While passive absorption techniques have their benefits, manyother strategies are being developed for the surface attachment of proteins. Dr Jean Emly looks at one such strategy, covalent coupling of molecules, proteins, as well as peptides and DNA, and the success it is having with anthraquinone involved in the immobilisation process.
Traditionally proteins have been attached to microtitre plates and other polymer surfaces by passive adsorption. This technique has been used by scientists performing ELISAs and similar techniques in many areas including clinical, pharmaceutical, food safety and pesticide analysis.
Although passive adsorption benefits from the ability to use low-cost plates, the weaknesses such as non-specific binding, non-uniform binding, high reagent consumption and the sheer hands on time that is required has led to the search for alternative methods for surface attachment of proteins.
Covalent coupling of molecules, proteins as well as peptides and DNA is an emerging technology which is rapidly gaining acceptance in the scientific world. Anthraquinone is a photochemically-reactive molecule which is used in this process and is now demonstrating many advances and successes.
Covalent coupling
As a result of forming a covalent chemical bond between the sample and polymer there is an enhanced signal-to-noise ratio which enables greater sensitivity for detection of molecules.
Stringent washing and assay conditions can be employed which reduces or even eliminates non-specific binding.
This technology also enables the user to have greater flexibility since there can be a free choice of format, polymer, sample molecule and even some control of orientation.
As shown in Table 1, the methods for generating the covalent attachments have various benefits and disadvantages.
Significant advances in the development of photochemical methods for covalent coupling have been achieved by a new technology using a quinone as the basis of the coupling reaction. Exiqon, a Danish Biotechnology company, has developed and patented a coupling method using anthraquinone as the basis of the coupling process.
Exiqon manufactures a photoprobe consisting of the anthraquinone molecule (Fig. 1) that has been coupled to an electrophilic group (via an ethylene spacer). This photoprobe is covalently coupled to the polymer surface by a short exposure to UV light. This is basically using the same principle behind the use of suncreams, which are activated by UV light from the sun.
By optimising the spacer design and the density of the electrophilic groups the polymer surfaces can be optimised for sminated nucleic acids, peptides, or proteins.
This flexibility is a significant advantage over passive adsorption, which will work for detection of proteins but is not suitable for the measurement of DNA, RNA or polysaccharides.
In addition the user does not have to pre-activate the surface before introduction of the target molecule and has some control over the orientation of the molecule.
The anthraquinone technology has been incorporated into the Immobilizer range of products. These incorporate DNA, Peptide and Protein Immobilizer surfaces on 96-well, 384-well plates and strips in transparent, black and white polystyrene (Fig. 2).
Flexibility and the future
The Anthraquinone technology, as described above, is already being used for a variety of applications including detection of antibodies against Borrelia burgdorferi (causative agent of Lyme disease) and use in ELISA for detection of human placental lactogen (hPl).
The anthraquinone photocoupling method has furthermore demonstrated its usefulness in the development of extremely sensitive and specific hybridisation assays for single base mutations (for example Factor V Leiden and Apolipoprotein B3500 mutations), and in the development of a Mix -ELISA for detection of Salmonella antibodies in pig meat. Most recently, the method has been used to develop an ELISA for detection of a frequently seen pesticide metabolite in drinking water.
Of great relevance is the flexibility that this technology offers to the researcher-the Anthraquinone technology can be applied to virtually any polymer (PS, PP, PE, PC) , any format (microplates, tubes, dishes, membranes, beads) and is not destructive towards the mechanical and optical properties of the polymer (Fig. 3).
In addition to DNA, peptides or proteins, target molecules such as small organic molecules, haptens and polysaccharides can be used.
Dr Jean Emly is market development manager with Flowgen.
Acknowledgments are due to Dr Hans Monberg of Exiqon for his contribution to the material used in preparation of this article.
For information about Immobilizer products please contact Flowgen on (+44) 0870 6000152
