Characterization of different substrates for Raman spectroscopic imaging of eukaryotic cells

For Raman spectroscopic analyses of the cells and other biological samples, the choice of the right substrate material is very important to avoid loss of information in characteristic spectral features because of competing background signals. In the current study, Raman spectroscopy is used to characterize several potential Raman substrates. Raman vibrational bands of the substrate material are discussed. The surface topography is analyzed by atomic force microscopy, and the root mean square surface roughness values are reported. Biocompatibility of the substrates is tested with Hep G2 cells evaluating cellular morphology as well as live/dead staining. Calcium fluoride, silicon, fused silica, borofloat glass, and silicon nitride membranes support cell growth and adherence. Silicon, borofloat glass, and fused silica give rise to Raman signals in the region of interest. Calcium fluoride substrate (UV grade) is suitable for Raman spectroscopic investigation of living cells. Nickel foil is suitable substrate for Raman spectroscopic investigation but cellular adherence and viability depend on the quality of the foil. Silicon nitride membranes coated with nickel chrome is a suitable Raman substrate in closed microfluidic systems. Copyright © 2016 John Wiley & Sons, Ltd.     

Nano-high-performance liquid chromatography in combination with nano-electrospray ionization Fourier transform ion-cyclotron resonance mass spectrometry for proteome analysis

Fourier transform ion-cyclotron resonance (FTICR) mass spectrometry offers several advantages for the analysis of biological samples, including excellent mass resolution, ultra-high mass measurement accuracy, high sensitivity, and wide mass range. We report the application of a nano-HPLC system coupled to an FTICR mass spectrometer equipped with nanoelectrospray source (nano-HPLC/nano-ESI-FTICRMS) for proteome analysis. Protein identification in proteomics is usually conducted by accurately determining peptide masses resulting from enzymatic protein digests and comparing them with theoretically digested protein sequences from databases. A tryptic in-solution digest of bovine serum albumin was used to optimize experimental conditions and data processing. Spots from Coomassie Blue and silver-stained two-dimensional (2D) gels of human thyroid tissue were excised, in-gel digested with trypsin, and subsequently analyzed by nano-HPLC/nano-ESI-FTICRMS. Additionally, we analyzed 1D-gel bands of membrane preparations of COS-6 cells from African green monkey kidney as an example of more complex protein mixtures. Nano-HPLC was performed using 1-mm reverse-phase C-18 columns for pre-concentration of the samples and reverse-phase C-18 capillary columns for separation, applying water/acetonitrile gradient elution conditions at flow rates of 200 nL/min. Mass measurement accuracies smaller than 3 ppm were routinely obtained. Different methods for processing the raw data were compared in order to identify a maximum number of peptides with the highest possible degree of automation. Parallel identification of proteins from complex mixtures down to low-femtomole levels makes nano-HPLC/nano-ESI-FTICRMS an attractive approach for proteome analysis.     

A family of macrocyclic antibiotics with a mixed peptide-peptoid beta-hairpin backbone conformation

Macrocyclic peptidomimetics having a mixed peptide-peptoid backbone have been synthesized and shown to possess antibiotic activity against gram-positive and -negative bacteria with a low hemolytic activity against human erythrocytes; one is shown to adopt a regular beta-hairpin conformation by NMR in aqueous solution.     

High Loading Efficiency and Controlled Release of Bioactive Immunotherapeutic Proteins Using Vaterite Nanoparticles

Nanoparticles may limit off-tumor/on-target ubiquitous activation of signaling by protein-based drugs. However, many challenges still exist in the design of a nanoparticle for protein delivery. In this study, conditions to establish vaterite nanoparticles as a pH-sensitive drug delivery system (DDS) for encapsulated protein drugs are comprehensively evaluated. Low coprecipitation pH of vaterite and protein prevents protein denaturation and yields high loading efficiency. Unprotected vaterite recrystallizes in aqueous solutions within 3 h to calcite and releases the loaded protein completely, but surface-modified particles with carboxyl groups containing polymers prove stable for more than 5 months. Notably, modification of vaterite with sulfonated polymers increases the loading of cationic proteins by a multiple. A system is developed for vaterite exposure to (pH) conditions under body-like-flow rates, with the dissolution of vaterite and simultaneous release of active proteins at tumor microenvironmental pH reaching up to 80% and only 20% at physiological pH within 2 h. Importantly, the immunomodulatory protein tumor necrosis factor preserves its native structure and fully retains functional activity in vitro after release from the particles. In conclusion, the studies described here provide a framework for the development of vaterite-based DDS as a carrier for bioactive protein-based therapeutics.     

Aluminacyclopropene: syntheses, characterization, and reactivity toward terminal alkynes

Reactions of LAl with ethyne, mono- and disubstituted alkynes, and diyne to aluminacyclopropene LAl[eta2-C2(R1)(R2)] ((L = HC[(CMe)(NAr)]2, Ar = 2,6-iPr2C6H3); R1 = R2 = H, (1); R1 = H, R2 = Ph, (2); R1 = R2 = Me, (3); R1 = SiMe3, R2 = C[triple bond]CSiMe3, (4)) are reported. Compounds 1 and 2 were obtained in equimolar quantities of the starting materials at low temperature. The amount of C2H2 was controlled by removing an excess of C2H2 in the range from -78 to -50 degrees C. Compound 4 can be alternatively prepared by the substitution reaction of LAl[eta2-C2(SiMe3)2] with Me3SiC[triple bond]CC[triple bond]CSiMe3 or by the reductive coupling reaction of LAlI2 with potassium in the presence of Me3SiC[triple bond]CC[triple bond]CSiMe3. The reaction of LAl with excess C2H2 and PhC[triple bond]CH (<1:2) afforded the respective alkenylalkynylaluminum compounds LAl(CH=CH2)(C[triple bond]CH) (5) and LAl(CH=CHPh)(C[triple bond]CPh) (6). The reaction of LAl(eta2-C2Ph2) with C2H2 and PhC[triple bond]CH yielded LAl(CPh=CHPh)(C[triple bond]CH) (7) and LAl(CPh=CHPh)(C[triple bond]CPh) (8), respectively. Rationally, the formation of 5 (or 6) may proceed through the corresponding precursor 1 (or 2). The theoretical studies based on DFT calculations show that an interaction between the Al(I) center and the C[triple bond]C unit needs almost no activation energy. Within the AlC2 ring the computational Al-C bond order of ca. 1 suggests an Al-C sigma bond and therefore less pi electron delocalization over the AlC2 ring. The computed Al-eta2-C2 bond dissociation energies (155-82.6 kJ/mol) indicate a remarkable reactivity of aluminacyclopropene species. Finally, the 1H NMR spectroscopy monitored reaction of LAl(eta2-C2Ph2) and PhC[triple bond]CH in toluene-d8 may reveal an acetylenic hydrogen migration process.     

Microscale sample deposition onto hydrophobic target plates for trace level detection of neuropeptides in brain tissue by MALDI-MS

A sample preparation method that combines a modified target plate with a nanoscale reversed-phase column (nanocolumn) was developed for detection of neuropeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A gold-coated MALDI plate was modified with an octadecanethiol (ODT) self-assembled monolayer to create a hydrophobic surface that could concentrate peptide samples into a approximately 200-500-microm diameter spot. The spot sizes generated were comparable to those obtained for a substrate patterned with 200-microm hydrophilic spots on a hydrophobic substrate. The sample spots on the ODT-coated plate were 100-fold smaller than those formed on an unmodified gold plate with a 1-microl sample and generated 10 to 50 times higher mass sensitivity for peptide standards by MALDI-TOF MS. When the sample was deposited on an ODT-modified plate from a nanocolumn, the detection limit for peptides was as low as 20 pM for 5-microl samples corresponding to 80 amol deposited. This technique was used to analyze extracts of microwave-fixed tissue from rat brain striatum. Ninety-eight putative peptides were detected including several that had masses matching neuropeptides expected in this brain region such as substance P, rimorphin, and neurotensin. Twenty-three peptides had masses that matched peaks detected by capillary liquid chromatography with electrospray ionization MS.     

Optical tweezers applied to a microfluidic system

We will demonstrate how optical tweezers can be combined with a microfluidic system to create a versatile microlaboratory. Cells are moved between reservoirs filled with different media by means of optical tweezers. We show that the cells, on a timescale of a few seconds, can be moved from one reservoir to another without the media being dragged along with them. The system is demonstrated with an experiment where we expose E. coli bacteria to different fluorescent markers. We will also discuss how the system can be used as an advanced cell sorter. It can favorably be used to sort out a small fraction of cells from a large population, in particular when advanced microscopic techniques are required to distinguish various cells. Patterns of channels and reservoirs were generated in a computer and transferred to a mask using either a sophisticated electron beam technique or a standard laser printer. Lithographic methods were applied to create microchannels in rubber silicon (PDMS). Media were transported in the channels using electroosmotic flow. The optical system consisted of a combined confocal and epi-fluorescence microscope, dual optical tweezers and a laser scalpel.