Versatile bio-ink for covalent immobilization of chimeric avidin on sol-gel substrates

A bio-ink for covalent deposition of thermostable, high affinity biotin-binding chimeric avidin onto sol-gel substrates was developed. The bio-ink was prepared from heterobifunctional crosslinker 6-maleimidohexanoic acid N-hydroxysuccinimide which was first reacted either with 3-aminopropyltriethoxysilane or 3-aminopropyldimethylethoxysilane to form silane linkers 6-maleimide-N-(3-(triethoxysilyl)propyl)hexanamide or -(ethoxydimethylsilyl)propyl)-hexanamide. C-terminal cysteine genetically engineered to chimeric avidin was reacted with the maleimide group of silane linker in methanol/PBS solution to form a suspension, which was printed on sol-gel modified PMMA film. Different concentrations of chimeric avidin and ratios between silane linkers were tested to find the best properties for the bio-ink to enable gravure or inkjet printing. Bio-ink prepared from 3-aminopropyltriethoxysilane was found to provide the highest amount of active immobilized chimeric avidin. The developed bio-ink was shown to be valuable for automated fabrication of avidin-functionalized polymer films.     

Comparison of two algorithmic data processing strategies for metabolic fingerprinting by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry

The alignment algorithm Statistical Compare (SC) developed by LECO Corporation for the processing of comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) data was validated and compared to the in-house developed retention time correction and data alignment tool INCA (Integrative Normalization and Comparative Analysis) by a spike-in experiment and the comparative metabolic fingerprinting of a wild type versus a double mutant strain of Escherichia coli (E. coli). Starting with the same peak lists generated by LECO's ChromaTOF software, the accuracy of peak alignment and detection of 1.1- to 4-fold changes in metabolite concentration was assessed by spiking 20 standard compounds into an aqueous methanol extract of E. coli. To provide the same quality input signals for both alignment routines, the universal m/z 73 trace of the trimethylsilyl (TMS) group was used as a quantitative measure for all features. The performance of data processing and alignment was evaluated and illustrated by ROC curves. Statistical Compare performed marginally better at the lower fold changes, while INCA did so at the higher fold changes. Using SC, quantitative precision could be improved substantially by exploiting the signal intensities of metabolite-specific unique (U) m/z ion traces rather than the universal m/z 73 trace. A list of 56 features that distinguished the two E. coli strains was obtained by the SC alignment using m/z U with an estimated false discovery rate (FDR) of <0.05. Ultimately, 23 metabolites could be identified, one additional and five less than with INCA due to the failure of SC to extract unitized m/z U's across all fingerprints with suitable spectral intensities for the latter metabolites.     

Superhydrophobic elastomer surfaces with nanostructured micronails

New approaches to the fabrication of microstructures of special shape were developed for polymers. Unusual superhydrophobic surface structures were achieved with the use of flexible polymers and hierarchical molds.Flexible polyurethane?acrylate coatings were patterned with microstructures with use of microstructured aluminum mold in a controlled UV-curing process. Electron microscope images of the UV-cured coatings on polymethylmethacrylate (PMMA) substrates revealed micropillars that were significantly higher than the corresponding depressions of the mold (even 47 vs. 35 μm). The elongation was achieved by detaching the mold from the flexible, partially cured acrylate surface and then further curing the separated microstructure. The modified acrylate surface is superhydrophobic with a water contact angle of 156° and sliding angle of < 10°.Acrylic thermoplastic elastomers (TPE) were patterned with micro?nanostructured aluminum oxide molds through injection molding. The hierarchical surface of the elastomer showed elongated micropillars (57 μm) with nail-head tops covered with nanograss. Comparison with a reference microstructure of the same material (35 μm) indicated that the nanopores of the micro?nanomold assisted the formation of the nail-shaped micropillars. The elasticity of the TPE materials evidently plays a role in the elongation because similar elongation has not been found in hierarchically structured thermoplastic surfaces. The hierarchical micronail structure supports a high water contact angle (164°), representing an increase of 88° relative to the smooth TPE surface. The sliding angle was close to zero degrees, indicating the Cassie–Baxter state.     

Orthogonal Click Conjugation to the Liposomal Surface Reveals the Stability of the Lipid Anchorage as Crucial for Targeting

Synthetic access to multiple surface decorations are a bottleneck in the development of liposomes for receptor mediated targeting. This opens a complex multiparameter space, exploration of which is severely limited in terms of sample numbers and turnaround times. Here, we unlock this technological barrier by a combination of a milligram‐scale liposome formulation using dual centrifugation and orthogonal click chemistry on the liposomal surface. Application of these techniques to conceptually new amphiphilic compounds, which feature norbornene and alkyne groups at the apex of sterically stabilizing, hyperbranched polyglycerol moieties, revealed a particular influence of the membrane anchor of functional amphiphiles. Folic acid residues clicked to cholesterol‐based amphiphiles were inefficient in folate‐mediated cell targeting, while dialkyl‐anchored amphiphiles remained stable in the liposomal membrane and imparted efficient targeting properties. These findings are of specific importance considering the popularity of cholesterol as a lipophilic anchor.     

Local control of protein binding and cell adhesion by patterned organic thin films

Control of the cell adhesion and growth on chemically patterned surfaces is important in an increasing number of applications in biotechnology and medicine, for example implants, in-vitro cellular assays, and biochips. This review covers patterning techniques for organic thin films suitable for site-directed guidance of cell adhesion to surfaces. Available surface patterning techniques are critically evaluated, with special emphasis on surface chemistry that can be switched in time and space during cultivation of cells. Examples from the authors’ laboratory include the use of cell-repellent self-assembled monolayers (SAM) terminated by oligoethylene glycol (OEG) units and the lifting of the cell repellent properties by use of electrogenerated Br₂/HOBr which can be performed with positionable microelectrodes. Structural changes of the SAM were analyzed by polarization-modulated infrared reflection absorption spectroscopy (PM IRRAS). Use of a soft array system of individually addressable microelectrodes enables formation of flexible and complex patterns in a short time and has the potential for further acceleration of probe-induced local manipulation of cell adhesion.     

Rapid MALDI-MS Assays for Drug Quantification in Biological Matrices: Lessons Learned,New Developments,and Future Perspectives

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has rarely been used in the field of therapeutic drug monitoring, partly because of the complexity of the ionization processes between the compounds to be quantified and the many MALDI matrices available. The development of a viable MALDI-MS method that meets regulatory guidelines for bioanalytical method validation requires prior knowledge of the suitability of (i) the MALDI matrix with the analyte class and properties for ionization, (ii) the crystallization properties of the MALDI matrix with automation features, and (iii) the MS instrumentation used to achieve sensitive and specific measurements in order to determine low pharmacological drug concentrations in biological matrices. In the present hybrid article/white paper, we review the developments required for the establishment of MALDI-MS assays for the quantification of drugs in tissues and plasma, illustrated with concrete results for the different steps. We summarize the necessary parameters that need to be controlled for the successful development of fully validated MALDI-MS methods according to regulatory authorities, as well as currently unsolved problems and promising ways to address them. Finally, we propose an expert opinion on future perspectives and needs in order to establish MALDI-MS as a universal method for therapeutic drug monitoring.     

Modification of polycarbonate surface properties by nano-, micro-, and hierarchical micro–nanostructuring

Polycarbonate surfaces were patterned with nanopillars, microbumps, or nanopillars superimposed on microbumps. Patterning was achieved by applying nanoporous anodized aluminum oxide (AAO) membranes, microstructured aluminum foil, or anodic alumina on microstructured aluminum as mold inserts in injection molding. The effect of the different-sized structures on properties of the polycarbonate surface was investigated in contact angle measurements with water and oleic acid. The water contact angle increased from 82° on the smooth surface to 139° on the hierarchical micro–nanostructure. The transmittance of the polycarbonate increased with nanopatterning, while the reflection properties of the polycarbonate surface decreased. Reflection was lowest for the nanostructure with 53 nm pillar diameter and 77 nm interpillar distance. Values ranged from 0.6 to 1.1% over the whole wavelength range of visible light, which was 4–5% units lower than the corresponding values for the smooth polycarbonate.     

Wavelength dependence of the orientation of optic axes in KGW

We report on the wavelength dependence of the orientation of the optic axes in monoclinic crystals using undoped and neodymium-doped KGd(WO₄)₂ (KGW) samples. Measurements have been performed over a wide wavelength range from 420 up to 1,580 nm with more than 70 measurement points. This provides the widest measurement range of the optic axis variation ever reported in literature for KGW crystals. With such a measurement, which can be performed in principle in all biaxial crystals, the refractive indices values can be checked with a precision down to 10^?3.