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Biosensor and method

Biosensor and method description/claims

The following patent applications disclose related subject matter: Ser. No. 09/______, filed ______ (______). These referenced applications have a common assignee with the present application.

The invention relates to (bio)chemical sensors, and more particularly to surface plasmon resonance (SPR) sensors and related methods.

Optical sensors for (bio)chemical detection can provide real-time analysis and typically rely on phenomena such as absorption lines, refractive index changes, and specific binding events. A surface plasmon resonance (SPR) sensor measures changes of refractive index in a dielectric biointerface on a thin conductor using the dependence of the surface plasmon wave vector on the refractive index. Thus with a biointerface including specific binding sites for an analyte, the analyte can be detected quantitatively in a fluid contacting the biointerface due to the change in refractive index by addition of the analyte to the biointerface. Green et al, Surface Plasmon Resonance Analysis of Dynamic Biological Interactions with Biomaterials, 21 Biomaterials 1823-1835 (2000) and Homola et al, Surface Plasmon Resonance Sensors: Review, 54 Sensors and Actuators B 3-15 (1999) describe various types for SPR sensors and applications. Useful pairs of analyte and analyte-specific ligand include antibody-antigen, lectin-carbohydrate, receptor-ligand, DNA-DNA, et cetera.

FIG. 6 illustrates a total internal reflection SPR sensor which illuminates a thin (e.g., 50 nm thick) gold sensor film from the inside with light of wavelength about 800 nm and detects the reflected light with a linear photo-diode array. The refractive index of the biointerface (boundary layer) on the outside of the gold sensor film determines the wave vector of surface plasmon waves at the gold-biointerface: the surface plasmon wave decays exponentially in the dielectric biointerface with a 1/e decay distance of roughly 200 nm. The inside illumination of the gold film covers a range of incident angles due to the geometry of the sensor; and for the angle at which the light's wave vector component parallel the gold film matches that of a surface plasmon wave, the illumination will be resonantly absorbed to excite surface plasmon waves. And the linear photo-diode array detects the angle at which resonant absorption occurs and, inferentially, the refractive index of the biointerface. Indeed, monitoring the refractive index as a function of time during introduction to the biointerface of a fluid containing an unknown quantity of analyte allows analysis of the reaction of analyte with the specific binding sites in the biointerface.

Various biointerface structures functionalized with specific analyte-binding sites have been proposed: self-assembled monolayer (SAM) assembled from thiols with functionalized tail groups, covalently immobilized derivatized carboxymethyl dextran matrix, streptavidin monolayer immobilized with biotin and functionalized with biotinylated biomolecules, functionalized polymer films, and so forth. For example, U.S. Pat. No. 5,242,828 (Biacore) discloses a gold surface with a SAM linker (barrier) film bound to the gold and with analyte-affinity ligands bound (immobilized) to either the SAM directly or to a hydrogel which, in turn, is linked to the SAM. The SAM units have the structure X—R—Y where X binds to the gold and may be a sulfide, R is a hydrocarbon chain of length 12-30 carbons (e.g., (CH2)16) and preferably without branching for close packing, and Y is —OH which binds to derivatized dextran (the hydrogel). Similarly, Lahiri et al, A Strategy for the Generation of Surfaces Presenting Ligands for Studies of Binding Based on an Active Ester as a Common Reactive Intermediate: A Surface Plasmon Resonance Study, 71 Analytical Chemistry 777-790 (1999), shows SAMs with units having structure X—R—Y with X a sulfide, R a hydrocarbon chain, and Y an ethylene glycol chain. And U.S. Pat. No. 6,197,515 discloses a SAM having unit structure X—R-Ch where Ch is a chelating group which binds a metal ion that, in turn, binds an analyte-affinity ligand (binding partner).

Linking an extended hydrogel to the linker film increases the binding capacity of the surface. For example, derivatized carboxymethyl dextran (molecular weight from 10 to 500 kDa) may be covalently linked to the linker film as in Löf{dot over (a)}s et al, Methods for Site Controlled Coupling to Carboxymethylated Surfaces in Surface Plasmon Resonance Sensors, 10 Biosensors and Bioelectronics 813 (1995). This hydrogel increases the binding capacity of the surface by as much as 10-fold. It requires charge preconcentration of the ligand into the hydrogel. This is done by suspending the ligand to be immobilized in a low ionic strength buffer at a pH below the isoelectric point of the ligand. The ligand will be positively charged in this buffer and will rapidly accumulate within the negatively charged hydrogel. Pre-activation of the hydrogel matrix by activating a fraction of the carboxyl groups results in efficient coupling of ligand. The most common activation chemistry employs a mixture of N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS). This produces NHS esters that react with amines (indigenous to most pertinacious ligands), and this is most efficient under basic conditions (e.g. pH 8-9). However, many ligands are acidic and will only become positively charged at very low pH. Therefore, immobilization yields for these ligands are very low to negligible.

SPR-based biosensors monitor the refractive index that results from binding of a target analyte at the biointerface. This refractive index change is proportional to the molecular mass of the target analyte and the number of molecules bound. Hence, for small molecules, or low binding levels, it is sometimes necessary to amplify this primary binding response by using a particle labeled secondary reagent. Gu et al, Enhancement of the Sensitivity of Surface Plasmon Resonance Biosensor with Colloidal Gold Labeling Technique, 5 Supramolecular Science, 695-698 (1998), studies the interaction of Fab′ (human IgG fragment) with a mixture of human IgG and sheep anti-human IgG with SPR; Gu enhances the signal by attaching colloidal gold to the sheep anti-human IgG. The SPR sensor has a biointerface made of a 2-mercapto-ethylamine SAM which amide connects to propionate that, in turn, disulfide connects to the Fab′. The colloidal gold particles increase the SPR signal by a factor of up to 300 as the sheep anti-human IgG binds to the Fab′. However, the stability of colloidal gold and other popular colloidal particles is often poor and stabilizers are required. Also linkage of molecules to these particles is commonly complicated with moderate results.

Cao et al, Preparation of amorphous Fe2O3 powder with different particle sizes, 7 J. Mater. Chem. 2447-2451 (1997) describes extension of Suslick's method of sonication of metal carbonyls to form amorphous iron oxide nanoparticles.

The present invention provides biointerfaces and SPR sensors plus related methods with one or more of the features of a SAM with rigid units adjacent to the assembly surface, reversible entrapment for ligand loading of an interaction layer, and detection amplification or preconcentration with amorphous iron oxide nanoparticles.

These have advantages including increased SPR sensor detection ease and/or sensitivity.

The drawings are heuristic for clarity.

FIG. 1 illustrates in cross-sectional elevation view a first preferred embodiment self-assembled monolayer.

FIG. 2 depicts a functionalized SAM interacting with target analyte.

FIG. 3 is a cross-sectional elevation view of a preferred embodiment biointerface and self-assembled monolayer.

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