A Versatile Protein and Cell Patterning Method Suitable for Long-Term Neural Cultures.

Herein, we present an easy-to-use protein and cell patterning method relying solely on pipetting, rinsing steps and illumination with a desktop lamp, which does not require any expensive laboratory equipment, custom-built hardware or delicate chemistry. This method is based on the adhesion promoter poly(allylamine)-grafted perfluorophenyl azide, which allows UV-induced cross-linking with proteins and the antifouling molecule poly(vinylpyrrolidone). Versatility is demonstrated by creating patterns with two different proteins and a polysaccharide directly on plastic well plates and on glass slides, and by subsequently seeding primary neurons and C2C12 myoblasts on the patterns to form islands and mini-networks. Patterning characterization is done via immunohistochemistry, Congo red staining, ellipsometry, and infrared spectroscopy. Using a pragmatic setup, patterning contrasts down to 5 µm and statistically significant long-term stability superior to the gold standard poly(l-lysine)-grafted poly(ethylene glycol) could be obtained. This simple method can be used in any laboratory or even in classrooms and its outstanding stability is especially interesting for long-term cell experiments, e.g., for bottom-up neuroscience, where well-defined microislands and microcircuits of primary neurons are studied over weeks.

First steps in combining modulation excitation spectroscopy with synchronous dispersive EXAFS/DRIFTS/mass spectrometry for in situ time resolved study of heterogeneous catalysts.

The synchronous combination of time resolved (energy dispersive) EXAFS, diffuse reflectance infrared spectroscopy (DRIFTS), and mass spectrometry (MS), applied within the general framework of concentration modulation spectroscopy, is demonstrated for in situ and time-resolved study of the behaviour of Rh/Al(2)O(3) and Pd/Al(2)O(3) catalysts during CO/NO redox cycling at 573 K. We show that by applying a phase sensitive detection technique the quality of information arising from the experiment is significantly improved. Moreover, in the case of the dispersive EXAFS the acuity of this technique is greatly enhanced and a surface sensitivity in the normally bulk sensitive EXAFS measurement can be induced. Lastly we apply this approach to a system (0.3 wt% Rh/Al(2)O(3)) that cannot normally be studied in any meaningful way with transmission based and highly time resolved EXAFS, and show that this method may provide a novel experimental window through which it is possible to restore highly time resolved structural-kinetic information from previously intractable systems.

An atomistic picture of the regeneration process in dye sensitized solar cells.

A highly efficient mechanism for the regeneration of the cis-bis(isothiocyanato)bis(2,2'-bipyridyl-4,4'-dicarboxylato)-ruthenium(II) sensitizing dye (N3) by I(-) in acetonitrile has been identified by using molecular dynamics simulation based on density functional theory. Barrier-free complex formation of the oxidized dye with both I(-) and , and facile dissociation of and from the reduced dye are key steps in this process. In situ vibrational spectroscopy confirms the reversible binding of I(2) to the thiocyanate group. Additionally, simulations of the electrolyte near the interface suggest that acetonitrile is able to cover the (101) surface of anatase with a passivating layer that inhibits direct contact of the redox mediator with the oxide, and that the solvent structure specifically enhances the concentration of I(-) at a distance which further favors rapid dye regeneration.

Enantioselective interactions at the solid-liquid interface of an HPLC column under working conditions.

A technique is presented which allows studying the enantioselective interactions occurring at the solid-liquid interface of a chiral stationary phase (CSP) and a racemate relevant to high performance liquid chromatography (HPLC). A conventional chiral column (Chiralpak AS) was mounted on an attenuated total reflection-infrared (ATR-IR) cell mimicking an HPLC setup equipped with an ATR-IR detector. Racemic pantolactone (PL) was used as the selectand. This setup in combination with modulation excitation spectroscopy (MES) allows for the identification of inter- and intramolecular hydrogen bonds being crucial for enantioseparation under HPLC operation conditions. The method is based on a two step strategy. In a first step, the enantiomers are separated by the chiral column similar to a standard HPLC experiment and upon adsorption on the identical CSP deposited on the internal reflection element (IRE), they are detected by ATR-IR spectroscopy. This experiment provides a retention time for each enantiomer. From the difference in retention, a suitable frequency is calculated which is used in a second experiment where the racemate concentration is varied alternately (modulation) in a way that the pulses of ( R)-PL and ( S)-PL exhibit a phase lag of 90 degrees after elution through the column. This procedure allows one to gain separate information of the enantioselective selectand-CSP interaction after performing a demodulation similar to a phase sensitive detection (PSD). A further benefit of this method is the strong enhancement of the signal-to-noise ratio. The effectiveness of the method is demonstrated by investigating the observed faster decrease in retention time of the later-eluted ( R)-PL, as compared to ( S)-PL, when separating at higher temperatures (from 12 to 36 degrees C). The origin is attributed to a weakening of a specific hydrogen bond between the C=O of ( R)-PL and the N-H of the CSP.

ATR-IR spectroscopy of pendant NH2 groups on silica involved in the Knoevenagel condensation.

The liquid-phase Knoevenagel condensation between benzaldehyde and ethyl cyanoacetate catalyzed by aminopropyl-modified silica has been investigated using in situ attenuated total reflection infrared (ATR-IR) spectroscopy. The aim of the work was to demonstrate the different levels of information on the reaction mechanism that can be achieved by operating the spectroscopic cell in the absence and in the presence of a solvent, in flow-through and stop-flow modes and in combination with concentration modulation spectroscopy. The reaction mechanism involves the formation of an imine intermediate whose existence has been verified in situ by combining in one experiment continuous and stop-flow operations. Identical information has been gained more elegantly using concentration modulation spectroscopy, which additionally provided information on the possible origin of the solvent effect observed in the Knoevenagel reaction. Faster production and consumption of the imine intermediate was observed in cyclohexane solvent than in toluene. Identification of other species evolving on the catalyst surface and monitoring of the effluents of the spectroscopic cell provided some insight in possible catalyst deactivation.

Absolute configuration modulation attenuated total reflection ir spectroscopy: an in situ method for probing chiral recognition in liquid chromatography.

A method to selectively probe the different adsorption of enantiomers at chiral solid-liquid interfaces is applied, which combines attenuated total reflection infrared spectroscopy and modulation spectroscopy. The spectral changes on the surface are followed while the absolute configuration of the adsorbate is changed periodically. Demodulated spectra are calculated by performing a subsequent digital phase-sensitive data analysis. The method is sensitive solely to the difference of the interaction of the two enantiomers with the chiral surface, and the small spectral changes are amplified by the phase-sensitive data analysis. Its potential is demonstrated by investigating an already well-studied system in liquid chromatography, namely, the enantiomer separation of N-3,5-dinitrobenzoyl-(R,S)-leucine (DNB-(R,S)-Leu) using tert-butylcarbamoyl quinine (tBuCQN) as the chiral selector immobilized on the surface of porous silica particles. The performed experiments and density functional theory calculations confirm an interaction model that was proposed earlier based on solution NMR and XRD in the solid state. It emerges that the ionic interaction is the strongest one, but the main reason for the potential for enantioseparation of the chiral stationary phase (CSP) is the distinct formation of a hydrogen bond of the (S)-enantiomer with the chiral selector. This H-bond is established between the amide N-H of DNB-(S)-Leu with the carbamate C=O of the CSP. The (R)-enantiomer instead shows no specific hydrogen bonds. Only the unspecific ionic bonding between the protonated quinine part of the tBuCQN and the carboxylate of the DNB-(R)-Leu (holds also for DNB-(S)-Leu) is observed.