IVD Technology Magazine | IVDT Article Index
Originally published March 1996
Covalent immobilization of bio-molecules to preactivated surfaces
Jill K. Veilleux and Lise W. Duran
Development of a solid-phase environment that provides optimum bioactivity without biomolecule loss, displacement, or surface migration is a common goal of research scientists, clinical laboratories, and diagnostic kit manufacturers. In this article, researchers from two manufacturers describe preactivation of multiple-well polystyrene plates by means of a patented photochemical coupling technique.
Choosing the correct surface as a solid phase is a critical step in assay development. Molecules may be immobilized either passively through hydrophobic or ionic interactions or covalently by attachment to activated surface groups. Noncovalent surfaces are effective for many applications; however, passive adsorption fails in many cases.1 Covalent immobilization is often necessary for binding molecules that do not adsorb, adsorb very weakly, or adsorb with improper orientation and conformation to noncovalent surfaces. Covalent immobilization may result in better biomolecule activity, reduced nonspecific adsorption, and greater stability.14
There are a number of ways to modify solid supports for the covalent immobilization of biomolecules. This article focuses on preactivation of surfaces by a patented process that covalently attaches a specific reactive group to the solid phase via a photolinkable spacer arm (see Figure 1). (Figures not yet available on-line.)
Surface modification can be accomplished by a variety of chemical and physical approaches (see Table I), including the following:
* Addition of amino groups by fuming of nitrous acid.5
* Bromoacetylation.6
* Oxidation by use of plasma, ultraviolet (UV) light, or electron beams as energy sources in the presence of oxygen and air.710
* Chemical grafting.11
* Glutaraldehyde coating.12,13
* Latex paint coating.
* Noncovalent attachment of an affinity spacer to aromatic groups.14
The first four processes listed produce covalent bonds but are inconvenient, hazardous, or expensive, and require high energy or harsh reaction conditions. The last three produce noncovalent bonds to solid supports that usually provide poor reproducibility and/or unstable association with the surface.
Photochemical Coupling
The photochemical coupling technique permits the covalent bonding of reagents to most organic plastics quickly under gentle reaction conditions and does not require expensive equipment.15 The photochemical groups used in this technique are stable to most synthetic organic reaction conditions. Upon UV irradiation, the photogroups form highly reactive intermediates that couple readily to surfaces that intrinsically contain or that have been pretreated to contain carbon-hydrogen bonds.
The coupling occurs via abstraction of the hydrogen from the surface and recombination of the resulting two radicals. The process allows synthesis of a variety of heterobifunctional cross-linking reagents containing a latent reactive group (usually a photochemical one, such as benzophenone), a spacer group, and a thermochemical group. The latter group is used for coupling to the biomolecule to be immobilized (see Figure 2).
Selection of the proper spacer group (length, polarity, etc.) allows covalent immobilization of biomolecules on most plastic surfaces. Hydrophilic spacers shield the biomolecule from the aromatic, hydrophobic surface, greatly reducing nonspecific adsorption (background signal) and biomolecule inactivation over time. This method of immobilization overcomes the problems of slow release (leaching) and lateral migration (clustering) of biomolecules that may occur with noncovalent bonding.
Selecting the Solid Phase
Solid phases come in a variety of forms--membrane filters, beads (including latex and paramagnetic particles), glass slides, silicon wafers, and multiple-well plates. Each has unique surface characteristics that can affect the performance of an assay.
The goal of surface modification is maximization of performance. The critical assay parameters that determine the success of the surface modification and, ultimately, the commercial success of the assay are as follows:
* The ability to achieve targeted sensitivity.
* Maximum specificity with minimal nonspecific adsorption.
* Adequate reactions and kinetics of the ligand with its target molecule.
* Stability of the ligand, which increases reproducibility and extends shelf life.
* Ease of use.
The photochemical coupling technique can modify all types of the solid supports used in assays. This effect includes modification of inorganic surfaces such as silicon, glass, and most metals that can be pretreated with silane to provide the carbon-hydrogen bonds necessary for the photochemical coupling reaction. The remainder of this article focuses on the photochemical modification of 96-well plates.
Selection of the solid phase is often influenced by the availability of compatible instrumentation and robotic systems. Polystyrene multiple-well plates have gained widespread acceptance in part because pipetting, washing, and signal detection are easily automated. Other advantages include the ability to analyze multiple samples simultaneously and compatibility with a number of different detection systems (e.g., colorimetric, fluorescent, and chemiluminescent).
Surfaces
Using BSI Corp.'s (Eden Prairie, MN) photochemical modification technology, Corning Costar (Kennebunk, ME) is able to preactivate polystyrene multiple-well plates for the specific and covalent immobilization of biomolecules. This covalent attachment of reactive groups produces the following four very stable surfaces:
* The N-oxysuccinimide (NOS) surface that covalently couples to amine groups.
* The maleimide surface for covalently coupling sulfhydryl (SH) groups.
* The hydrazide surface that is reactive toward periodate-activated carbohydrates.
* The universal for covalently immobilizing any molecule pos- sessing aliphatic carbon-hydrogen bonds.
N-oxysuccinimide. The NOS surface reacts almost exclusively with primary amine groups, with the exception of mercaptans. This nucleophilic substitution reaction covalently immobilizes biomolecules via available amine moieties by forming stable amide bonds, as shown in Figure 3. Covalent immobilization can be achieved in as little as 30 minutes. No surface preparation or sample preparation is required. The NOS surface is useful for binding small antigens, peptides, enzymes, and aminated DNA.
As interest in DNA probes for the rapid and specific diagnosis of genetic and infectious diseases has grown, finding the right surface for immobilizing DNA has gained importance. Use of the NOS-activated surface minimizes the challenges associated with immobilization of DNA for use in DNA probe assays. This surface has been shown to immobilize 5' amine-modified oligonucleotides (oligos), providing an ideal template for hybridization and amplification. The DNA is bound at one end rather than at numerous sites along the molecule, which results in high specificity and extremely low background.
The data shown in Figure 4 demonstrate the covalent immobilization of a synthetic oligo, aminated at the 5' end via a C6 linker, to the NOS surface. The oligonucleotide was immobilized for 1 hour at 37°C in 50-mM phosphate buffer (pH 8.5) containing 1-mM ethylene diamine tetraacetic acid (EDTA). After the wells were washed three times with 100-mM Tris (pH 7.5) and 150-mM NaCl, the unreactive NOS groups were blocked with 10-mM Tris and 1-mM EDTA buffer for 30 minutes at 37°C. A biotinylated antisense detection probe was used to examine capture-probe coupling. Measurements were made using a standard streptavidinalkaline phosphatase detection system.
The results indicate that there is a tenfold increase in absorbance for DNA covalently bound to the NOS surface as compared with that which is passively bound to a raw polystyrene surface. Hybridization is more efficient when the capture probe is covalently bound, suggesting that NOS-immobilized oligos are suitable for the rapid detection of DNA in diagnostic assays.
Maleimide. The second surface is the maleimide, which is intended to immobilize biomolecules through available SH moieties. This surface has proved useful for the site-specific immobilization of antibodies, Fab fragments, peptides, and SH-modified DNA.
Sample preparation for the maleimide surface involves the simple reduction of disulfide bonds between two cysteine residues on a protein by use of a mild reducing agent, such as dithiothreitol, 2-mercaptoethanol or tris(2-carboxyethyl)phosphine hydrochloride. The modification of primary amine groups with 2-iminothiolane hydrochloride (Traut's reagent) to introduce sulfhydryl groups is an alternative for biomolecules lacking them. Free sulfhydryls are immobilized to the maleimide surface as shown in Figure 5.
The maleimide surface was tested for the site-specific immobilization of sulfhydryl groups by using a goat IgG Fc fragment, either unmodified or modified with Traut's reagent. Specific immobilization through sulfhydryl groups was detected with a horseradish peroxidase (HRP)labeled rabbit antigoat IgG Fc. The site-specific immobilization of Traut's modified Fc is demonstrated in Figure 6. There is a clear indication of sulfhydryl-specific immobilization on the maleimide surface. Results show a sevenfold increase in absorbance for Traut's modified Fc as compared to unmodified Fc.
Hydrazide. The hydrazide surface is designed for covalent coupling of periodate-activated carbohydrates or glycosylated biomolecules. This surface has proved beneficial for the site-specific immobilization of antibodies, carbohydrates, glycolipids, glycoproteins, and many enzymes.
Site-specific immobilization can greatly increase sensitivity by orienting the molecule (e.g., antibody) so that the receptor sites are available for antigen binding. Antibodies are immobilized to the hydrazide surface through the carbohydrate moieties on the Fc region, which allows the Fab regions to orient properly (Figure 7).
The hydrazide surface was tested for the site-specific immobilization of periodate-activated carbohydrates by immobilization of alkaline phosphatase, a glycoprotein that does not readily adsorb to raw polystyrene. The results of immobilization of periodate-activated and nonactivated alkaline phosphatase to the hydrazide surface and to a high-binding surface are shown in Figure 8. Binding of periodate-activated alkaline phosphatase to the hydrazide surface is 16 times that of nonactivated alkaline phosphatase. The amount of alkaline phosphatase covalently bound to the hydrazide surface is also considerably more than can be passively bound to the high-binding surface.
Universal. The fourth covalent surface, the universal, contains a proprietary photoactivatable linker. This surface can immobilize any molecule possessing aliphatic carbon- hydrogen bonds from which hydrogen can be abstracted by the activated photogroup via UV illumination. Although the linkage is nonspecific and does not allow for site-directed orientation of a biomolecule, this surface is useful for the covalent immobilization of cell lysate, antigens of unknown structure, double-stranded DNA, and nonproteinaceous molecules, such as lipids.
The universal surface was tested for the immobilization of insulin, a small polypeptide that binds weakly via passive adsorption. The insulin was incubated for one hour on two universal plates and one raw-polystyrene high-binding plate. One of the universal plates was then subjected to UV cross-linking, which covalently immobilizes the ligand.
The insulin immobilized without UV activation to the universal surface is bound through hydrophobic interactions alone. The insulin immobilized to the polystyrene high-binding surface is bound through both hydrophobic and ionic interactions.
The bound insulin was then detected with an HRP-labeled sheep antiinsulin antibody. As is shown in Figure 9, the strongest signal was obtained for insulin covalently immobilized via UV cross-linking to the universal covalent surface.
Conclusion
Collectively, these findings show that covalent immobilization by BSI Corp.'s photochemical coupling approach on Corning Costar multiple-well plates increases the activity of DNA, antibodies, enzymes, and small polypeptides over that seen on unmodified polystyrene. These preactivated surfaces can be incorporated by diagnostic device manufacturers to better serve the end-user's needs.
References
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Jill K. Veilleux is immunology and molecular biology project manager at Corning Costar (Kennebunk, ME). Lise W. Duran, PhD, is the director of microbiology R&D at BSI Corp. (Eden Prairie, MN).
