Sensor chip, immobilization

The amount of antibody immobilized on the sensor chip decreased with increasing concentration of methyl viologen. To enlarge the difference in the sig-... 

Fig. 7. A proposed structure of the complex of the antibody with the trivalent antigen immobilized on the surface of the sensor chip...

Fig. 12a,b. The sensorgrams for the binding of the antibody dendrimer (a) or IgG (b) to the anionic porphyrin immobilized onto the surface of the sensor chip. Phosphate borate buffer (0.1 M, pH 9.0) was used. TCPP was immobilized via hexamethylenediamine spacer onto the sensor chip and then a solution of IgG or the dendrimer was injected to the flow cell. After 60 s from the injection of the antibody solutions, flow ceU was filled with buffer... 

Fig. 13a-e. The increase of the signal intensities by the addition of the dendritic complexes composed of IgGs and protein A. The hapten was immobilized to the surface of the SPR sensor chip. The increase of the signal intensities on the complex formation of hapten with the antibodies were monitored. The addition of mouse IgG specific for hapten (Abl) (a), the complex of the Abl with protein A (b), one to one complex of Abl with anti-mouse IgG (Fc) antibody (Ab2) (c), two to one complex of Abl with Ab2 (d), and two to one complex of Abl with Ab2 in the presence of protein A (e)... 

An enhancement of SPR signal intensity was observed by the addition of the antibody to the divalent antigen-antibody complex immobilized onto the surface of the sensor chip, indicating the formation of linear supramolecules. An amplification method of the detection signals for a target molecule has been... 

The instrument consists of a procesmg unit, reagents for Ugand immobilization, exchangeable sensor chips and a personal con uter for control and evaluation. 

Figure 7.9. Schematic diagram of a surface plasmon resonance biosensor. One of the binding partners is immobilized on the sensor surface. With the BIACORE instrument, the soluble molecule is allowed to flow over the immobilized molecule. Binding of the soluble molecule results in a change in the refractive index of the solvent near the surface of the sensor chip. The magnitude of the shift in refractive index is related quantitatively to the amount of the soluble molecule that is bound.

SPR systems also showed encouraging results with their ability to detect mycotoxins. The BIACORE was used to detect a mycotoxin, DON, produced by Fusarium species, from spiked wheat sample in a competitive inhibition assay (Schnerr et ah, 2002). Biotinylated DON was immobilized on the sensor chip which was previously coated with strep-tavidin. Mycotoxin extracts from wheat samples were first allowed to react with the antibody and then injected into the BIACORE. The detection range was established to be 0.13-10 pg/ml. In a slightly modified format, DON was also detected by SPR at a range of 2.5-30 ng/ml (Tudos et ah, 2003). 

Fig. 12 (a) Schematic representation of the preparation of a molecularly imprinted polymer with immobilized Au nanoparticle and the detection of an analyte upon selective swelling of the MIP. Reprinted with permission from [123]. Copyright (2004) American Chemical Society, (b) Schematic representation of Au-MIP/MIP-coated SPR sensor chip. Reprinted with permission from [124]. Copyright (2005) American Chemical Society... 

The avidin-biotin interaction has also been used to immobilize antibodies and proteins, especially in commercial systems based on surface plasmon resonance (SPR) measurements (e.g., the BIAcore). The extraordinary affinity (Kl 10-15 M) of avidin (or its bacterial relative, streptavidin) for the vitamin biotin is the basis of this immobilization procedure. A solid support (e.g., glass beads, sensor chip, optical fiber) covered with avidin can be used as an activated carrier for a very sturdy immobilization of previously biotinylated antibodies. In spite of the many methods for biotinylating proteins described in the literature, the use of biotinyl N-hydroxysuccinimide ester (BNHS) and similar derivatives, remains the most useful [65]. 

An example of the immobilization of antibodies on channel surfaces was presented by Eteshola and Leckband [395]. A microfluidic sensor chip was developed to quantify a model analyte (sheep IgM) with sensitivities down to 17 nM. This was achieved by first immobilizing a layer of bovine serum albumine (BSA) onto the channel wall, followed by specific adsorption of protein A to which the primary antibody for IgM was coupled covalently. This antibody could capture IgM, which was detected with the secondary antibody, labeled with horseradish peroxidase (Scheme 4.91). This enzyme catalyzes the conversion of the fluorogenic substrate 3-(p-hydroxyphenyl)propioni c acid into a fluorophore, which was quantified off-chip with a spectrofluorometer. The measured fluorescence signal was proportional to the analyte concentration in the test sample. 

Binding kinetics were determined using surface plasmon resonance. Human integrin a,/3i was immobilized on the sensor chip. Kd was calculated from koS/k0 . Clones 11, 7, 4, 24, and 2 represent five humanized LM609 versions. Fab were produced by E. coli except for mouse which was prepared from IgG by papain digestion. 

Acetylcholineesterase, urease, glucose oxidase and butyryl chloinesterase. Immobilized enzyme on to the sensor chip by corsslinking with glutaraldehyde and BSA. Conductivity changes, produced by the enzyme-catalyzed hydrolysis of ACh were measured for the analysis. Detection limits for ACh was 0.07 mM with corresponding sensitivity of 5.6 0.2 pS/mM. The device could be also used for apparent Michaelis constant determination. [85]... 

The first consideration when setting up an AlphaScreen assay is the choice of an assay format In most cases, the decision will depend on the interaction partners under investigation and on the biological tools available for their detection. The interaction partners can be coupled to the beads directly via reductive amination of reactive aldehyde groups, similar to the immobilization on a Biacore sensor chip (see above). The usefulness of this approach is limited by the reaction conditions, which may not be appropriate for maintaining the biologically active conformation of the biomolecule. Therefore the biomolecule of interest is usually not coupled to the beads directly, but instead captured via an antibody, also preventing steric hindrance. While not strictly necessary, it is often convenient to use a biotinylated molecule which can be captured by streptavidin-coated donor beads. 

Nakamura, C. Inuyama, Y. Shirai, K. Sugimoto, N. Miyake, J., Detection of porphyrin using a short peptide immobilized on a surface plasmon resonance sensor chip. Biosens. 

Fig. 5.8 Schematic setup of the surface plasmon resonance (SPR) detection unit in a Bia-core instrument. One interaction partner is immobilized on a modified gold surface, whereas the other flows by in solution. On the other side of the sensor chip, a beam of polarized light is reflected by the gold film. The optical phenomenon SPR leads to a re-...

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