Immobilization of biomolecules on substrates by attaching them to adsorbed bridging biomolecules

Abstract

An assay article for detection first biomolecules contained in a sample is described. The assay article includes a substrate having a modified surface and a first biomolecule directly adsorbed and immobilized on the modified surface of the substrate without linking moieties. A second biomolecule is bound to or adsorbed on the first biomolecule. Also disclosed is a method of making the assay article. A first biomolecule (other than an adhesive protein) is contacted with a modified surface of a substrate. The substrate is dried to directly adsorb the first biomolecule and immobilize it on the modified surface of the substrate without additional fixing steps to form an activated substrate. Then, a second biomolecule is contacted with the activated substrate under conditions sufficient for the first biomolecule to bind the second biomolecule.

Description

[0001] This application is a continuation-in-part of application Ser. No. 09 / 694,701, filed on Oct. 23, 2000.AREA OF THE ART[0002] The present invention relates generally to solid substrates with immobilized biomolecules. In particular, the invention relates to assay articles with adsorbed first biomolecules to which second biomolecules are bound. The invention also relates to methods of construction of such assay articles and methods of their use in detection of target biomolecules.DESCRIPTION OF THE PRIOR ART[0003] Analysis of unknown biomolecular targets often involves their specific binding to known biomolecular probes. The most common technique employing immobilized biomolecules is the Southern blot hybridization technique, in which a set of DNA targets is immobilized on a membrane and a solution containing labeled DNA probe molecules is used to bathe the membrane under conditions where complementary molecules will anneal. In an analogous technique called Northern blot hybridiz...

AI Technical Summary

Benefits of technology

0029] The present invention provides many advantages. Since the invention allows for the adsorption of first biomolecules directly on a solid substrate without chemical crosslinking, costly production of modified first biomolecules, such as thiol- or amino-modified DNA, may be avoided. Also, the task of making arrays is greatly simplified and the production costs are significantly reduced, because the first biomolecules are simply air-dried on the substrate. Furthermore, even if a particular biomolecule doesn't adsorb well on a particular substrate, it still may be attached to the substrate via first biomolecule. Thus, a broad range of assay devices with various biomolecules attached can be easily prepared in accordance with the present invention.

Problems solved by technology

The in situ method, however, suffers from certain shortcomings.

Consequently, it is hard to obtain accurate and reproducible data with arrays formed by direct synthesis.

However, the derivatization of a glass surface creates a positive electrostatic net charge, which results in undesirable non-specific electrostatic binding of nucleic acids to the solid substrate during subsequent hybridization procedures.

These methods, however, also entail unspecific binding of nucleic acids on the substrates.

Such unspecific binding of DNA makes interpretation of the hybridization results difficult.

However, the derivatization of polynucleotides and their subsequent covalent binding to solid substrates are long and tedious processes.

Consequently, the arrays produced by conventional techniques are fairly expensive ($6-$36 per slide).

Similar problems exist in respect to immobilization of other biomolecules on solid substrates.

In summary, the conventional immobilization methods do not provide desirable fast hybridization and high specificity of binding of targets to probes.

Additionally, currently available methods of manufacturing assay articles for use in the detection of biomolecules are slow, tedious, and economically unfavorable.

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