Applications of array biosensor for detection of food allergens

Although food is a necessity, compounds within food products can be dangerous and life-threatening for people with food allergies. These allergy-causing compounds, such as proteins from eggs and milk, must be identified on the labels of commercial products. Unintentional contamination of food with these compounds occurs as a result of storage, manufacturing procedures, or cleaning procedures. A sensitive, specific, and rapid method to identify foods containing allergens is required by the food industry. The array biosensor, a rapid detection system, may provide a solution to this need. The array biosensor performs fluorescent immunoassays on the surface of a planar waveguide by first running samples, then fluorescently labeled antibodies, over a surface patterned with capture antibodies. An optical image is captured by a charged-coupled device camera and converted into fluorescence values. Signal intensity and spot location provide information on the compound and its concentration. The array biosensor has been successfully demonstrated for toxin, bacteria, and virus detection at low levels in under 20 min in food, clinical samples, and environmental matrixes. An assay for detection of ovalbumin as an indicator of egg contamination has been developed with limits of detection of 25 pg/mL in buffer and 1.3 ng/mL (13 ng/g) in non-egg pasta extract (buffer:pasta 10:1, v/w).     

Synthesis of cyclic oxoenaminoesters

Ammonia and primary amines react with dimethyl 3-oxo-1,ω-alkanedioates to give cyclic,N-acylated enaminoesters.     

Impact of prefractionation using Gradiflow on two-dimensional gel electrophoresis and protein identification by matrix assisted laser desorption/ionization-time of flight-mass spectrometry

Prefractionation of protein samples prior to two-dimensional electrophoresis (2-DE) has the potential to increase the dynamic detection range for proteomic analysis. We evaluated a membrane-based electrophoretic separation technique (Gradiflow) for its ability to fractionate an exoproteome sample from the filamentous fungus Trichoderma reesei. The sample was separated on the basis of size and charge. Buffer optimization was found to be necessary for successful size fractionation. Fractionation by charge was used to resolve the sample into four fractions that were subjected to analysis by two-dimensional electrophoresis (2-DE). Enhanced detection of low-abundance proteins with selective removal of high-abundance species was achieved. Fractionated and unfractionated samples were examined for differences in the ability to identify proteins following 2-DE using trypsin in-gel digestion followed by peptide mass fingerprinting using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Fractionated samples showed marked improvement in protein identification ability and sequence coverage. This study demonstrates the utility of the Gradiflow for fractionation, resulting in an enhancement of resolution and characterization of a moderately complex proteome.     

PHYTOCHROME EVOLUTION: A PHYLOGENETIC TREE WITH THE FIRST COMPLETE SEQUENCE OF PHYTOCHROME FROM A CRYPTOGAMIC PLANT: (Selaginella martensii SPRING)

We have sequenced cDNA and genomic clones coding for phytochrome of the fern Selaginella. On the amino acid level, this phytochrome shares sequence homologies with phytochromes of higher plants which range between 62 (phytochrome B of Arabidopsis) and 55 (56)% [phytochrome C of Arabidopsis (Avena)]. Introns in the Selaginella gene are short and occupy positions known from phytochrome sequences of higher plants. A rooted phylogenetic tree based on mutation distances puts Selaginella phytochrome closest to the hypothetical ancestor. A similar tree arises if the tree is constructed with partial sequences (about 200 amino acids) around the chromophore attachment site. An extension of this tree by sequences of other cryptogamic plants (Mougeotia, Ceratodon, Psilotum) shows all these sequences including those of the phytochromes B and C of Arabidopsis on a branch, well separated from the branch formed by phytochromes known to accumulate in etiolated plants. The rooted phytochrome phylogenetic tree, however, is difficult to reconcile with the fossil record.     

De novo synthesis of 4'-ethoxy nucleoside analogues

Butenolides 5a and 13 were used as optically active templates in the de novo synthesis of 4'-disubstituted nucleoside analogues. The butenolides were reduced and acylated in situ to give acetates 10 and 14. Vorbrüggen coupling gave the protected nucleoside analogues 11 and 15. Reduction of 11 gave 4'-ethoxy-2',3'-dideoxythymidine (6) and deprotection of 15 gave 4'-ethoxy-2',3'-dideoxydidehydrothymidine (7). The cis-dihydroxylation of a variety of butenolides occurred with the major product formed from oxidation of the beta-face.     

XRD-Studies of SiC/Si layers on carbon substrates

Structural investigations of thin films of SiC, SiC with free silicon and various titanium suicides (TiSi₂, TiSi and Ti₅Si₃) are described. The crystal phases have been identified using X-ray diffractometry. The growth of reaction products from surface reactions between silicon and deposited titanium can be observed.Dedicated to Professor Dr. rer.nat. Dr. h.c. Hubertus Nickel on the occasion of his 65th birthday     

Tuning single nucleotide discrimination in polymerase chain reactions (PCRs): synthesis of primer probes bearing polar 4'-C-modifications and their application in allele-specific PCR

There are many methods available for the detection of nucleotide variations in genetic material. Most of these methods are applied after amplification of the target genome sequence by the polymerase chain reaction (PCR). Many efforts are currently underway to develop techniques that can detect single nucleotide variations in genes either by means of, or without the need for, PCR. Allele-specific PCR (asPCR), which reports nucleotide variations based on either the presence or absence of a PCR-amplified DNA product, has the potential to combine target amplification and analysis in one single step. The principle of asPCR is based on the formation of matched or mismatched primer-target complexes by using allele-specific primer probes. PCR amplification by a DNA polymerase from matched 3'-primer termini proceeds, whereas a mismatch should obviate amplification. Given the recent advancements in real-time PCR, this technique should, in principle, allow single nucleotide variations to be detected online. However, this method is hampered by low selectivity, which necessitates tedious and costly manipulations. Recently, we reported that the selectivity of asPCR can be significantly increased through the employment of chemically modified primer probes. Here we report further significant advances in this area. We describe the synthesis of various primer probes that bear polar 4'-C-modified nucleotide residues at their 3' termini, and their evaluation in real-time asPCR. We found that primer probes bearing a 4'-C-methoxymethylene modification have superior properties in the discrimination of single nucleotide variations by PCR.     

Enantioselective, chirally templated sol-gel thin films

Enantioselective surfactant-templated thin films were fabricated through the sol-gel (SG) process. The enantioselectivity is general in the sense that it discriminates between pairs of enantiomers not used for the imprinting process. The chiral cationic surfactant (-)-N-dodecyl-N-methylephedrinium bromide (1) was used as the surfactant template, and after its extraction chiral domains were created. The chiral discriminative feature of these films was examined by challenging with pure enantiomer solutions for rebinding. Selective adsorption was shown using (R)- and (S)-propranolol, (R)-2 and (S)-2, respectively, and (R)- and (S)-2,2,2-trifluoro-1-(9-anthryl)ethanol, (R)-3 and (S)-3, respectively, as the chiral probes. The selective adsorption was measured by fluorescence analysis, and the chiral selectivity factors were found to be 1.6 for 2 and 2.25 for 3. In both cases, (R)-enantiomer was adsorbed preferably. The resulting material was characterized by transmission electron microscopy, by diffraction, and by surface area measurements, and was found to be semicrystalline with short-range ordered domains (50 A) of hexagonal symmetry.     

The modulation of face-face π-π interactions in Lewis acid catalysis

The enantiomeric excess observed for the exo-adduct from the Lewis acid catalysed Diels-Alder reaction between cyclopentadiene and methacrolein can be increased up to 21% by simple modification of the electronics of the aromatic ring in a series of stilbene-derived diol ligands, suggesting that the proposed face-face π-π interaction between the catalyst and the dienophile can be modulated by altering the electron density on the aromatic ring.     

Effect of ionic strength on the self-assembly, morphology and gelation of pH responsive β-sheet tape-forming peptides

Molecular self-assembly is an intrinsic property of proteins central to their biological functionality. One important industrially interesting property is the ability to control and switch on and off self-assembly using a variety of external chemical and physical triggers. Model peptides have been developed with significantly reduced chemical and structural complexity compared to biological proteins. These are ideal systems for exposing the fundamental principles that drive protein-like self-assembly, as well as for establishing in a quantitative manner their structure-function relationship. We investigate simple, short model peptides that adopt a purely β-strand conformation, align in an antiparallel manner and self-assemble in one dimension in solution into long β-sheet nanotapes and higher order aggregates with no other conformation (i.e., helices, turns or random coils) present in the aggregates. These micrometre-long nanostructures gel in solutions at concentrations as low as 0.2% v/v. Their gel-fluid transition has been previously shown to be controlled by pH, temperature, or by mixing with complementary peptides. Here we show the dramatic effect of another chemical trigger, that of physiological-like salt concentration, on the self-assembly, morphology and gelation of a series of systematically designed charged self-assembling tape-forming peptides, each 11 amino acid residues in length, in the pH range of 2-14. This study provides a detailed understanding of the self-assembly of this class of peptides in aqueous solutions of biologically relevant pH and ionic strength. This insight has led to the development of injectable self-assembling peptide lubricants as potential therapeutics for the treatment of early stage knee joint osteoarthritis.