Medical Device Link.

Originally Published IVD Technology November/December 2001

Membrane immobilization of nucleic acids:
Part 3: Covalent linkage and system manufacturability

The simplicity and rapidity of membrane-based nucleic acid tests are improving but have a long way to go.

Kevin D. Jones

Figure 1. DNA binding to cellulose fibers after cell lysis and washing.

The second part of this article discussed the techniques available to assay developers for using membrane substrates to immobilize nucleic acids in rapid diagnostic applications (IVD Technology, September 2001, page 53). Covalent linkage was identified as a category of techniques having controlled-chemistry qualities with significant potential for use in membrane-based nucleic acid diagnostic systems. This concluding installment describes methods of covalent linkage in some detail (see Figure 1). It ends with an examination of manufacturability issues that present challenges to aspiring developers of membrane-based rapid assays using nucleic acid target materials.

Methods of Covalent Linkage

Direct Coupling. Nucleic acids are inherently unreactive; that is, they will not ordinarily react under normal conditions. If sufficient energy is introduced into the system, however, a nucleic acid can be forced to react with other groups. The direct cross-linking of nucleic acid with proteins under visible light (in the presence of a photosensitizer such as methylene blue) and in UV light (without the need for a sensitizer) has been demonstrated.^1,2

Reactive Membranes. A great deal of interest has been shown in the production of activated membranes, that is, membranes that have direct reaction chemistry available on their surfaces. The ability to apply an aqueous sample directly to the membrane, and to have binding occur naturally without the need for further reagents, offers significant advantages, especially with respect to the production of microarrays. Several different activation chemistries have been tried. The group of chemistries used has typically been something like azide, with generally reactive characteristics; however, all the groups suffer from similar problems.

One problem is loss of activity. Over time, the membrane’s chemically active surface can react with atmospheric gases and lose activity. The more active the chemistry (i.e., the faster the linkage to the target), the greater the rate of loss of activity. To overcome this problem, manufacturers now use less-reactive groups that show a specific reaction with the target probe itself. A typical example would be the reaction between an aldehyde and an amine. Both functionalities are relatively stable.

Another issue involves nonspecific binding. The activated membrane must be blocked prior to use. Otherwise, there will be additional binding of the sample to the membrane. The blocking step used must be simple, quick, and highly efficient. Ideally, it would result in the production of a hydrophilic, uncharged residue on the membrane surface. However, this is not always possible. The blocking step must also be performed after attachment of the target to the membrane. Passivation of the membrane surface will introduce a new chemical functionality. While being unreactive to a nucleic acid, this chemistry may be characterized by an increase in charge or hydrophobic attraction that could result in an increased level of nonspecific signal. The cause of this can be either an increase in binding of the sample or else binding of the detection system.

Available Surface Chemistry. Owing to problems attending the production, storage, and handling of a solid phase that is inherently reactive, reactive membranes are no longer seen as an answer to the goal of covalently linking a probe to a membrane. It is far more common to have a substrate provided with chemistry that will react only with specific chemistry attached to the probe. Such a membrane surface is less reactive and less open to loss of activity with time and storage. The majority of membranes now in the marketplace appear to have either amine or aldehyde groups available for reaction on their surface.

Figure 2. The Schiff’s base reaction between a primary amine and an aldehyde. Subsequent reduction of the Schiff’s base results.

The linkage of amine-containing biomolecules to aldehyde surfaces has been understood for many years. However, the method of linkage to aldehyde membranes is less well known. The advantage of an aldehyde-activated surface is that a significant amount of binding takes place—presumably through the formulation of a Schiff’s base—without the need to add other reagents (see Figure 2). There has been a great deal of discussion about the need to reduce the Schiff’s base purportedly formed. A Schiff’s base is relatively stable; however, the reaction can be reversed under physiological conditions. To ensure the formation of a nonreversible bond, the intermediate Schiff’s base should be reduced with a mild reducing agent, for example, sodium borohydride.

It has been determined, however, that under normal conditions it is unnecessary to reduce the Schiff’s base. Two possible reasons have been put forward to account for this. One suggestion is that an effect similar to the Z principle (discussed in part 2 of this article in connection with physical adsorption) operates for Schiff’s base binding.³ Even though the Schiff’s base reaction is reversible under physiological conditions, because multiple bases have been formed, one base may reverse while other bases continue to provide binding. Thus, the molecule will remain attached. The other hypothesis is based on the observation that there is little amine character in the adduct formed by the addition reaction.⁴ It must be understood that Schiff’s base addition occurs through amine groups. However, it has been shown that the amine groups present upon the native nucleic acids are unreactive because of the presence of purine or pyrimidine ring systems. Any reaction that does occur will therefore be between a terminal amine (e.g., N6-6-aminohexyl(d)ATP or N6-6-aminohexyl(d) CTP) introduced during probe production, and an aldehyde. The orientation of the linked probe is away from the membrane surface, which allows free interaction with any incoming sample.

An oligonucleotide can be produced when either a reactive amine or a reactive thiol group is present. The amine or thiol can be linked to another reactive group. The two common strategies for this are to link to a similar reactive moiety (amine to amine or thiol to thiol), which is called homobifunctional linkage, or to link to the opposite group (amine to thiol or thiol to amine), known as heterobifunctional linkage. Both techniques have been used in the production of labeled nucleic acid probes, and with the availability of membranes with free amine or thiol groups on their surface, other similar applications could be devised. Membranes with amine or thiol surface groups can be purchased from any of several manufacturers or activated by means of standard techniques.

Figure 3. The reaction between amines and glutaraldehyde highlighting the problem with homobifunctional linkers. The second linking reaction would be a competition reaction between any unreacted material from the first amine (R-NH2) as well as the desired reaction with the second amine (R'-NH2).

Traditional attachment techniques, especially for amine groups, have relied upon homobifunctional linkages. One of the most common has been the use of bisaldehydes such as glutaraldehyde (see Figure 3). Glutaraldehyde itself is no longer routinely used. The most common homobifunctional routes now utilize either disuccinimydyl suberate (DSS), which was commercialized by Syngene (Frederick, MD) as synthetic nucleic acid probe (SNAP) technology, or the reagent p-phenylene diisothiocyanate.^5,6 The use of N,N'-o-phenylenedimaleimide supplies an example of the homobifunctional cross-linking of thiol groups.⁷ With all of the homobifunctional cross-linking agents, the probe is initially activated and then added to the secondary material. Although successfully employed to manufacture labeled probes, homobifunctional linkers are losing popularity owing to problems associated with their application in the production of dimeric probes. The nature of a homobifunctional linker—the fact that the same reactive group is at both ends—means that there will inevitably be some reaction between probes during the initial probe activation step. For this reason heterobifunctional linkers have become more popular.

Figure 4. The use of a disulfide exchange reaction in a bifunctional linkage. N-succinidimidyl-3-(2-pyridyldithio)proprionate (SPDP) initially links to a primary amine to give a dithiol-modified compound. This can then react with a thiol to exchange the pyridylthiol (a very good leaving group) with the incoming thiol.

An alternative approach for thiol use has been a thiol-exchange reaction (see Figure 4). If a thiolated probe is introduced onto a disulfide membrane, a disulfide-exchange reaction can occur that leads to the probe being held to the membrane surface via a disulfide bond. This has been demonstrated for the reaction between a thiolated probe and an aldrithiol-treated membrane surface.⁸ The reaction is instantaneous and quantitative. The only problems with this approach relate to the potential for dimerization of the thiolated probe before use. The inclusion of any reagents to stop dimerization via disulfide bond formation would also interfere with the formation of the disulfide bond required to immobilize the probe.

A multitude of potential cross-linking chemistries are available for the heterobifunctional cross-linking of amines and thiols. Generally, these procedures have been used with a thiolated nucleotide. Reagents typically employed have been NHS (N-hydroxysuccinimide ester), MBS (m-maleimidobenzoyl-N-succinimide ester), and SPDP (a pyridyldisulfide-based system).^9,10 Use of SPDP produces a disulfide bond in much the manner of the aldrithiol linkage described above; however, the disulfide bond is internal to the linker rather than between the linker and the probe. The heterobifunctionals now most commonly used rely upon an aminated oligonucleotide. The reagent sulfo-SMCC is finding very wide application owing to its relative stability (see Figure 5).¹¹

Whatever the specific method of achieving the linkage, except for disulfide exchange, covalent linkage is inherently a batch process. The time required for the linking of the target to the membrane and for the subsequent membrane block is finite. Typical reaction times range from about 30 minutes to several hours. This variation makes automation and large-scale production difficult. The situation has been somewhat eased through the introduction of photoactive linking chemistries. A photoactive chemistry can facilitate the use of simple mass-production techniques, however, the chemistry is expensive and relatively rare.

Manufacturability of Membrane Systems

Ideally, any technique used for large-scale manufacturing would be simple, quick, and inexpensive but also reproducible, and it would not interfere with the interactions that allow hybridization. Also essential is that any technique be amenable to quality control; tests subject to FDA regulation would have difficulty passing inspection if the QC of the capture line or testing of incoming substrate materials were overly complicated.

Figure 5. The heterobifunctional linkage reaction between succinimydyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), a primary amine (R-NH2), and a thiol (R'-SH). In a heterobifunctional linkage reaction there can be no opportunity for competition reactions to occur, as seen in homobifunctional reactions.

Because of the shelf-life challenges of surface passivation in highly reactive membranes, it is often more desirable to employ a nucleic acid that has been activated to react with the surface than a surface that is generally reactive toward nucleic acids. The use of available surface chemistry has facilitated simplified protocols and procedures, as it is the membrane itself that undergoes bulk modification. The probes may require some modification before application, but this step can often be carried out during probe production or in the solution phase prior to application.

Judging from protein-based immunoassays and the immature nucleic acid assay market, the manufacture of simple membrane-based systems is most straightforward when the materials can be applied in a continuous process and allowed to bind by drying. Control of the system is easy—for example, inclusion of a dye will show the line coverage—and can quantify the amounts applied. Simple spray application and air-drying methods are well known from the production of traditional lateral-flow assays. Suitable production equipment and a great deal of experience in process control, optimization, and troubleshooting should streamline large-scale manufacture. Employment of either simple or, where suitable, enhanced physical adsorption should result in cheap and rapid production. A bead-based assay, in which a nucleic acid is immobilized onto a large bead that is trapped by size, would also be relatively uncomplicated to produce, provided that the beads were available.

Despite the desirability of spray application and drying from a process and control point of view, there will inevitably be cases where no technique based upon that technology is acceptable. Alternative processes will need to be used. This is especially true for the production of arrays.

The addition of a UV cross-linker to a standard continuous production system should make photolinking of nucleic acids through traditional base-oriented bonding or the use of a specific photochemistry relatively easy. A light source deteriorates with time, so, in order to ensure consistency of cross-linking throughout the lifetime of the production equipment, some kind of photodetector to measure and control the illumination is necessary. Too little illumination will result in incomplete binding. Too much could overlink the nucleic acid and destroy activity. Design of the necessary control mechanism should not be too difficult.

Chemical cross-linking is currently performed as a batch process. The membrane, nucleic acid, and immobilization reagents are incubated for a designated time before termination of the reaction. Although desirable in many ways, this batch step is difficult to justify in a production environment. Costs would probably be significantly higher than for the continuous processes proposed above. There may be some way that this procedure could be modified via plasma or gaseous treatments, but the initial equipment cost would be much greater.

The production of microarrays and bead-based assays represents an obvious exception to the generalization about batch processing. Beads (or microparticles) can be produced very efficiently and cost-effectively in large batches using covalent chemistry methods. The scale of production, and the volume of material required per test, means that the batch processes can cope.

Micro- and macroarrays are assembled in a batch rather than a continuous process, so the requirement to perform covalent linkage in batch fashion is not a limitation. Microarrays are also the only type of system in which the probe can be assembled stepwise upon the surface of the material. The techniques pioneered by Affymetrix (Santa Clara, CA) involve each base being added individually to the surface in the correct sequence. The manufacturing features of microarrays—high cost and batch production—are unique. Their processing methods cannot be seen as a viable alternative for the majority of applications.

Conclusion

Several membrane-based nucleic acid tests are on the market today. The probes are immobilized on a membrane strip that is placed in a solution containing the products of a polymerase chain reaction (PCR). While this technology may be simple and rapid in comparison with some other techniques, it is still a long way from the simplicity and rapidity of familiar test strips. Lateral-flow test strips have been demonstrated for nucleic acid testing by the companies Xtrana (Broomfield, CO) and PanBio Ltd. (Brisbane, Australia), but again, the sample applied is normally a PCR product. Although the test method itself is a lateral-flow type, the problematical steps are performed prior to sample introduction. The tests are therefore multistep in nature and best suited for the professional marketplace.

It is possible to firmly attach a suitable nucleic acid probe to a membrane substrate, by strong physical or covalent binding, and to orient it so that the bases are free to interact with an incoming probe. The problems that must be solved in order to enable the production of a simple, one-step rapid assay are now related to sample collection and preparation, amplification, and the provision of suitably sensitive detection technologies.

Some rapid techniques are now available for sample preparation and amplification; however, many of them are better suited for microfabricated de-vices than for membrane-based assays. Systems for sample preparation (e.g., FTA from Whatman, Isocode from Schleicher & Schuell, or micromachined cell filters) and simple integrated amplification (e.g., flow thermocyclers) are available, but these techniques are not used in traditional lateral-flow assays. A membrane might be serviceable as the detection substrate in a single test or an array. Other techniques, however, may very well be preferable.

The major apparent advantages of membrane-based assays are simplicity and low cost. If membrane-based applications could be developed that couple the simplicity and economy of membrane-based assays with the functional requirements of nucleic acid tests, they would find a large potential market. But if membrane-based assays can be used only as part of a multistep system, it may well be that alternative technologies such as microfabrication and microarrays—despite offering their technological advantages at a cost—will be preferred over the simple immunochromatographic test strip.

Whatever the base technology, the likelihood is that any system will be instrument read. Overcoming the challenges of quantitative-test production may be the key to producing one-step nucleic acid tests. Once a membrane-based test can be made truly quantitative, then perhaps the problems of devising a nucleic acid test will appear less daunting.

References

1. R Lalwani et al., “Visible Light–Induced DNA-Protein Cross-Linking in DNA-Histone Complex and Sarcoma-180 Chromatin in the Presence of Methylene Blue,” Journal of Photochemistry and Photobiology 7 (1990): 57–73.

2. JW Hockensmith et al., “Laser Cross-Linking of Protein–Nucleic Acid Complexes,” in Methods in Enzymology, vol. 208, ed. RT Sauer (London: Academic Press, 1991), 211–236.

3. MB Meza, “Covalent Binding in Diagnostic Test Design,” in The Latex Course 2000 (Indianapolis: Bangs Labs, 2000).

4. FA Quiocho and FM Richards, “The Enzymatic Behaviour of Carboxypeptidase A in the Solid State,” Biochemistry 5 (1966): 4062–4076.

5. E Jablonski et al., “Preparation of Oligodeoxynucleotide-Alkaline Phosphatase Conjugates and Their Use as Hybridization Probes,” Nucleic Acids Research 14 (1986): 6115–6128.

6. MS Urdea et al., “A Comparison of Non-Radioisotopic Hybridization Assay Methods Using Fluorescent, Chemiluminescent and Enzyme Labeled Synthetic Oligodeoxyribonucleotide Probes,” Nucleic Acids Research 16 (1988): 4937–4956.

7. SS Ghosh et al., “Use of Maleimide-Thiol Coupling Chemistry for Efficient Synthesis of Oligonucleotide-Enzyme Conjugate Hybridization Probes,” Bioconjugate Chemistry 1 (1990): 71–76.

8. KD Jones et al., “Comparative Study of Glass Slides versus Microporous Ceramic Slides for Nucleic Acid Arrays” (poster presented at the 32nd Oak Ridge Conference, Boston, May 5–6, 2000).

9. AM Alves et al., “Hybridisation Detection of Single Nucleotide Changes with Enzyme Labelled Oligonucleotides,” Nucleic Acids Research 16 (1988): 8722.

10. A Marukami et al., “Highly Sensitive Detection of DNA Using Enzyme DNA Probes: Colorimetric and Fluorometric Detection,” Nucleic Acids Research 17 (1989): 5587–5595.

11. RD Joerger et al., “Analyte Detection with DNA-Labeled Antibodies and Polymerase Chain Reaction,” Clinical Chemistry 41 (1995): 1371–1377.

Kevin D. Jones, PhD, is the manager for diagnostic technology at Whatman International Ltd. (Maidstone, Kent, UK). He can be reached via e-mail at kjones@whatman.co.uk.

Copyright ©2001 IVD Technology