BIAcore system

Fig. 10. A SPR Detection realized in a BIAcore system. A fan of polarized light passes a prism and is focused at the interface to an aqueous phase under conditions of total reflection. An evanescent wave enters the solvent phase. If the prism is coated with a thin gold layer at the interface the free electrons in the metal absorb energy from the evanescent wave for a distinct angle, depending on the refractive index of the solvent near the interface. B The gold layer can be modified with, e.g., a carboxydextrane matrix, where catcher molecules can be immobilized by standard chemistry. If a ligand is applied with the aqueous phase it may interact with the catcher and accumulate in the matrix, causing a shift in the resonance angle. If no specific binding occurs the refractive index in proximity of the sensor is less affected...

Pharmacia claims its BIAcore system can provide information on the affinity, specificity, kinetics, multiple binding patterns, and cooperativity of a biochemical interaction on line without the need of washing, sample dilution or labeling of a secondary interactant. Their scientists have mapped the epitope specificity patterns of thirty monoclonal antibodies (Mabs) against recombinant core HTV-l core protein. 

However, the study of hydrophobic proteins using Biacore systems is possible using protocols specially designed for the purpose. For example, proteins may be firstly incorporated into liposomes and then immobilized on hydrophobic sensor surfaces (Sect. 3.3). It has been reported that hpid bilayers have been tethered to a sensor surface via hydrophihc spacers immobilized on a plain gold chip into which membrane-spanning proteins are then inserted [57]. The emphasis of this technique rests on encasing sensitive protein domains within a hpid microenvironment in which they can assume a native, functional structure. 

SPR detection is highly adaptable to multiplexed configurations in miniaturized formats. The flow cells in the original Biacore systems had four measuring spots positioned within a few millimeters (Fig. 19). Prototype systems with eight parallel flow channels have also been described and applied to food analysis apphcations [64]. 

Direct detections of E. coli have been demonstrated since 1998, when Fratamico et al. demonstrated the detection of viable E. coli 0157 H7 using a Biacore system [18]. They compared two sensing platforms, both based on attachment via Ar-ethyl-ArC(diniethylaminopropyl)carbodiimide hydrochloride (EDC) and AT-hydroxy-succinimide (NHS) chemistry. Monoclonal or polyclonal antibody was bound directly to the sensing surface or was immo-Irilized on a bound layer of protein A or protein G. Using a sandwich assay, as seen in Fig. 1, a lower limit of detection of 5-7 x 10 cfu/mL was demonstrated. 

In addition, the Biacore system is fully automatic and incorporates a reliable microflnidic system that facilitates accurate and precise sample delivery and flow-rate manipnlation. Significantly, it also permits multichannel analysis and thns, reference-subtraction [71], which is useful for comparative analyses and is a prereqnisite for kinetic and affinity estimations. A significant advantage of SPR over optical detection techniques is that the incident light energy does not actnally penetrate the bnlk sample and thus, measurements can be made equally on colored or tnrbid solntions and on clear samples [72]. Typically Biacore and SPR/evanescent wave-based technologies have been routinely used for analysis of small molecnles. With specific reference to foodstuffs, these include hormones [9], antibiotic residnes [73], and small molecules that are indicative of microbial contamination snch as microbial toxins [10,46,74]. 

New BIAcore systems such as BIAcore 3000 or TlOO use a different flow cell geometry than the older systems BIAcore 2000 or BIAcore X, further reducing mass transport effects. 

Since the first commercially available Biacore system (Biacore AB, Uppsala, Sweden) was introduced back in 1990, the technology has advanced to become a standard method in studying biomolecular interactions. This Chapter describes the function and practical utilization of this technology, discusses typical user questions and offers suggestions for problem solving. Studies of oligosaccharide interactions are presented, while the whole application range includes all kinds of biomolecules. 

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