivity of an implant surface. In this study, polyurethane was treated with oxygen plasma, which allowed for a simultaneous modification of the surface chemistry and topography to modulate fibronectin adsorption. By varying the parameters of the treatment, human plasma fibronectin adsorbed on the surfaces in different conformations, orientations, and binding affinities, which was investigated by atomic force microscopy, fluorescence microscopy, monoclonal and polyclonal antibody staining and reflectometric interference spectroscopy. Apart from the most hydrophilic rough surfaces, the adsorbed fibronectin showed a lower binding affinity and less conformational change on the more hydrophilic surfaces. A large amount of exposed fibronectin-cell binding was detected on the rough treated and the smooth untreated surfaces. Primary isolated human umbilical vein and human microvascular endothelial cells showed a significantly higher cell adherence on the absorbed fibronectin with a low binding affinity and low conformational changes. Significant differences in the formation of mature focal adhesions and the reorganization of F-actin were identified on the rough treated and the smooth untreated surfaces. Our data suggest that oxygen plasma treatment is a reliable technique for the modulation of fibronectin adsorption in order to adjust fibronectin bioactivity and impact cell responses to implant surfaces.

Critical assessment of relevant methods in the field of biosensors with direct optical detection based on fibers and waveguides using plasmonic, resonance, and interference effects.

Direct optical detection has proven to be a highly interesting tool in biomolecular interaction analysis to be used in drug discovery, ligand/receptor interactions, environmental analysis, clinical diagnostics, screening of large data volumes in immunology, cancer therapy, or personalized medicine. In this review, the fundamental optical principles and applications are reviewed. Devices are based on concepts such as refractometry, evanescent field, waveguides modes, reflectometry, resonance and/or interference. They are realized in ring resonators; prism couplers; surface plasmon resonance; resonant mirror; Bragg grating; grating couplers; photonic crystals, Mach-Zehnder, Young, Hartman interferometers; backscattering; ellipsometry; or reflectance interferometry. The physical theories of various optical principles have already been reviewed in detail elsewhere and are therefore only cited. This review provides an overall survey on the application of these methods in direct optical biosensing. The "historical" development of the main principles is given to understand the various, and sometimes only slightly modified variations published as "new" methods or the use of a new acronym and commercialization by different companies. Improvement of optics is only one way to increase the quality of biosensors. Additional essential aspects are the surface modification of transducers, immobilization strategies, selection of recognition elements, the influence of non-specific interaction, selectivity, and sensitivity. Furthermore, papers use for reporting minimal amounts of detectable analyte terms such as value of mass, moles, grams, or mol/L which are difficult to compare. Both these essential aspects (i.e., biochemistry and the presentation of LOD values) can be discussed only in brief (but references are provided) in order to prevent the paper from becoming too long. The review will concentrate on a comparison of the optical methods, their application, and the resulting bioanalytical quality.

Blister-Actuated LIFT Printing for Multiparametric Functionalization of Paper-Like Biosensors.

Laser induced forward transfer (LIFT) is a flexible digital printing process for maskless, selective pattern transfer, which uses single laser pulses focused through a transparent carrier substrate onto a donor layer to eject a tiny volume of the donor material towards a receiver substrate. Here, we present an advanced method for the high-resolution micro printing of bio-active detection chemicals diluted in a viscous buffer solution by transferring droplets with precisely controllable volumes using blister-actuated LIFT (BA-LIFT). This variant of the LIFT process makes use of an intermediate polyimide layer partially ablated by the laser pulses. The expanding gaseous ablation products lead to blisters in the polyimide and ejection of droplets from the subjacent viscous solution layer. A relative movement of donor and receiver substrates for the transfer of partially overlapping pixels is realized with a custom-made positioning system. Using a specially developed donor ink containing bio-active components presented method allows to transfer droplets with well controllable volumes between 20 fL and 6 pL, which is far more precise than other methods like inkjet or contact printing. The usefulness of the process is demonstrated by locally functionalizing laser-structured nitrocellulose paper-like membranes to form a multiparametric lateral flow test. The recognition zones localized within parallel micro channels exhibit a well-defined and homogeneous color change free of coffee-ring patterns, which is of utmost importance for reliable optical readout in miniature multiparametric test systems.

Unraveling the Binding, Proton Blockage, and Inhibition of Influenza M2 WT and S31N by Rimantadine Variants.

Recently, the binding kinetics of a ligand-target interaction, such as the residence time of a small molecule on its protein target, are seen as increasingly important for drug efficacy. Here, we investigate these concepts to explain binding and proton blockage of rimantadine variants bearing progressively larger alkyl groups to influenza A virus M2 wild type (WT) and M2 S31N protein proton channel. We showed that resistance of M2 S31N to rimantadine analogues compared to M2 WT resulted from their higher koff rates compared to the kon rates according to electrophysiology (EP) measurements. This is due to the fact that, in M2 S31N, the loss of the V27 pocket for the adamantyl cage resulted in low residence time inside the M2 pore. Both rimantadine enantiomers have similar channel blockage and binding kon and koff against M2 WT. To compare the potency between the rimantadine variants against M2, we applied approaches using different mimicry of M2, i.e., isothermal titration calorimetry and molecular dynamics simulation, EP, and antiviral assays. It was also shown that a small change in an amino acid at site 28 of M2 WT, which does not line the pore, seriously affects M2 WT blockage kinetics.