Chip surface charge switch for studying histone-DNA interaction by surface plasmon resonance biosensor.
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Adsorption↗
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Poly(ethylene glycol)-based biosensor chip to study heparin-protein interactions.
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Animals↗
Using surface plasmon resonance to directly measure slow binding of low-molecular mass inhibitors to a VanX chip.
VanX, a d,d-dipeptidase, is one of five gene products responsible for vancomycin resistance in pathogenic bacteria and is an attractive drug target in circumventing clinical drug resistance. Our previous combinatorial search of VanX substrates in a dipeptide library of d-X(1)-d-X(2) (19(2)=361) forms has led to the discovery of three new compounds (d-Ala-d-Phe, d-Ala-d-Tyr, and d-Ala-d-Trp) having higher k(cat)/K(M) values than those of its natural substrate, d-Ala-d-Ala. Based on structures of newly identified substrates, two representative transition state analogs of substrates, d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) dipeptide phosphonates, used as VanX inhibitor were rationally designed and chemically synthesized. In the synthesis, eight synthetic steps in total were employed for preparing each VanX inhibitor, and their overall isolated yields were 21 and 11% for 6a and 6b, respectively. Binding interactions of d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) with VanX were confirmed unambiguously and measured quantitatively by surface plasmon resonance. The result reveals that both dipeptide phosphonates are slow-binding inhibitors of VanX (for 6a, k(on)=1.18 x 10(3)M(-1)s(-1), k(off)=2.31 x 10(-3) s(-1), K(D)=1.96 microM, chi(2)=0.0737; for 6b, k(on)=1.09 x 10(3)M(-1)s(-1), k(off)=1.80 x 10(-2)s(-1), K(D)=16.5 microM, chi(2)=0.0599). This suggests that only a fraction of the conformers of the inhibitors in solution adopts a conformation best suited for binding interaction with VanX and that the VanX-inhibitor complex may concomitantly undergo a conformational isomerization from an initial but fast weak-binding adduct to slowly convert to a tight-binding complex with a more stable bound geometry. Moreover, in comparison with 6b, an additional aromatic interaction of 6a with the Phe79 residue in the active site of the enzyme, through an energetically favorable face-to-face offset stacked orientation, may account for its higher affinity than 6b to VanX.
Bacterial Proteins↗
High-resolution electrophysiology on a chip: Transient dynamics of alamethicin channel formation.
Microstructured planar substrates have been shown to be suitable for patch clamp recording from both whole cells and isolated patches of membrane, as well as for measurements from planar lipid bilayers. Here, we further explore this technology with respect to high-resolution, low noise single-channel recording. Using solvent-free lipid bilayers from giant unilamellar vesicles obtained by electro-swelling, we recorded channels formed by the peptaibol alamethicin, a well-studied model system for voltage-dependent channels, focusing on the transient dynamics of single-channel formation upon application of a voltage step. With our setup, we were able to distinctly resolve dwell times well below 100 mus and to perform a thorough statistical analysis of alamethicin gating. Our results show good agreement with models that do not rely on the existence of non-conducting preaggregate states. Microstructured apertures in glass substrates appear promising with respect to future experiments on cellular ion channels reconstituted in suspended lipid membranes.
Alamethicin↗
Detection of DNA hybridization by ABEI electrochemiluminescence in DNA-chip compatible assembly.
The electrochemiluminescence (ECL) of a luminol derivate (ABEI) generated both by a carbon electrode and a polypyrrole-coated carbon electrode was examined. It was found that the polypyrrole film (ppy) did not inhibit the ECL. After that, ABEI anchored on a single stranded DNA target (ODNt) has been used for the ECL detection of the hybridization between a complementary single stranded DNA probe (ODNp) covalently linked to a polypyrrole support and the ODNt. The ECL detection has been performed using a DNA sensor having a low surface concentration of ODNp probes, constituted of a polypyrrole copolymer electrosynthesized from a pyrrole-ODNp/pyrrole monomer ratio of 1/20,000.
Electrochemistry↗
Ground wheat straw as a substitute for portions of oak wood chips used in shiitake (Lentinula edodes) substrate formulae.
Oak woodchips, used for production of shiitake Lentinula edodes (Berk) Pegler, are increasingly difficult to obtain due to dwindling supplies. We investigated the effect of adding ground wheat straw as a substitute for portions of oak woodchips in substrate formulae on mushroom yield and size. We also determined the effect of mushroom cropping on relative feed value (RFV) by chemical analysis of the substrate at spawning (AS) and after cropping (AC). Three formulae containing 0%, 8% and 16% ground wheat straw and 52%, 44% and 36% oak sawdust, respectively, were bulk pasteurized (111 degrees C for 20 min) in an autoclaving mixer, subjected to spawn run (21 d), browning (28 d) and a production cycle of three breaks (38 d). Mean (4 crops) mushroom yields were 11% higher when 8% wheat straw was used in the medium and 19% higher when 16% wheat straw was substituted for portions of oak sawdust. There were no significant differences in mushroom sizes between any of the treatments. Relative feed values of shiitake substrates AC increased more dramatically as more wheat straw was added to the formulae. Using mature alfalfa (full bloom) as a base value of 100%, RFVs for substrate AS were 98%, 92%, and 92% for 0%, 8% and 16% straw, respectively; RFVs AC were 118%, 120% and 133%, respectively. Substrate AC containing 16% straw had a RFV comparable to corn silage (well-eared). Fat contents of the substrates decreased by 50-62% AC, whereas potassium contents decreased by 40%. Use of ground wheat straw in synthetic medium would not only increase mushroom yield by up to 19%, but may help alleviate periodic shortages of oak sawdust. In addition, growers would avoid the added expense of aging the wheat straw (for 8-12 week) as is typically done for oak sawdust in the industry. This is the first report of RFVs for spent shiitake substrate (SSS) predicting its excellent potential for use as animal feed.
Agriculture↗
On-chip biosensing of estrogen receptor-alpha at single molecular level.
A novel method for detecting interaction between DNA and DNA-binding protein at single molecular level has been proposed. In this study, estrogen receptor-alpha (ER-alpha) was used for biosensing as the proof-example. A 518 bp-long (ca. 176 nm) DNA probe labeled with streptavidin at its 5'-terminus was prepared by inserting a consensus oligonucleotide sequence that binds to ER-alpha. A solution containing ER-alpha was dropped onto the Ni-treated mica substrate on which the DNA prove was previously immobilized, and it was observed by AFM. Specific binding of ER-alpha could be observed by measuring the distance between the site where binding occur, to the streptavidin label.
Binding Sites↗
Assessment of porous silicon substrate for well-characterised sensitive DNA chip implement.
A biochip approach based on porous silicon as substrate is presented. The goal is to enhance the sensitivity of the biochip by increasing the specific surface area on the support. The elaboration of porous silicon layers has been optimized to guarantee good accessibility for large bio-molecule targets. Oligonucleotide probes are synthesised directly on the surface using phosphoramidite chemistry. The high specific surface area of porous silicon allows the direct characterisation, by infrared spectroscopy, of the porous layer formation and the functionalisation steps. The monolayer grafting and derivatisation protocol is additionally characterized by wettability and fluorescence microscopy. The surface modification of porous layers (i.e. thermal oxidation and chemical derivatisation) ensures the stability of the structure against strong chemical reagents used during the direct oligonucleotide synthesis. Finally the protocol is successfully transferred to a flat Si/SiO(2) substrate, and validated by biological target specific recognition during hybridisation tests. In particular, radioactive measurements show a 10-fold enhancement of the oligonucleotide surface density on the porous silicon substrate compared to the flat thermal silica.
Crystallization↗
Miniaturized one-chip electrochemical sensing device integrated with a dialysis membrane and double thin-layer flow channels for measuring blood samples.
We have developed a microfluidic device consisting of a gold film working electrode modified with lactate oxidase and Os-poly(vinylpyridine) mediator containing horseradish peroxide, and reference and counter electrodes in a microflow detection channel separated by a microdialysis membrane from another microflow channel used for sample injection. The dialysis membrane is cellulose with a molecular weight cut off of 10 kDa. We achieved control over a wide recovery rate range of 3-94% because the device is capable of controlling both flow rates in the dual thin-layer channels. We were able to measure the lactate concentration in blood samples within a few minutes without any pretreatment because biomolecules are simultaneously separated by molecular weight and detected in the device. We achieved quantitative and reproducible measurements of the lactate concentration in blood samples, and obtained a relative standard deviation of 1.5% (n = 8). With our device, the lactate concentration in dog whole blood was measured with high stability without any pretreatment.
Animals↗
Electrochemiluminescent hybridization chip with electric field aided mismatch discrimination.
This paper describes a heterogeneous DNA-hybridization assay based on electrochemiluminescence (ECL) detection on gold electrodes. Short, 15-mer oligonucleotides were conjugated with a synthesized electrochemiluminescent label, bis(2,2'-bipyridine)-5-isothiocyanato-1,10-phenanthroline ruthenium(II) at the amino-modified 5'-end. Gold electrodes were derivatized with 15-mer oligonucleotide probes via 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross-linking reaction and hybridized with Ru-labeled strands. Two types of self-assembled-monolayers have been utilized for the immobilization reaction, 3-mercaptopropanoic acid (3-MHA) and 16-mercaptohexadecanoic acid (16-MHA). Longer thiols were more stable at high electrode potentials needed for the ECL generation. The system was sensitive down to 1 fmol of labeled complementary strand, detected in 30 microL of buffer. Mismatch discrimination was achieved both passively by washing and actively by application of negative electrode potential on electrodes prior to detection, but active denaturing lead to better results. Two base-pair mismatches were discriminated at room temperature.
Base Pair Mismatch↗
Direct electrochemical addressing of immunoglobulins: immuno-chip on screen-printed microarray.
An original immobilisation technology is presented for the development of chemiluminescent protein biochips, suitable for measurement in complex matrices. The immobilisation strategy involved is based on diazotated aniline derivatives, which could be electro-addressed, thus creating a covalent linkage with a conducting material surface. The present electrochemical system is a cost effective and mass-produced carbon paste screen-printed (SP) microarray composed of eight 0.2 mm2 working electrodes, one carbon pseudo-reference electrode and one auxiliary electrode. Rabbit immunoglobulins (IgG) were chemically modified with an aniline derivative (4-carboxymethylaniline) in order to be easily electro-grafted to the SP microarray surface. The possibility of successively electro-address the eight sensing layers of a particular array, with a good reproducibility (more than 80%) and without loss of reactivity was demonstrated. Moreover, these immobilised proteins were subsequently used as a capture agent for the determination of rheumatoid factor (RF) in human sera. The absence of non-specific signal or interference problem enabled the detection of RF values in complex samples in the 5.3-485 IU/ml range with a good correlation with the standard Auraflex ELISA test method.
Animals↗
A biomolecule friendly photolithographic process for fabrication of protein microarrays on polymeric films coated on silicon chips.
The last years, there is a steadily growing demand for methods and materials appropriate to create patterns of biomolecules for bioanalytical applications. Here, a photolithographic method for patterning biomolecules onto a silicon surface coated with a polymeric layer of high protein binding capacity is presented. The patterning process does not affect the polymeric film and the activity of the immobilized onto the surface biomolecules. Therefore, it permits sequential immobilization of different biomolecules on spatially distinct areas on the same solid support. The polymeric layer is based on a commercially available photoresist (AZ5214) that is cured at high temperature in order to provide a stable substrate for creation of protein microarrays by the developed photolithographic process. The photolithographic material consists of a (meth)acrylate copolymer and a sulfonium salt as a photoacid generator, and it is lithographically processed by thermal treatment at temperatures <or=50 degrees C, development with dilute aqueous basic developer solutions and exposure at wavelengths above 300 nm. Following this photolithographic procedure onto the polymeric layer coated silicon surface, protein spots with diameters ranging from 2 to 50 microm were created. The proposed methodology provided good intra-spot homogeneity (CV <or=5%) and inter-spot repeatability (CV <or=5%), as it was determined through epifluorescence microscopy after reaction of the immobilized proteins with their respective fluorescently labeled binding counterparts. Moreover, the polymeric film selected for immobilization of biomolecules presented high protein binding capacity, which was at least three folds higher than that obtained using aminosilanized surfaces. The proposed methodology is expected to facilitate considerably the fabrication of dense protein microarrays for bioanalytical applications.
Animals↗
Highly sensitive gold nanoparticles biosensor chips modified with a self-assembled bilayer for detection of Con A.
In this paper, an improved method for detection of Concanavalin A (Con A) with label-free optical biosensors is reported. 1-Dodecanethiol (DDT) was self-assembled onto gold nanoparticles which were deposited on glass slides, and then glycolipid molecules were inserted into dodecanethiol by physical interactions only. The recognition between Con A and carbohydrate was observed by UV-vis spectrophotometry. The absorption spectrum shifted when Con A was bound to the sugar residues of glycolipids immobilized onto nanogold slides, while almost no spectrum change was observed when another nonspecific protein molecule met the nanogold slides. The self-assembled bilayer on nanogold substrates had very high sensitivity for Con A, the minimum detection concentration of Con A can be down to 0.1 nM. In addition to the ultra sensitivity for investigating carbohydrate-lectin interaction, the self-assembled bilayer structure, is expected to replace many receptors which require time-consuming organic syntheses for the fixation to the transducer. The simplicity and sensitivity of this biosensor architecture once again show the prospect of nanogold application in biosensor.
Biosensing Techniques↗
Interferometric biosensor based on planar optical waveguide sensor chips for label-free detection of surface bound bioreactions.
A label free optical biosensor based on a free-space Young interferometer configuration is presented. Commercial planar Ta(2) O(5) waveguides are used as sensing elements and allow the investigation of surface bound bioreactions like immunoreactions or biological affinity systems. Design criteria are discussed and a detailed characterization of the sensor performance is presented. The developed interferometer yields an effective refractive index resolution of 9 x 10(-9), corresponding to a surface coverage of approximately 13 fg/mm(2). The performance of the system is characterized by two different affinity systems: the antibody-antigen complex protein G-immunoglobulin G is used as a model system for monitoring reaction kinetics. Further measurements on a silanized surface show the formation of a streptavidin monolayer on a biotinylated surface.
Biosensing Techniques↗
A phase-based stereo vision system-on-a-chip.
A simple and fast technique for depth estimation based on phase measurement has been adopted for the implementation of a real-time stereo system with sub-pixel resolution on an FPGA device. The technique avoids the attendant problem of phase warping. The designed system takes full advantage of the inherent processing parallelism and segmentation capabilities of FPGA devices to achieve a computation speed of 65megapixels/s, which can be arranged with a customized frame-grabber module to process 211frames/s at a size of 640x480 pixels. The processing speed achieved is higher than conventional camera frame rates, thus allowing the system to extract multiple estimations and be used as a platform to evaluate integration schemes of a population of neurons without increasing hardware resource demands.
Biomedical Engineering↗
Protein chip array profiling analysis of sera from neuroblastoma patients.
Neuroblastoma, the most common extracranial solid tumour in children, is characterised by highly heterogeneous clinical behaviour; patients are stratified into risk categories according to a combination of clinical and biological markers. However, identifying non-invasive prognostic markers predicting outcome independently from current risk-stratification features remains critical for better disease monitoring. Using the SELDI-TOF-MS technology (surface-enhanced laser desorption/ionization time-of-flight mass spectrometry), we found a serum biomarker that strongly correlates with prognosis in neuroblastoma patients. Subsequent peptide mapping identified this biomarker as SAA protein. In support of this observation, high SAA levels were detected by ELISA in the sera of patients with poor prognosis neuroblastoma. Based on this finding, promises and limitations of the approach are discussed.
Enzyme-Linked Immunosorbent Assay↗
An SPR immunosensor for human cardiac troponin T using specific binding avidin to biotin at carboxymethyldextran-modified gold chip.
BACKGROUND: The cardiac troponin T (TnT) is a cardiospecific, highly sensitive marker for myocardial damage and is immediately released to bloodstream during the acute myocardial infarction (AMI). Surface plasmon resonance (SPR) immunosensor was developed for the quick detection of human cTnT in real time. METHODS: An SPR sensor streptavidin terminated self-assembled monolayer, which was used to binding biotinylated anti troponin T monoclonal antibodies, was developed. RESULTS: The cTnT was determined from successive injections with a linear range from 0.03 up to 6.5 ng/ml. The detection limit was 0.01 ng/ml corresponding to a resonant angle change of 1.28 millidegrees. The system presented a good repeatability with 3.4% of variation between run after regeneration of the coated surface with a solution of 1% SDS. The effects of blocking of non-specific adsorption using different solutions were studied. It was possible to measure the cTnT without dilution of the human serum with good specificity and reproducibility. CONCLUSION: This sensor is practical and offer quick response in interval of 800 s.
Antibodies, Monoclonal↗