Biacore sensor chip

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. 

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). 

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]. 

The surface matrix of a carboxymethylated sensor chip CMS (Pharmacia Biosensor, Uppsala, Sweden) was activated by injection of 35 pL of 0.05 M N-hydroxy-succinimide (NHS)/0.2 M N-ethyl-N (dimethylaminopropyl)carbodiimide (EDC). The BIAcore IFC forms four parallel flow cells on the sensor chip. Three of them were derivatized with 40 mM cystamin-dihydrochloride/ethanolamine in the ratios... 

Studies of the interaction of IL-6 and the sIL-6R were monitored by SPR detection using a BIAcore instrument [1] (Pharmacia, Uppsala). Immobilisation of the respective proteins to the carboxymethylated dextran matrix coating the gold sensor chip was performed using the EDC/NHS coupling chemistry as previously described for IL-6 and sIL-6R [3, 7, 8]. Regeneration of the sensor surface for IL-6 and sIL-6R was performed with lOmM HCl for 3 min [8] and 4M MgCl2 in lOmM Tris-HCl buffer, pH 7.4 for 1 min respectively. 

The first commercial SPR was launched hy Pharmacia Biosensor AB (presently Swedish BIAcore AB) in 1990. Since then, the device has been refined and now BIAcore [37] offers several models (BIACORE 3000, BIACORE 2000, BIACORE 1000, BIACORE X, J, Q, S51, and C models). The biosensors of BIACORE 1000 to 3000 are fully automated instruments, with a disposable sensor chip, an optical detection unit, an integrated micro-fiuidic cartridge, an autosampler, method programming and control software. Less expensive manually controlled alternatives cU e the BIACORE x and BiacoreQuant . 

Another technique used for the analysis of receptor-ligand interaction is surface plasmon resonance (SPR), with its first commercially available application in the BIAcore instruments [44] (Fig. 5.8). Like PCS, it allows the determination of kinetics by monitoring the association and dissociation of a receptor-ligand complex in real time. The interaction partners do not necessarily have to be labeled, which is an advantage of the technique. The principle of SPR measurements is based on an optical phenomenon. The core unit in this technique is a sensor chip consisting of a thin gold film with a modified surface attached on one side. One reactant is attached to the modified sensor surface, whereas the other reaction partner flows past this surface in solution. When the two interaction partners form a com-... 

Biosensor instruments such as Biacore (General Electric) exploit the sensitivity of a surface plasmon resonance response to the mass localised near the surface of a sensor chip. Various approaches can be used for kinases. Inhibition in solution assays involve immobilisation of a target definition compound (TDQ on the sensor surface.13,30 A buffer containing the kinase is flowed over the surface so that the protein is able to bind to the immobilised TDC, giving a signal. When test compounds are included in the buffer, they can compete for the TDC-kinase interaction, allowing estimation of Kd. 

The first SPR immunosensor for detection of pesticides was developed by Mimmni et al. [22] in the early 1990s. They used an SPR sensor developed by Biacore AB, Sweden, with the atrazine derivative bound to dextran matrix on the sensor chip. The detection of atrazine was performed using the inhibition assay and monoclonal antibodies. The sensor response was subsequently amplified by secondary antibody, which was bound to the antibody captured by the atrazine derivative (sandwich assay, see Chap. 7 in this volume [54]). This biosensor was demonstrated to measure atrazine in distilled and tap water within the range 0.05-1 ng mL in 15 min and exhibited relatively low crossreactivity with simazine and tetrabutyl atrazine (20%). The sensor surface was regenerated with 100 mM sodium hydroxide in 20% acetonitrile. 

Nakamura et al. demonstrated direct detection of herbicides by using a heavy-subunit-histidine-tagged photosynthesis reaction center (HHisRC) from bacterium Rhodobacter sphaerodies [26] (Fig. 3). They used a Biacore X instrument (from Biacore AB, Sweden) and a sensor chip with dextran matrix... 

Shimomura et al. used a Biacore 2000 SPR sensor instrument (Biacore AB, Sweden) for detection of PCB 3,3 4,4, 5-pentachlorobiphenyl [32]. They employed competition assay format and the sensor chip with polyclonal antibodies immobilized in the dextran matrix. The sample was mixed with a conjugate of PCB-horseradish peroxidase (HRP) and injected into the sensor. The presence of the analyte was detected as a decrease in binding of PCB-HRP conjugate. The detection was performed in 15 min with a detection limit of 2.5 ngmL in buffer. The sensor was demonstrated to be regenerable by 0.1 M hydrochloric acid. 

Detection of heavy metals was demonstrated by Wu et al. who used a Bi-acore X instrument (Biacore AB, Sweden) with rabbit metallothinein coupled to dextran matrix on the sensor chip [48]. Metallothinein is a protein that can be found in the cells of many organisms and is known to bind to metals (especially cadmium and zinc). Model experiments in which metallothein was used as a receptor demonstrated the potential of this sensor to directly detect Cd, Zn, and Ni in buffer at concentrations down to 0.1 jigmL . ... 

An assay for diagnosing type I diabetes mellitus based on the detection of anti-glutamic acid decarboxylase (GAD) antibodies in buffer by a Biacore 2000, is presented in [47,48]. Biotinylated GAD was immobihzed on a streptavidin-coated surface. The effect of mixed SAM composition (differing in ratios of hydroxyl- and carboxyl-terminated alkanethiols) on the sensitivity of the sensor was investigated. On SPR sensor chips prepared with the optimized SAM composition (10 1 ratio of 3-mercaptopropanol to 11-mercaptoundecanoic acid), a concentration of anti-GAD as low as... 

Detection of estrone and estradiol in buffer using SPR sensors Biacore 2000 and Biacore 1000 was carried out by Coille et al. [53]. Analyte-BSA conjugates and BSA were immobilized in the sensing and reference channels of a sensor chip, respectively, using NHS-esters. Analyte concentrations detected using inhibition format in this work were in the range 0.01-3000 ngmL ... 

Nieba L., Nieba-Axmann S. E., Rersson A., Hamalainen M., Edebratt F., Hansson A., Lidholm J., Magnusson K., Karlsson A. F., and Rliickthun A., BIAcore analysis of histidine-tagged proteins using a chelating NTA sensor chip, Anal. Biochem., 252, 217-228, 1997. 

Researchers have recently explored the possibility of combining the BiaCore instrument with MS, in which analytes immobilized on the sensor chip were analyzed with MALDI-TOF directly from the area of the flow cells (Fig. 14.57a) [310,... 

Biacore Cl, FI, and CMS sensor chips (GE Healthcare Life Sciences Pittsburgh, PA)... 

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